ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group

Transcription

ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group - Immunology/Microbiology
111 Antibiotics and anti-microbial agents
Sunday, May 03, 2015 8:30 AM–10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 264–287/C0149–C0172
Organizing Section: Immunology/Microbiology
Contributing Section(s): Clinical/Epidemiologic Research, Cornea,
Eye Movements/Strabismus/Amblyopia/Neuro-Ophthalmology,
Physiology/Pharmacology, Retinal Cell Biology, Retina
Program Number: 264 Poster Board Number: C0149
Presentation Time: 8:30 AM–10:15 AM
The In Vitro and In Vivo Antibacterial Evaluation of Brilacidin
Regis P. Kowalski1, Eric G. Romanowski1, Robert M. Shanks1,
Kathleen Yates1, Francis S. Mah1, 2. 1Ophthalmology, The Charles T.
Campbell Lab - UPMC, Pittsburgh, PA; 2Ophthalmology, ScrippsHealth, San Diego, CA.
Purpose: Brilacidin (BRI) (PMX30063) is the first anti-infective
in a new class of defensin mimetics. The goals of the study were to
evaluate the in vitro and in vivo efficacy of BRI as an ocular antiinfective.
Methods: In vitro: MICs using broth dilution were determined
for clinical ocular isolates (n = 25 per bacteria) of Ciprofloxacin
Susceptible (CS) Staphylococcus aureus (CSSA), Ciprofloxacin
Resistant (CR) Staphylococcus aureus (CRSA), CS Staphylococcus
epidermidis (CSSE), CR Staphylococcus epidermidis (CRSE),
Streptococcus pneumoniae (SP), Streptococcus viridans group
(SV), Moraxella Species (MS), Haemophilus influenzae (HI),
Pseudomonas aeruginosa (PA), and Serratia marcescens (SM). In
vivo: In 24 NZW rabbits, corneal epithelial defects were created OS
(abraded corneas), while the epithelium OD remained intact (intact
corneas) to determine the effect of drug penetration. The corneas
were intrastromally injected with 1000 CFU of MRSA. Rabbits were
separated into 4 groups (n=6): A) BRI 0.5%, B) Vancomycin (VAN)
5%, C) saline (SAL), and D) no treatment (baseline CFU). Four hrs
after MRSA challenge, topical treatment of one drop every 15’ for 5
hrs was initiated. One hr after treatment the corneas were harvested
for CFU. The data were non-parametrically analyzed.
Results: In vitro: Data is expressed as MIC50, MIC90, and Range
of MICs in μg/ml. CSSA (0.25, 0.25, 0.125-0.5); CRSA (0.25, 0.5,
0.125-1.0); CSSE (0.125, 0.25, 0.03125-0.25); CRSE (0.125, 0.25,
0.03125-0.25); SP (1, 1, 0.5-128); MS (4, 64, 0.5-128); HI (8, 8,
2-32); PA (4, 4, 0.5-8); SM (8, 32, 0.25-32). In vivo: For abraded
corneas, VAN and BRI produced similar reductions in MRSA
CFU, and were less than saline (P<0.05, K-W). However, only
BRI demonstrated a 99.9% reduction compared to baseline CFU.
For intact corneas, VAN significantly reduced CFU compared with
BRI which demonstrated a slight but significant decrease vs. SAL
(P<0.05, K-W). BRI reduced CFU in abraded corneas significantly
more than intact corneas (P<0.05, M-W) suggesting the corneal
epithelium acts as a barrier for penetration. There was no difference
in CFU in abraded and intact corneas with VAN (P>0.05, M-W)
suggesting high penetration through the corneal epithelium.
Conclusions: BRI demonstrated broad spectrum in vitro activity
against ocular pathogens. BRI was equally efficacious as VAN in
a MRSA keratitis model only when the corneal epithelium was
removed.
Commercial Relationships: Regis P. Kowalski, PolyMedix (F);
Eric G. Romanowski, PolyMedix (F); Robert M. Shanks, None;
Kathleen Yates, None; Francis S. Mah, PolyMedix (F)
Support: Industry - PolyMedix
Molecular typing and antimicrobial resistance of ocular
methicillin-resistant Staphylococcus aureus
Ana L. Hofling-Lima1, Katiane Santin2, Smairah F. Abdallah2,
Roberta C. Mingrone2, Marcus V. Gaspari2, Antonio C. Pignatari2,
Paulo J. Bispo2. 1Ophthalmology, Universidade Federal de Sao Paulo,
Sao Paulo, Brazil; 2LEMC, Universidade Federal de Sao Paulo, Sao
Paulo, Brazil.
Purpose: To evaluate the molecular epidemiology and antimicrobial
resistance profile of methicillin-resistant Staphylococcus aureus
(MRSA) isolated from conjunctivitis, keratitis, dacryocystitis and
blepharoconjunctivitis.
Methods: 42 MRSA isolates from patients treated at the Visual
Sciences and Ophthalmology Department, Federal University of
Sao Paulo from January 2007 to May 2013 were included. PCR to
nuc, mecA and luk genes was performed in order to confirm species
identification, methicillin resistance and presence of the PantonValentine leukocidin (PVL). Staphylococcal Cassette Chromosome
(SCCmec) typing was assessed by multiplex PCR. Macrorestriction
profile of chromosomal DNA was performed by pulsed field gel
electrophoresis (PFGE). Minimum inhibitory concentration (MIC)
to oxacillin, ciprofloxacin, moxifloxacin, gatifloxacin, linezolid and
vancomycin was determined by E-test.
Results: SCCmec type IV (40.5%) and type II (35.7%) were the
most prevalent. MRSA type IV grouped in 4 different clusters by
PFGE, while type II isolates formed only 2 clusters, with 73.3%
belonging to the cluster A. Only 1 isolate was SCCmec type I; 2 type
V; 3 type III; and 4 were non-typable. MRSA types IV and V were
most frequently recovered from conjunctivitis (58.8%) and presented
higher susceptibility to the fluoroquinolones compared with types
I, II and III. MRSA type II were predominant in keratitis (80%) and
were 100% resistant to the fluoroquinolones tested. MRSA type III
were recovered from previously hospitalized patients and were all
related to the brazilian epidemic clone. All isolates were susceptible
to vancomycin and linezolid. All specimens were negative for the
presence of luk gene.
Conclusions: MRSA types IV and V usually correlated to
community-acquired infections (CA-MRSA) were the most
susceptible to fluoroquinolones. We demonstrated that SCCmec
types II and III, which are mainly associated with hospital-acquired
infections (HA-MRSA), may be causative agents of ocular infections.
The last were susceptible only to vancomycin and linezolid.
Commercial Relationships: Ana L. Hofling-Lima, None; Katiane
Santin, None; Smairah F. Abdallah, None; Roberta C. Mingrone,
None; Marcus V. Gaspari, None; Antonio C. Pignatari, None;
Paulo J. Bispo, None
Support: do not use
Comparison of the efficacy of 5% or 10% povidone-iodine (PVI)
irrigation in combination with moxifloxacin 0.5% as prophylaxis
in patients undergoing cataract surgery
Adriana Caballero1, Veronica E. Castillo1, Margarita Samudio3,
Norma Farina3, Agustin Carron1, Herminia Mino de Kaspar2,
Florentina Laspina3, Sonia Abente3, M.Rosa Sanabria3, Diogenes
Cibils1. 1Department of Ophthalmology, National University of
Asunción, Asuncion, Paraguay; 2Ludwig-Maximillians University,
Munich, Germany; 3Research Institute for Health Sciences. National
University of Asunción., Asuncion, Paraguay.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
Purpose: To compare efficacy of 5% or 10% PVI irrigation in
combination with moxifloxacin to decrease bacteria recovery on the
conjunctiva before cataract surgery.
Methods: After approval by the IRB of Faculty of Medicine of the
National University of Asunción, Paraguay, cataract patients were
asked to participate in this prospective study from september 2011 to
august 2013. All patients received in the eye to be operated topical
moxifloxacin 4 times one day before surgery, then were randomly
assigned to receive 5% PVI (5% PVI-Group ), or 10% PVI (10%
PVI-Group). In both groups the conjunctiva was flush-irrigated with
10ml of the respective PVI concentration. Conjunctival specimen was
obtained at 3 timepoints: T0 = no-surgery eye (control), T1 = surgeryeye, both before PVI irrigation, T2 after PVI irrigation, T3 at the end
of surgery. All specimens were inoculated into thioglycolate-broth.
Results: A total of 120 patients completed the study: 61 (5%PVIGroup ), 59 (10%PVI-Group). Preoperative use of moxifloxacin (T1)
reduced bacteria recovery in 19% (n=82/120, 68.3%) as compared to
the eye control T0 (59/120, 49.2%), p=0.009. After PVI irrigation, a
strong reduction of conjunctival bacterial recovery was observed in
both groups, but difference in positive cultures was not significant
comparing 5%PVI-Group (7/61, 11.5%) to 10%PVI-Group (10/59,
16.9% ) (P = 0.39). At the end of surgery, bacteria recovery was
similar in both groups (14/61, 23.0% and 14/59, 23.7%, (P = 0,92) .
Coagulase-negative Staphylococcus was the most isolated bacteria
with similar frequency in both groups. No corneal defect due to PVI
occurred in any patients.
Conclusions: The preoperative irrigation with 10% PVI in
combination with moxifloxacin showed similar effectiveness to 5%
PVI in decreasing bacteria recovery.
Commercial Relationships: Adriana Caballero, None; Veronica
E. Castillo, None; Margarita Samudio, None; Norma Farina,
None; Agustin Carron, None; Herminia Mino de Kaspar, None;
Florentina Laspina, None; Sonia Abente, None; M.Rosa Sanabria,
None; Diogenes Cibils, None
Susceptibility and resistance of Staphylococcus epidermidis ocular
strains in a Hispanic population
Jaime Torres1, Alejandro F. Ibarra-Lozano1, Pedro M. Gonzalez1,
Jorge E. Valdez2, 1. 1Ophthalmology Institute, Tecnologico de
Monterrey, Monterrey, Mexico; 2Ophthalmology Research Chair.
School of Medicine and Health Sciences, Tecnologico de Monterrey,
Monterrey, Mexico.
Purpose: To identify the pattern of susceptibility of ocular
Staphylococcus epidermidis strains to antibiotics in the Northeast
region of Mexico in order to offer most effective treatments for
ocular infections.
Methods: This is an observational retrospective series of cases study
of the microbiological cultures obtained from different ocular sites in
which S. epidermidis was isolated, in the Microbiology Department
of Hospital San Jose, Tecnologico de Monterrey, Mexico. The time
lapse studied was from January 2002 to December 2012. A total of 62
positive cultures for S. epidermidis were obtained, in which antibiotic
testing was reported as susceptible or resistant with the results
expressed as percentages. Classifications for the antibiotics were
made according to the Minimum Inhibitory Concentration (MIC)
reported by the Microbiology Department.
Results: A total of 62 eyes were included in the study. The average
age of the patients was 28.5 (range 0-79 years old). The most
effective antibiotics according to the percentage of the susceptible
strains were: Rifampin 100%, Vancomycin 97%, TrimethoprimSulfamethoxazole 82%. The most potent antibiotics according to the
MIC average of susceptible cases: Oxacilin 0.3 mg/mL, Clindamycin
0.41 mg/mL, and Erythromycin 0.6 mg/mL. The most common sites
of sample isolation were: eye secretion (72.4%, 45 cases), vitreous
(9.6%, 6 cases), and corneal ulcer (4.8%, 3 cases). A smaller number
of samples were collected from corneal scrape, eyelid border, contact
lens, conjunctiva, and aqueous humor.
Conclusions: The most effective antibiotic against Staphiloccoccus
epidermidis isolated from ocular infection sites in the Northeast of
Mexico was Oxacilin, followed by Clindamycin and Erythromycin.
This is the first study to determine ocular S. epidermidis susceptibility
to antibiotics in our population. The results obtained from this
research, will allow to offer most effective treatment for ocular
infections.
Commercial Relationships: Jaime Torres, None; Alejandro F.
Ibarra-Lozano, None; Pedro M. Gonzalez, None; Jorge E. Valdez,
None
Blue Light /Riboflavin-mediated Elimination of Methicillinresistant Staphylococcus aureus, Using an In Vitro Model of
Keratitis Treatment
Anders Backman1, Raymond Goodrich2, Karim Makdoumi3, 4.
1
Clinical Research Center, University Hospital, Orebro, Sweden;
2
TERUMO BCT, Denver, CO; 3Ophthalmology, University Hospital,
Orebro, Sweden; 4Centre for Health Care Sciences, Orebro, Sweden.
Purpose: Antibiotic resistant bacteria could cause keratitis treatment
failures. Blue light has better properties than the used 365nm
light, used in collagen crosslinking (CXL) and suggested therapy
of keratitis. We wanted to investigate the in vitro effect of two
different Blue lights/Riboflavin-mediated photo-oxidative stress
on a pathogenic bacteria with antibiotic resistance properties in a
crosslinking (CXL) treatment model.
Methods: Staphylococcus aureus(MRSA)(CCUG41586/
ATCC43300, Methicillin resistant), were cultured and suspended
in PBS to around 4 x 105/mL. The suspension (10 mL) was mixed
with 50 mL of riboflavin (RF)(Sigma) solution in RPMI (GIBCO) to
0.01% RF. This was pipetted (15 mL) onto 7 mm wells on microscope
slides (CEL-30-2325BLHTC, Thermo Scientific), forming a 0.4
mm layer, exposed 9-10 min to 28.4J or 5.4J of blue light; 412 nm
(TRIA-Beauty lamp Mod: SPBL, CA,USA) or 450 nm ( prototype
lamp,TERUMO BCT, Denver, Colorado). The effect on a deeper fluid
layer (1.14mm) was tested in a 40 mL well similarly. Samples were
withdrawn (5 mL) from the wells and diluted in PBS and spread on
Blood agar plates, incubated, and the CFU- counts were calculated.
The mean results (n=8) were compared to simultaneous controls with
and without light treatment(T-test).
Results: MRSA was efficiently eliminated similarly by 412nm or
450nm+ RF at high dose (99%/98%). A lower dose of 412nm/450nm
+RF reduced MRSA much less significantly (55%/44%)(P0.0002).
The effect of light only treatment reduced the CFU equally efficient
at high dose (79 and 81%). A low dose of light only (412 or
450nm) had no significant effect (P= 0.1). Blue light of 412nm was
effective on 1.14mm layers and reduced bacteria at high dose+/-RF
(93%/83%) and low dose +RF (67%)(P0.00001).
Conclusions: MRSA could be reduced almost completely by
exposure to Blue light (412 or 450nm) at high dose (28J) +/- RF, in
this treatment model. The safe CXL dose of 5.4J (365nm) applied
with 412nm +RF (10 min) reduced bacteria by 55%-67%. A longer
exposure (CXL =30 min.) and lower irradiation setting would
probably increase the efficacy(x 1.5) as seen previously. This could
perhaps also be a possible way to improve treatment of keratitis in
cases involving antibiotic resistant bacteria, perhaps with superior
effect compared to standard CXL in deeper infections. Further studies
are needed to evaluate this.
Commercial Relationships: Anders Backman, None; Raymond
Goodrich, TERUMO BCT (E), TERUMO BCT (F); Karim
Makdoumi, None
Role of lysozyme as an antibacterial in tears
Katie da Silva-Antunes, Poonam Mudgil, John Whitehall. University
of Western Sydney, Camden, NSW, Australia.
Purpose: Tears contain a number of antimicrobial proteins that help
in protecting the ocular surface from microbial pathogens. Among
these lysozyme is the major protein. Given its abundance in tears, it is
expected to play a major role in tear defence. To investigate it further,
this study aimed at studying the efficacy of lysozyme against ocular
pathogenic bacteria to evaluate its role in antimicrobial protection of
the ocular surface.
Methods: The inhibition of growth of the ocular pathogenic bacteria
(Staphylococcus aureus 31 and Pseudomonas aeruginosa 19) by a
range of concentrations of lysozyme was tested in Mueller-Hinton
broth using the broth dilution method. Time-kill assay was performed
to observe total viable counts of bacteria surviving the lysozyme
treatment. Scanning electron microscopy was used to observe the
morphology of cells treated with lysozyme.
Results: Lysozyme inhibited bacterial growth only to a small extent.
A low percentage of growth inhibition (9 to 18% for 2.5 to 10mg/
mL of lysozyme) was observed for Staphylococcus aureus 31,
and a very little growth inhibition (2 to 6% for 2.5 to 10mg/mL of
lysozyme) was observed for Pseudomonas aeruginosa 19. Time kill
assay also showed that the bacterial cell death was not much affected
by lysozyme. Scanning electron microscopy showed similar cell
morphology for control and treated cells except that there was loss of
aggregation in the treated cells in case of Staphylococcus aureus 31.
Conclusions: In spite of its abundance in tears, lysozyme in not an
efficient antimicrobial of the ocular surface by itself. This suggests
that other proteins either individually or in combination with
lysozyme might be more effective in protecting the ocular surface
from pathogens. This will be tested in further investigations to
understand the role of different proteins in tear defence.
Commercial Relationships: Katie da Silva-Antunes, None;
Poonam Mudgil, None; John Whitehall, None
The spectrum and antibiotic resistance profile of bacteria found
on the ocular surface –
a 10 year report from the University Eye Hospital Düsseldorf
Mathias Roth1, David Finis1, Kristina Spaniol1, Colin MacKenzie2,
Gerd Geerling1. 1Ophthalmology, University of Dusseldorf,
Dusseldorf, Germany; 2Microbiology, University Düsseldorf,
Düsseldorf, Germany.
Purpose: Effective initial treatment is of great importance in patients
with infectious keratitis, since if inadequately treated the duration of
the disease might be prolonged and the visual outcome potentially
severely decreased. Optimal empiric antibiotic therapy requires
knowledge of the current bacterial spectrum and susceptibility of
these bacteria to the antibiotics in use. We thus analyzed the data
from the last 10 years at our clinic.
Methods: All ocular surface specimens worked up in the medical
microbiology department of the University Düsseldorf between 2005
and 2014 were analyzed. Special attention was paid to resistance
to antibiotics relevant in ophthalmology and the possible change in
both bacterial spectrum and resistance profile over the study period.
Statistical analysis was performed with SPSS 21.0 applying nonparametric tests (level of significance: p % 0,05)
Results: Over the 10 year period the number of swabs increased
significantly. In total, 430 of 2897 collected specimens from 1913
patients showed a positive culture result (14.8 %). Gram-positive
species were found in 252 specimens, Gram-negative species in 145
specimens and fungi in 33 specimens. The most common organisms
were: Staphylococcus aureus (22 %), Pseudomonas spp. (14%),
coagulase-negative Staphylococcus (14%) and Streptococcus spp.
(10%). The resistance profile over the study period only changed for
erythromycin from 2009 – 2010 (p=0.031).
For the most frequently used antibiotics, the fluoroquinolones
and aminoglycosides, a resistance rate of ~ 10 % was found. A
combination of those 2 antibiotic agents yielded a resistance rate of
~ 5 % (gentamicin + levofloxacin: 4,6%; gentamicin + moxifloxacin:
5.8%).
Conclusions: Our analysis revealed that the number of
microbiological tests increased over the 10 year period, which
underlines the growing importance of quality assurance and
evidence-based targeted therapy. The bacterial spectrum in ocular
surface swabs in the University eye clinic Düsseldorf is similar
to the spectrum reported in other comparable centers. Before
determination of the pathogen a combination of aminoglycosides and
fluoriquinolone seems to be the most effective for the initial therapy
in suspected bacterial ocular surface infection.
Commercial Relationships: Mathias Roth, None; David Finis,
None; Kristina Spaniol, None; Colin MacKenzie, None; Gerd
Geerling, None
Molecular and Mupirocin Profiles of MSSA and MRSA Isolates
Associated with Chronic/Recalcitrant Ocular Disease
Darlene Miller, Jorge Maestre, Edith Perez, Ben D. Wilson, Harry
W. Flynn, Eduardo C. Alfonso. Bascom Palmer Eye Institute, Univ of
Miami Miller Sch of Med, Miami, FL.
Purpose: To document the molecular characteristics and mupirocin
profiles of Staphylococcus aureus isolates recovered from chronic
and or recalcitrant ocular disease.
Methods: We used three separate multiplex PCR reactions to
characterize molecular profiles for SCCmec, Panton Valentine
leukocidin (PVL) toxin and the global accessory gene regulator
(ARG) for 135 isolates recovered (2011-2014) from 52 patients with
chronic staphylococcal disease (persistent colonization/recovery of
S. aureus after a treatment course of at least 14 days). Mupirocin
MIC profiles (N=60) were documented using E tests. Resistance was
categorized as high level (HL), >512 ug/ml, low level (LL), 8-256 ug/
ml and susceptible (SL), less than 8 ug/ml.
Results: .The average number of repeat isolates per patient averaged
2.9 with an average time interval of 174 days (range 10-810 days).
Reservoirs for repeat infections included conjunctiva (38.5%), cornea
(16.3%), lacrimal system (11.1%), lids (10.4%), socket (9.6%) and
ocular other (14.1%). Methicillin susceptible isolates were recovered
from 25.9% (n=35) of patients.. The majority of the MRSA isolates
were healthcare associated (n=66/75, 88%). SCCmec profiles
included SCCmec I (4.5%), SCCmec II-95.5% (N= 66/75, 88%,) .
Community associated MRSA profiles (n=28) were all SCCmec IV,
(IVa-39.3%, IVd-60.7%). 4,4% of isolates were untypable. The PVL
toxin was present in 94.8% of isolates. ARG type 2(51.8%, n=70)
was the most common ARG genotype followed by ARG 1 (29.6%),
ARG 3 (14.8%), ARG 4 (0.07%) and nontyable (3%). Isolates with
genotype: SSCmec II, ARG2 and PVL + (50.3%, n=68) served as
markers for chronic ocular disease. High level mupirocin resistance
was found in 6.7% (4/60) of isolates (conjunctiva-2, cornea-2).
Low level resistance was document in 2 (3.3%-conjunctiva, lids).
Resistance occurred more frequently in MRSA isolates (5/6, 83.3%).
General in vitro susceptibility for mupirocin was 90% (n=54/60).
Conclusions: Unique molecular profiles may serve as markers for
chronic ocular staphylococcal disease and high level resistance to
mupirocin. Mupirocin could be useful in decolonizing and reducing
ocular surface reservoirs.
Commercial Relationships: Darlene Miller, None; Jorge Maestre,
None; Edith Perez, None; Ben D. Wilson, None; Harry W. Flynn,
None; Eduardo C. Alfonso, None
Support: RPB Unrestricted Award, NIH Core Grant P30EY014801
Biocidal Efficacy of a New Hydrogen Peroxide Disinfecting
Solution Against Clinical Bacterial and Yeast Isolates, and
Acanthamoeba Species
Manal M. Gabriel, Cindy McAnally, Rhonda Walters, Linda Clark,
Monica Crary, John Bartell, Bradley Catalone. Vision Care, Alcon
Labs, Fort Worth, TX.
Purpose: Lens care products are currently evaluated for efficacy
against a panel of microorganisms, which includes five laboratory
maintained strains representing keratitis-causing species. This study
evaluated the efficacy of a new Hydrogen Peroxide lens care solution
with clinical isolates derived from corneal ulcers and microbial
keratitis, including strains of Acanthamoeba spp.
Methods: The antimicrobial efficacy of a new 3% Hydrogen
Peroxide lens care solution was evaluated by the Stand-Alone test
method (FDA 510[k] guidance/EN ISO 14729 standard). Clinical
bacteria and yeast isolates were obtained from patients with
microbial keratitis or corneal ulcers including cytotoxic and invasive
strains of Pseudomonas spp., emerging Gram-negative organisms
Stenotrophomonas spp. and Achromobacter, clinical ocular species of
Staphylococcus (MRSA), Enterobacter, Candida and Acanthamoeba
spp. Three lots of the test solution were inoculated to contain a final
concentration of 1.0 x 105 - 1.0 x 106 CFU/mL for bacteria and yeast.
For Acanthamoeba spp., the test solution was inoculated with 1.0 x
104 - 1.0 x 105 cells/ml. The inoculated solutions were evaluated for
viable counts at 6 hours disinfection time. Test controls (no product)
provided a baseline organism count by which log reduction values
were calculated.
Results: Clinically relevant Pseudomonas, Stenotrophomonas,
Achromobacter, Staphylococcus and Enterobacter had an average
log reduction of >4.0 logs (0.0-0.3 ±SD) following 6 hour exposure
(disinfection time) to the new 3% Hydrogen Peroxide lens care
solution. Candida had an average log reduction of 2.7 logs (0.1
±SD) and Acanthamoeba spp. had an average log reduction of >1.0
logs (0.2 ±SD) at 6 hours. The new Hydrogen Peroxide solution,
challenged with ocular clinical isolates, meets the primary criteria of
the EN ISO 14729 Stand-alone test, requiring >3 log reduction for
bacteria and >1 log reduction for yeast. There are no criteria currently
for activity of contact lens care solutions against Acanthamoeba spp.
Conclusions: These results demonstrate that the new 3%
Hydrogen Peroxide solution was effective at eliminating or
reducing populations of relevant clinical ocular isolates including
Acanthamoeba spp.
Commercial Relationships: Manal M. Gabriel, Alcon Laboratories
(E); Cindy McAnally, Alcon Laboratories (E); Rhonda Walters,
Alcon Laboratories (E); Linda Clark, Alcon Laboratories (E);
Monica Crary, Alcon Laboratories (E); John Bartell, Alcon
Laboratories (E); Bradley Catalone, Alcon Laboratories (E)
Selenium Contact Lens Hydrogel Polymer: Inhibition of Both
Gram-Negative and Gram-Positive Bacterial Biofilm Formation
Phat Tran1, Patrick Pham1, Abdul Hamood3, Robert Hanes2, Blake
Lackey1, Ted W. Reid1, 3. 1Ophthalmology & Visual Sciences, Texas
Tech Univ Hlth Sciences Ctr, Lubbock, TX; 2Sparx Engineering,
Pearland, TX; 3Department of Microbiology and Immunology, Texas
Tech University Health Sciences Center, Lubbock, TX.
Purpose: Biofilm formation on contact lenses has been cited as
a possible cause of corneal infection and acute red eye. A contact
lens that blocks biofilm formation should reduce the frequency of
these clinically significant problems. Selenium compounds have the
ability to catalyze the formation of superoxide radicals in the tear
film, which are cytotoxic to bacteria. Thus, this study investigated
the effectiveness of a covalent organo-selenium polymerized into a
hydrogel, against bacterial biofilm formation.
Methods: Organo-selenium compounds were polymerized directly
into a hydrogel. The inhibition of biofilm formation with the organoselenium hydrogel was investigated by incubating organo-selenium
hydrogels and selenium free hydrogel in a nutrient broth containing
Staphylococcus aureus GFP and Pseudomonas aeruginosa GFP
for 24 hours at 37oC. Biofilms were examined by the Confocal
Laser Scanning Microscopy (CLSM) and quantified by determining
the Colony Forming Unit (CFU) per lens. To determine the CFU/
lens, each lens was gently rinsed twice with PBS and placed into
a microcentrifuge tube containing 1 ml phosphate buffered saline
(PBS), and then vigorously vortexed three times for 1 minute, to
detach the cells. Suspended cells were serially diluted 10-fold in PBS
and 10-μl aliquots of each dilution were spotted on LB agar plates.
The plates were incubated at 37oC for 24 hours and the CFU were
counted. For CLSM. we used the S. aureus and P. aeruginosa strains
which carries the gene that encodes the green fluorescent protein. All
the experiments were conducted at least in triplicate.
Results: Colony forming unit assays showed total inhibition,
representing over 6 logs of Staphylococcus aureus and Pseudomonas
aeruginosa killing on organo-selenium polymerized hydrogels.
Confocal laser scanning microscopy confirmed these results.
Conclusions: The organo-selenium hydrogel polymer successfully
blocked the formation of a bacterial biofilm on the polymer by
Staphylococcus aureus and Pseudomonas aeruginosa in vitro.
Commercial Relationships: Phat Tran, None; Patrick Pham,
None; Abdul Hamood, None; Robert Hanes, None; Blake Lackey,
None; Ted W. Reid, Selenium, Ltd (P)
Cornea Bacterial Cultures, Susceptibility Report and
Epidemiological Analysis for University of Nebraska Medical
Center, Omaha, NE, 2000-2014
Jonathan Crews1, John Halgren2, Robin High3. 1Residency program,
UNMC Ophthalmology, Omaha, NE; 2Ophthalmology, Truhlsen Eye
Institute, UNMC, Omaha, NE; 3Biostatistics, University of Nebraska
Medical Center, Omaha, NE.
Purpose: To investigate the frequency of different species of bacteria
grown from cornea cultures during a 15 year period. To establish
a susceptibility report or antibiogram for cornea bacterial cultures
obtained over a 15 year period. To analyze relationship of age,
gender, and setting for the cornea bacterial cultures obtained during
this time period.
Methods: This is a retrospective analysis. Data was obtained
from the microbiology department from results of cornea cultures
from August 12, 2000 to October 26, 2014. Patients’ charts were
not reviewed and protected health information was not disclosed;
therefore, institutional review board was not utilized.
Results: 159 cornea bacterial cultures and antibiotic susceptibility
were analyzed. The percent susceptibility to various antibiotics are
outlined in the chart below.
Frequency for each species is as follows: S. pneumonia (13/159,
8.2%), Viridans group Streptococcus (11, 6.9%), S. agalactiae (1,
0.6%), S. pyogenes (1, 0.6%), Enterococcus faecalis (2, 1.3%), S.
aureus (23, 14.5%), MRSA (12, 7.5%), S. epidermidis (36, 22.6%),
Coagulase-negative Staph (17, 10.7%), Bacillus species (2, 1.3%),
Diphtheroids (3, 1.9%), Micrococcus species (2, 1.3%), Serratia
marcescens (7, 4.4%), Serratia liquefaciens (1, 0.6%), Proteus
mirabilis (1, 0.6%), E. coli (1, 0.6%), P. aeruginosa (15, 9.4%),
Pseudomonas fluorescens-putida group (3, 1.9%), Stenotrophomonas
maltophilia (1, 0.6%), Acinetobacter species (3, 1.9%), Moraxella
species (2, 1.3%), and Achromobacter xylosoxidans (2, 1.3%).
Of the cornea cultures, 52.2% were from male and 45.9% from
female patients. 3 of the specimens had unknown gender listed. Of
the cornea cultures, 1.9% were <15 years of age, 10.1% were 15-35
years of age, 28.9% were 36-60 years of age and 59.1% were 60+
years of age. The outpatient setting accounted for 90.6%, inpatient
accounted for 5.0% and emergency department accounted for 4.4% of
the specimens obtained.
Conclusions: Establishing a susceptibility report for the hospital
provides valuable data for ophthalmic practitioners who practice in
this region. Specifically, it will assist in selecting effective antibiotics
and potentially narrowing the antibiotic choice based on preliminary
culture results. It also provides data that can be compared to cornea
bacterial cultures from other regions.
Commercial Relationships: Jonathan Crews, None; John
Halgren, None; Robin High, None
Treatment of surgical sutures with antiseptic or antibiotic to
reduce suture contamination in strabismus surgery: an in vitro
experiment
Namratha Turlapati, Mark Ruttum, Sue Kehl. Ophthalmology,
Medical College of Wisconsin, Milwaukee, WI.
Purpose: Bacterial contamination of suture material has been
reported following strabismus surgery even with current standard
prevention techniques of surgical field sterilization and disinfection
with povidone iodine. Pretreatment of these sutures with antiseptic
or antibiotics may reduce the bacterial load and theoretically further
reduce incidence of endophthalmitis following strabismus surgery.
The purpose of this study is to determine if treatment of sutures used
in strabismus surgery with antibiotic or antiseptic can significantly
reduce suture contamination.
Methods: This was an in vitro experiment designed to compare
suture contamination following exposure to stock solutions of
bacteria. Sutures (6-0 polyglactin) were divided in four groups:
untreated (control) group 1, povidone iodine treated group 2,
gentamicin 2.5% solution treated group 3, and gentamicin ophthalmic
ointment treated group 4. Treated sutures were soaked in the
antibiotic or antiseptic for 5 minutes. Suture from each group was
exposed to a bacterial solution of 5.0 log concentration of four
types of bacteria (S. pneumoniae, S. aureus, S. epidermidis, and H.
influenzae), sonicated, and cultured on blood agar and chocolate agar
plates. Growth at 24 hours was measured in colony forming units.
Results: The untreated control, gentamicin 2.5% solution treated,
and gentamicin ophthalmic ointment treated groups had growth after
exposure to each of the four bacterial solutions. The povidone iodine
10% treated group did not demonstrate any growth which was found
to be statistically significantly different (p<0.0001). As analyzed
by the Kruskal-Wallis Exact Test, the relative growth among the
other treatment groups was not found to be statistically significant
(p=0.8432).
Conclusions: Contamination of suture during strabismus surgery
has been reported and is the suspected mechanism of infection for
post-operative endophthalmitis following strabismus surgery. Lack
of bacterial growth from the povidone iodine treated suture group is
an important finding because this suggests pretreating sutures with
antiseptic will decrease suture contamination and theoretically rates
of infectious endophthalmitis following strabismus surgery.
Commercial Relationships: Namratha Turlapati, None; Mark
Ruttum, None; Sue Kehl, None
Antibiotic Treatment Exacerbates Focal Retinal Degeneration in
a Mouse Model of AMD
Liliana Guedez1, Nicholas Popp1, DeFen Shen1, Haohua Qian2,
Yichao Li2, Chi-Chao Chan1. 1Laboratory of Immunology, National
Eye Institute, Bethesda, MD; 2Visual Function Core, National Eye
Institute, Bethesda, MD.
Purpose: Age-related macular degeneration (AMD) is a leading
cause of irreversible central vision loss in the elderly. Inflammation,
oxidative stress, and cell death are involved in the pathogenesis
of retinal degeneration in AMD. Board spectrum antibiotics have
been reported to decrease inflammation in autoimmune uveitis
by modulating the gut’s microflora. In this study, we investigated
whether antibiotics might have a similar anti-inflammatory effect in
AMD.
Methods: Age-matched Ccl2/Cx3cr1 double knock-out (DKO)
mice, an AMD mouse model, were treated or untreated for three
months with antibiotics (Vancomycin, Ampicillin, Neomycin and
Metronidazole) delivered orally in their drinking water. Funduscopy
was performed monthly; visual function was assessed by ERG. Three
months post-treatment, the eyes were enucleated for histology and
RNA extraction. Transcripts for Il-1β, Tnf-α and iNos were detected
by qRT-PCR.
Results: After 3 months, antibiotic treated mice showed significantly
higher clinical grading scores, indicative of increased focal retinal
degeneration. Treated mice demonstrated a decline in their darkadapted ERG response. In contrast to controls, ocular histology
from treated mice revealed a prominent thinning and extensive focal
atrophy of their photoreceptors. Unlike Il-1β mRNA levels, Tnf-α
and iNos mRNA were significantly elevated in antibiotic treated eyes.
Conclusions: This study has demonstrated that antibiotics exacerbate
retinal degeneration in an AMD mouse model. The key pathological
aspects of focal photoreceptor and RPE degeneration were severely
worsened by antibiotics, with an associated decline in visual function.
This contrasts against the beneficial anti-inflammatory effects of
antibiotics in uveitis. We found that antibiotics significantly increased
ocular expression of Tnf-α and the oxidative stress marker iNos.
These findings support a role for inflammatory and oxidative stress
pathways in retinal degeneration, warranting potential therapeutic
intervention with oxidative stress inhibitors.
Commercial Relationships: Liliana Guedez, None; Nicholas Popp,
None; DeFen Shen, None; Haohua Qian, None; Yichao Li, None;
Chi-Chao Chan, None
Microbiological Isolates in Persistently Culture Positive
Endophthalmitis
Ella H. Leung1, Ajay E. Kuriyan1, Harry W. Flynn1, Laura C. Huang2,
Darlene Miller1. 1Department of Ophthalmology, Bascom Palmer
Eye Institute/ University of Miami, Miami, FL; 2University of Miami,
Miami, FL.
Purpose: To determine the incidence of persistent endophthalmitis,
to identify the most common microorganisms that are present after
initial treatment with intravitreal antibiotics, and to compare the
infectious etiologies in repeat positive vitreous cultures.
Methods: Non-comparative case series of patients with positive
vitreous cultures obtained at the Bascom Palmer Eye Institute from
1981-2011.
Results: Out of 1,079 patients with positive vitreous cultures, there
were 76 patients (7.05%) who continued to have signs and symptoms
of endophthalmitis and had repeat intraocular cultures that were still
positive after one injection of intravitreal antibiotics. Gram positive
bacteria were identified in 48.6% of patients (n=37), gram negative
bacteria in 9.21% (n=7), polymicrobial in 19.2% (n=15), and
fungi in 15.8% (n=12). Thirteen percent (9 of 76 patients) had a
mixture of fungi and bacteria, and 15% (11 of 76 patients) had a
different organism identified on subsequent cultures. The most
commonly identified organisms in persistent endophthalmitis were
Staphylococcus species (39.5%, n=30), Streptococcus species
(26.3%, n=20), Enterococcus species (13.2%, n=10), Candida species
(7.89%, n=6), Propionibacterium species (7.89%, n=6), Pseudomonas
species (6.57%, n=5), Aspergillus species (5.26%, n=4), and
Acremonium species (3.95%, n=3).
Conclusions: Persistently culture endophthalmitis is uncommon but
not rare. Overall, gram positive bacteria were the most commonly
identified organism. Specifically, Staphylococcus species and
Streptococcus species remained the most common infectious
etiologies.
Commercial Relationships: Ella H. Leung, None; Ajay E.
Kuriyan, None; Harry W. Flynn, None; Laura C. Huang, None;
Darlene Miller, None
Coagulase-Negative Staphylococcus (CoNS) Causing
Endophthalmitis: A Decreasing Trend of Susceptibility Among
Vancomycin and Fluoroquinolones
Jack D. Stringham, Darlene Miller, Harry W. Flynn. Ophthalmology,
Bascom Palmer Eye Institute, Sunny Isles, FL.
Purpose: To evaluate the susceptibility of CoNS causing
endophthalmitis to vancomycin and fluoroquinolones over time.
Methods: Microbiology records were reviewed to document trends
in automated vancomycin MIC90 and percent inhibited using Vitek
2 data on vitreous isolates recovered from (1990-1994-baseline) in
five-year increments to 2010-Oct2014. Parallel resistance for the
fluoroquinolones (ciprofloxacin, levofloxacin and moxifloxacin) were
also documented. Data for levofloxacin and moxifloxacin were only
available for the last 4 periods (1995-1999, 2000-2004, 2005-2009,
2010-Oct2014).
Results: Vancomycin MIC90 at base line (1990-1994, N=90) was
<1ug/ml. 96% of the all isolates were inhibited at this concentration.
The MIC90 (% susceptible) for the remaining periods were 19951999, N=75, <1 mg/ml (100%), 2000-2004, N=59, <2 mg/ml (90%),
2005-2009, N=73, <2 mg/ml (95%), and 2010-Oct2014, N=55, <2 mg/
ml (100%). A significant decline in the percent of isolates susceptible
to a vancomycin concentration of 1 ug/ml was observed in the last 3
periods when compared to baseline (96%), 2000-2004 (11%; p=0.00),
2005-2009 (8%; p=0.00) and 2010-Oct2014 (40%; p=0.00).
Ciprofloxacin resistance increased from 14.6% (N=82) at baseline
to 62.3% (N=61) for the last test period (p=0.00). Similar significant
increases in resistance to levofloxacin and moxifloxacin were
documented from the initial test period 1995-1999 to 2010-Oct2014.
Resistance to levofloxacin increased from 17.4% (N=23) to 60.7%
(N=61; p=0.00). Moxifloxacin resistance increased from 21.7%
(N=23) to 62.3% (N=61; p=0.00).
Conclusions: CoNS isolates remain susceptible to vancomycin,
but the concentration required to kill or inhibit 90% of the isolates
has doubled over the last 24.5 years. Less than 40% of our CoNS
isolates are susceptible in vitro to the fluoroquinolones, moxifloxacin
included.
Commercial Relationships: Jack D. Stringham, None; Darlene
Miller, None; Harry W. Flynn, None
Comparative efficacy of two different regimens of povidoneiodine 5% eye drops instillation in reducing conjunctival
bacterial flora
Leticia Barroso, Antonio Brunno Nepomuceno, Sarah P. Cazella,
Luiza Toscano, Jefferson Ribeiro, Andre Messias, Rodrigo Jorge.
Ophthalmology, School of Medicine of Ribeirao Preto, University of
São Paulo, Brasilia, Brazil.
Purpose: To compare the efficacy of 1 PI povidone-iodine 5% eye
instillation 2 minutes before or 3 drops 30 minutes before the last
swab in reducing conjunctival bacterial flora culture positivity.
Methods: Patients were randomly divided to receive 1 PI drop
starting 2 minutes before the last swab (PI group) or 3 drops starting
30 minutes before the last swab (PIplus group). Conjunctival swab
was obtained five minutes before, and 30 minutes after the first
povidone iodine drop was instilled into the conjunctival sac. Corneal
paquimetry was performed before and after procedure using OcuScan
Alcon RXP (Alcon, Fortworth, Texas). Conjunctival swabs were
incubated aerobically in enriched Thioglycolate liquid medium (meat
broth) and in three solid culture media (Agar Chocolate, Trypticase
Soy Agar with 5% sheep blood, and Agar Sabouraud).
Results: A total of 120 patients (120 eyes) were included: 59 eyes in
the group PI and 61 in PIplus. There were no significant differences
between groups regarding age and gender. A similar proportion of
patients had microorganisms isolated from the conjunctival swab
before treatment: 18/61 (29.5%) for PIplus group and 14/59 (23.7%)
for PI group (P=0.5386). Also, no significant difference was found
for the number of positives cultures that became negative after
treatment between groups (p=0.4225). When comparing groups, a
similar proportion of positive cultures after treatment was found, 9/61
(14.7%) for PIplus group and 12/59 (20.3%) for PI group (P=0.4768),
relative risk of 0.7254 (CI95%= 0.3303 to 1.593). Staphylococcus
epidermidis were the most frequently isolated microorganisms in
cultures from both groups. Mean corneal thickness didn’t change
significantly in both groups after treatment.
Conclusions: This data indicates that using 3 drops 30 minutes
before or 1 PI drop 2 minutes before are associated with similar
redunction of conjunctival bacterial flora.
Commercial Relationships: Leticia Barroso, None; Antonio
Brunno Nepomuceno, None; Sarah P. Cazella, None; Luiza
Toscano, None; Jefferson Ribeiro, None; Andre Messias, None;
Rodrigo Jorge, None
Support: Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) 2010/17350-6
Clinical Trial: NCT01739920
Treatment of bacterial and viral conjunctivitis with topical
ultrapure stable hypochlorous acid (HOCl): a clinical evaluation
and treatment response in 79 cases
Peter S. Adamson1, Hendrik Roos2, Jon von Holdt3. 1ORBIT, UCL,
Institute of Ophthalmology, London, United Kingdom; 2HPAScientific, Port Louis, Mauritius; 3Classique Optical, Johannesburg,
South Africa.
Purpose: Determine the efficacy and tolerability of ultrapure
stablised hypochlorous acid as a general anti-microbial agent in the
treatment of bacterial and viral conjunctivitis. Hypochlorous acid
is one of the most efficient anti-microbial agents, effective against
numerous bacteria and viruses including MRSA and HIV, as well as
fungi. Hypochlorous acid mediates its action on contact and is selfsterilising and with no possibility of emerging resistance. Previous
methods of manufacture contain other chorine compounds, such as
chlorine gas and hypochlorite which are noxious and irritating to the
eye. We have developed an ultrapure and stabilized HOCl solution
and have undertaken studies to determine the utility in treating
infective eye disease.
Methods: 65 cases of bacterial and 14 cases of viral conjunctivitis
(which included one case of severe viral corneal endothelitis) were
treated with a solution of Ultrapure Stabilized HOCl at 80mg/l, pH
5.4. Tolerability and efficacy were noted during the treatment period.
HOCl eye drops were administered two drops in each eye 3 times
per day and prior to sleep. The clinical response and tolerability was
examined at day 1, 3 and 7.
Results: Total resolution of the bacterial conjunctivitis occurred in
64/65 cases examined, including one subject who had previously
failed Tobramax, Tobradex, Maxitrol and Fuse ointment. One
bacterial case had a poor response, possibly due to compliance issues
and the presence of a systemic infection. All viral conjunctivitis cases
showed complete and rapid resolution of symptoms. In addition to
resolution of the infection, all patents showed rapid improvement
of redness, eye discharge, photophobia and impaired vision. In the
one case with associated viral corneal endothelitis, corneal clouding
resolved with normal vision returning. No tolerability issues were
identified.
Conclusions: A new treatment method for infective eye conditions is
proposed which does not rely on the use of topical antibiotics. HOCl,
when applied at 80mg/l pH5.4, is profoundly effective and well
tolerated in cases of bacterial or viral conjunctivitis.HOCl is cheap to
manufacture and is stable at ambient temperatures and may be useful
in the treatment of infective eye disease in locations where access to
antibiotics is difficult.
Commercial Relationships: Peter S. Adamson, HPA Scientific (I);
Hendrik Roos, HPA-Scientific (I); Jon von Holdt, None
Support: Investigator
Antimicrobial performance of preservative agents in
antiglaucoma ophthalmic solutions
Taiichiro Chikama, Nur Putri Irmayasari, Latief Miftahul Akhyar,
Yoshiaki Kiuchi. Ophthalmology, Hiroshima Univ Grad Sch of
Biomed Sci, Minami-ku, Japan.
Purpose: We examined the antimicrobial performance of
preservatives—benzalkonium chloride (BAC) at three different
concentrations and the SofZia system—present in topical ocular
hypotensive agents.
Methods: Four antiglaucoma ophthalmic solutions distributed
in Japan—latanoprost (0.005%) with 0.02% BAC (Xalatan),
bimatoprost (0.03%) with 0.005% BAC (LUMIGAN), tafluprost
(0.0015%) with 0.001% BAC (TAPROS), and travoprost (0.004%)
with SofZia (TRAVATANZ)—were tested for their antimicrobial
activity against Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli, Propionibacterium acnes, Streptococcus
pneumoniae, and Candida albicans. Each drug solution (10 mL)
was inoculated with 1 × 106 colony-forming units of each organism
and incubated at 20° to 25°C. Samples of each mixture collected at
various times from 6 h to 28 days were serially diluted in MuellerHinton broth (for S. aureus, P. aeruginosa, E. coli and C. albicans),
BL liquid medium (for S. pneumonia) or GAM liquid medium (for
P. acnes) and plated in triplicate on tryptic soy agar (for S. aureus, P.
aeruginosa, E. coli), BL agar plus horse blood (for S. pneumonia),
GAM agar (for P. acnes) or Sabouraud dextrose agar (for C.
albicans). Culture plates were incubated at 30° to 35°C for >3 days or
at 20° to 25°C for >5 days, respectively, and the number of colonies
was then counted.
Results: Latanoprost with 0.02% BAC completely inhibited bacterial
growth after incubation for 24 h as well as fungal growth at 6 h.
Bimatoprost with 0.005% BAC completely inhibited bacterial and
fungal growth after incubation for 6 h. Tafluprost with 0.001% BAC
completely inhibited bacterial growth after incubation for 7 days as
well as fungal growth after incubation for 14 days. Travoprost with
SofZia completely inhibited bacterial growth at 6 h but had no effect
on fungal growth.
Conclusions: All four ophthalmic solutions performed well in tests
of their antimicrobial activity. Only travoprost with the SofZia
preservative system did not inhibit the growth of C. albicans within
28 days.
Commercial Relationships: Taiichiro Chikama, None; Nur Putri
Irmayasari, None; Latief Miftahul Akhyar, None; Yoshiaki
Kiuchi, Alcon (F), Pfizer (F), Santen (F), Senju (F)
Etiology and susceptibility pattern of isolates in endophthalmitis
over a 5-year period
Juliana M. Kato1, Maura S. Oliveira3, Anna S. Levin3, João Nobrega
de N. Almeida2, Flavia Rossi2, Luiza Manhezi de Freitas Oliveira2,
Aline D. Ruppert2, Tatiana Tanaka2, Yoshitaka Nakashima2, Sergio
L. Pimentel2. 1Faculty of Medicine of University of Sao Paulo, Sao
Paulo, Brazil; 2Ophthalmology, University of São Paulo, São Paulo,
Brazil; 3Department of Infection Control, University of Sao Paulo,
Sao Paulo, Brazil.
Purpose: Despite prompt referral and treatment just after diagnosis
suspicion, endophthalmitis outcomes remain poor. Current attempts
of prevention and management depend on empiric antibiotic therapy.
Supervising causative agents and microbial spectrum may be
important to identify trends in antibiotic susceptibility and improve
guidelines. Purpose: To analyze the etiology and antimicrobial
susceptibility profile of isolates in endophthalmities in a cohort of
consecutive cases of patients referred to a tertiary health center.
Methods: Approval from the Ethics Committee of the Clinical
Hospital of University of Sao Paulo was obtained and a retrospective
study of all endophthalmities seen at a tertiary care center in
Brazil between 2010 and 2014 was conducted. Samples obtained
from aqueous and/or vitreous fluid were subjected to culture at
the microbiological laboratory. Identification and susceptibility
testing was performed by an automated broth microdilution method
(bioMerieux Vitek 2, Hazelwood, MO, USA). Breakpoints were
those defined by the CLSI.
Results: Among 91 patients diagnosed of endophthalmitis, 45 had
culture-positive results (49%). The most prevalent gram-positive
isolated was Staphylococcus epidermidis (19%) and viridans group
streptococci (19%), followed by Staphylococcus aureus (8%) and
Enterococcus faecalis (8%). Coagulase-negative staphycococcus and
Streptococcus pneumoniae counted for 6% each. The most common
gram-negative was Pseudomonas aeruginosa (6%). 6% of the
culture-positive were fungi. Susceptibility pattern is given on table 1.
Out of 3 Enterococcus faecalis tested, 1 was resistant to vancomycin.
Moxifloxacin obtained only 50% susceptibility and levofloxacin,
75%. Samples tested for moxifloxacin between 2010 and 2012 had
67% susceptibility, whereas two years later it decreased to 29%
susceptibility. (Graphic 1)
Conclusions: Moxifloxacin is a fourth-generation fluoroquinolone
widely used in Ophthalmology as a postoperative topical antibiotic
therapy. On this short series, we identified a decrease in microbial
susceptibility for moxifloxacin.
Miller, JJ, Scott, IU, Flynn, HW, Jr. Smiddy, WE, Newton, J, Miller,
D. Acute-onset endophthalmitis after cataract surgery (2000–2004):
incidence, clinical settings, and visual acuity outcomes after
treatment. Am. J. Ophthalmol. 139:983–987, 2005.
N: number of isolates tested; S: number of isolates sensitive to the
antibiotic.
Commercial Relationships: Juliana M. Kato, None; Maura
S. Oliveira, None; Anna S. Levin, None; João Nobrega de N.
Almeida, None; Flavia Rossi, None; Luiza Manhezi de Freitas
Oliveira, None; Aline D. Ruppert, None; Tatiana Tanaka, None;
Yoshitaka Nakashima, None; Sergio L. Pimentel, None
Netilmicin for antibiotic prophylaxis before cataract surgery
Vincenzo Papa1, Cristina Cannatella1, Anna Rita Blanco1, Marcello
Santocono2. 1SIFI SPA, Lavinaio, Italy; 2Ophthalmology Unit,
DiStefano Private Hospital, Catania, Italy.
Purpose: The most common source of pathogens in postoperative
endophthalmitis is the patient’s own ocular surface flora. Topical
applied antibiotics used for a brief preoperative regimen should
eradicate conjunctival and eyelid bacteria. A cross-sectional case-only
observational study (NCT02124603) was performed to determine the
antibiotic susceptibility of the ocular surface bacterial flora isolated
from patients undergoing cataract surgery and the effectiveness of
topical antibiotic prophylaxis in eliminating preoperative bacteria.
Methods: 120 consecutive patients scheduled for cataract surgery
were enrolled (49 males and 71 females, mean age 72.3±8.4 years).
Two weeks before surgery, conjunctival and lid margin cultures were
obtained from the eye to be operated. Patients were treated starting
2 days before surgery with a topical broad spectrum antibiotic (0.3%
netilmicin,1 drop tid). Cultures were repeated the day of surgery,
just before povidone-iodine application. Bacteria were isolated using
selective culture media and identified by biochemical tests. In vitro
susceptibility for the commercially available topical antibiotics
netilmicin (NET), moxifloxacin (MFX), chloramphenicol (CAF),
levofloxacin (LFX), tobramycin (TOB), ofloxacin (OFX) and
azythromicin (AZY) was determined using the disk susceptibility
test. Eradication was defined as the disappearance of identified
bacteria after antibiotic treatment. A descriptive analysis was
performed.
Results: Among 120 patients, 87 (72.5%) had positive bacterial
growth: 32 were positive for lid margin, 14 for conjunctiva and 41 for
both. A total of 131 isolates (125 Gram+ and 6 Gram-) were obtained:
92 (70%) were Coagulase-negative staphylococci (CNS). The overall
susceptibility rate was: NET 94%, MFX 86%, CAF 74%, LFX 70%,
TOB 59%, OFX 57%, AZY 15%. A detailed susceptibility rate for
NET is shown in the figure. The 3-days application of NET was able
to eradicate 98% and 91% of conjunctival and lid margin isolates,
respectively (p<0.001, binomial test).
Conclusions: Despite controversy exists regarding the translation
of in vitro data into clinical outcome, our data indicate that NET is
the only tested antibiotic with an overall 90-plus percent in vitro
susceptibility rate and that a 3-days preoperative course with NET is
able to eliminate the majority of bacteria.
NT: not tested
and high levels of multi-drug resistance in the already problematic
staphylococci. Continued surveillance of ocular pathogens is
necessary in order to provide healthcare professionals with the
awareness of evolving trends in antimicrobial susceptibility patterns
to effectively guide the treatment of ocular infections.
Commercial Relationships: Penny A. Asbell, Bausch + Lomb (F);
Christine M. Sanfilippo, Bausch + Lomb (E); Daniel F. Sahm,
IHMA (E); Heleen H. DeCory, Bausch + Lomb (E)
Commercial Relationships: Vincenzo Papa, SIFI SpA (E); Cristina
Cannatella, SIFI SpA (E); Anna Rita Blanco, SIFI SpA (E);
Marcello Santocono, SIFI SpA (C)
Antibiotic Resistance Among Ocular Pathogens – Results from
the ARMOR Surveillance Study 2013-Present
Penny A. Asbell1, Christine M. Sanfilippo2, Daniel F. Sahm3, Heleen
H. DeCory2. 1Ophthalmology, Icahn School of Medicine at Mount
Sinai, New York, NY; 2Pharmaceuticals, Bausch & Lomb, Rochester,
NY; 3IHMA, Schaumburg, IL.
Purpose: The Antibiotic Resistance Monitoring in Ocular
micRoorganisms (ARMOR) study was initiated in 2009 to survey
resistance levels among ocular pathogens on a nationwide scale.
Here we report the complete study results for 2013 compared to
preliminary 2014 data.
Methods: Isolates of Streptococcus pneumoniae, Staphylococcus
aureus, coagulase-negative staphylococci (CoNS), Pseudomonas
aeruginosa, and Haemophilus influenzae were subjected to antibiotic
susceptibility testing. In 2013, 543 isolates were collected from
22 sites; 140 isolates have been collected from 7 sites to date in
2014. Minimum inhibitory concentrations were determined by
broth microdilution for up to 16 representative antibiotics per CLSI
methods. Systemic breakpoints (where available) were used to
categorize isolates as susceptible or non-susceptible (intermediate
and resistant).
Results: With the exception of a fluoroquinolone-resistant isolate
and an azithromycin-resistant isolate observed in 2014, all H.
influenzae isolates were susceptible to all drugs tested. Isolates of
P. aeruginosa in 2013 were non-susceptible to polymyxin B (27%),
imipenem (16%), and ciprofloxacin (9%); no resistance was detected
in 2014. Compared to 2013, non-susceptibility rates for 2014 S.
pneumoniae isolates more than doubled for penicillin (26% vs.
53%), azithromycin (31% vs. 63%), and chloramphenicol (2% vs.
11%). Isolates of S. aureus in 2014 were non-susceptible to oxacillin
(28%), ciprofloxacin (22%), and azithromycin (53%), showing
decreases from the previous year. From 2013 to 2014, tobramycin
non-susceptibility among CoNS increased substantially (35%) while
azithromycin and oxacillin resistance rates only slightly increased
(65% and 63%, respectively); other CoNS susceptibilities generally
remained steady. While multi-drug resistance (MDR) in 2014
decreased among S. aureus (16%) and methicillin-resistant (MR) S.
aureus (50%), MDR among CoNS and MRCoNS increased to 55%
and 84%, respectively.
Conclusions: To date, the 2014 ARMOR surveillance data show
increased drug non-susceptibility rates among S. pneumoniae isolates
2.5, 5, and 10% Betadine solution is not effective in inhibiting the
growth of different Gram Negative and Gram Positive Pathogens
in vitro
Akash Desai1, Phat Tran1, Abdul Hamood2, Kelly T. Mitchell1, Ted W.
Reid1. 1Opthalmology, Texas Tech Health Sciences Center School of
Medicine, Lubbock, TX; 2Immunology and Molecular Microbiology,
Texas Tech Health Sciences Center, Lubbock, TX.
Purpose: Injections of intravitreal medications have become
routine care in ophthalmology officies throughout the world for the
treatment of several retinal diseases. Studies estimate that the rate of
endophthalmitis from intraocular injections ranges from .006% to
1.67%. Providone-iodine (Betadine) is widely accepted as the main
antiseptic to decrease this risk. This study was undertaken to measure
the effectiveness of different Betadine concentrations in inhibiting the
growth of both Gram negative and Gram positive bacteria.
Methods: The bacteria tested were laboratory strains of
Staphylococcus aureus GFP and Pseudomonas aeruginosa GFP,
clinical isolates of Staphylococcus aureus and Pseudomonas
aeruginosa, and two strains of Methicillin-resistant Staphylococcus
aureus (MRSA). Betadine disks were prepared by adding 20 μl of
2.5, 5, or 10% Betadine solution onto 6 mm diameter BBL blank
paper disks. Disks were allowed to dry before use, and three disks
were distributed evenly onto a lawn of bacteria on the LB Agar
surface. The plates were incubated at 37oC for 24 h. The diameters of
the zones of inhibition were measured to the nearest millimeter with a
ruler. Moreover, the disks from the S. aureus GFP and P. aeruginosa
GFP plates were examined under the Confocal Laser Scanning
Microscopy (CLSM). In addition, the remaining microorganisms on
the disks were quantified by the colony forming unit (CFU) assay. All
experiments were done at least in triplicate.
Results: All the bacteria except P. aeruginosa GFP laboratory strain
showed zones of inhibition when using 2.5% Betadine. At 5 and 10%
Betadine concentrations, all bacteria showed zones of inhibition.
However, when the disks were tested for bacteria after the zone
of inhibition study using CFU assays, only Gram positive bacteria
showed killing at 10% Betadine. The CLSM confirmed the CFU
results for S. aureus GFP AH133 and P. aeruginosa GFP.
Conclusions: The three main conclusions are: 1) the zone of
inhibition assay does not give a realistic assessment of the ability of
an antimicrobial to kill bacteria; 2) In vitro, 2.5 and 5% Betadine are
not effective at killing all the bacterial species associated with post
procedure endophthalmitis; and 3) In vitro, 10% Betadine is only
effective against the Gram positive bacteria tested.
Commercial Relationships: Akash Desai, None; Phat Tran, None;
Abdul Hamood, None; Kelly T. Mitchell, None; Ted W. Reid, None
Conjunctival Bacterial Resistance Patterns to Antibiotics for
Intravitreal Injection: Effects of Maxitrol and Betadine in
Clinical Practice
Enoch Kassa1, Jonathan Gambrell1, 2, Yang Sun1, Charles Barr2.
1
Ophthalmology, Indiana University School of Medicine,
Indianapolis, IN; 2Ophthalmology, Louisville University, Louisville,
KY.
Purpose: To determine if intravitreal injections are conducted in a
properly sterilized environment. Endophthalmitis is the most feared
complication of intravitreal injections. Conventionally, anti-septics
without antibiotics are used to sterilize the conjunctival surface
before injections. This study investigates the effect of Maxitrol
(neomycin and polymyxin B sulfates and dexamethasone ophthalmic
suspension) on bacterial isolates when used in addition to betadine
5%.
Methods: This is a randomized prospective study from 2011-2012
at the University of Louisville School of Medicine Department of
Ophthalmology. Eighty-six patients requiring intravitreal injections
were enrolled in the study, with 110 eyes randomized into 55 control
sample and 55 treatment sample. A swab culture was obtained on all
eyes before any anti-bacterial or anti-septic application. A second
swab culture was obtained post application of the sterilizing agents
and/or antibiotic. Sensitivities were obtained and the cultures from
each group were counted and compared. Statistical analysis was
performed using SASS.
Results: Conjunctival swab cultures were initially obtained from
110 eyes. 50 swabs from untreated eyes grew bacteria (n=110, 45%).
55 eyes were then treated with betadine 5% and the other 55 eyes
were treated with a combination of Maxitrol and Betadine 5%. 20
swabs from eyes treated without Maxitrol grew bacteria while 8
swabs treated with Maxitrol grew bacteria (n=110, p-value=0.0084).
The most common bacteria isolated was coagulase negative
staphylococcus (CNS) (63 %). 15 (75%) swabs from eyes treated
without Maxitrol grew CNS while 4 (50%) swabs from Maxitrol
treated eyes grew CNS (n=110, p-value=0.0098).
Conclusions: Significant reduction in endophthalmitis causing
organisms were observed in the group treated with both Maxitrol
and betadine. In patients receiving intravitreal injections, preparing
the injection site with a combination of betadine and Maxitrol may
reduce the incidence of inadvertent infectious complications.
Commercial Relationships: Enoch Kassa, None; Jonathan
Gambrell, None; Yang Sun, American GLX society (R), Indiana
university BRG (R), Lowe syndrome society (R), NIH (R), Research
to prevent blindness (R), Ziegler Foundation (R); Charles Barr,
None
The effect of the linezolid and the vancomycine on biofilm
production that formed on two different acrylic hydrophobic
intraocular lenses
Sertac Argun Kivanc1, Berna Akova Budak1, Meral Yildiz1, Merih
Kivanc2. 1Ophthalmology, Uludag University, Bursa, Turkey;
2
microbiology, Anadolu University, Eskiehir, Turkey.
Purpose: To investigate the effect of linezolid and vancomycine on
biofilm formation on two different types of hydrophobic intraocular
lenses.
Methods: Ica A, icaD and bap positive Staphylococcus epidermidis
was used in this study for biofilm production. Biofilms were
cultivated on disks of two different types of acrylic hydrophobic
lenses( 25 % water content as Lens A, 2 % water content as Lens
B) with different water contents.In the first protocol, the lenses
were incubated for 24 hours after bacterial contamination. Then,
10 μl vancomycin from 1mg/0.1 ml or 15 μl linezolid from 2mg/
ml solution was added to media containing the lenses. In the
second protocol, the lenses were contaminated with bacteria and
10 μl vancomycin from 1mg/0.1 ml or 15 μl linezolid from 2mg/
ml solution were added at the same time. In both protocols, after
24 hours incubation of the plates, the lenses were evaluated by
spectrophotometry ( OD 620 nm) and number of bacteria was
counted. The lenses were examined with scanning electron
microscopy.
Results: In the first protocol, the number of bacteria was
5.1(Log10CFU/mL) for lens A and 5.3 for lens B with linezolid and
5.2 for lens A and 5.2 for lens B with vancomycin. In the second
protocol, the number of bacteria was 1.4(Log10CFU/mL) for lens A
and 1.4 for lens B with linezolid and 1.4 for lens A and 1.2 for lens B
with vancomycin.
Conclusions: The effect of the linezolid and the vancomycine on
biofilm formation on acrylic hydrophobic intraocular lenses was
found similar with different water contents. Both were less effective
when added on preformed biofilm.
Commercial Relationships: Sertac Argun Kivanc, None; Berna
Akova Budak, None; Meral Yildiz, None; Merih Kivanc, None
147 AMD immunobiology, autoimmunity, and immunoregulation
Sunday, May 03, 2015 1:30 PM–3:15 PM
Program #/Board # Range: 838–889/D0300–D0351
Contributing Section(s): Cornea, Physiology/Pharmacology, Retinal
Cell Biology
Program Number: 838 Poster Board Number: D0300
Presentation Time: 1:30 PM–3:15 PM
IFIT3/P60 Expression Reflects Cell Specific Response to Viral
Infection
Nathaniel Sears, Steffen Christoffersen, George Hoppe, Jonathan E.
Sears. Cleveland Clinic, Cole Eye Institute, Cleveland, OH.
Purpose: Extracellular RNA viruses are recognized by Toll-like
receptor 3, and when intracellular by retinoic acid inducible gene I
(RIG-I)/MDA5 receptors, stimulating formation of the mitochondrial
antiviral signaling complex that leads to NFkB and interferon
response factor 3 (IRF3) activation. The cell phenotype then has a
divergent response that can include either programmed cell death or
chronic inflammation. The purpose of this study is to determine how
cells regulate their fate between these two phenotypes by analyzing
IRF-3 dependent genes.
Methods: Cultured ARPE-19 cells and human Muller cells were
challenged with dsRNA. Immunoanalysis of IRF-3, pIRF3,
caspase-8, and interferon-induced proteins with tetratricopeptide
repeats (IFIT3/P60) were performed.
Results: Both Muller cells and ARPE-19 cells demonstrate similar
induction of IRF3 and pIRF3 expression in response to dsRNA.
Caspase 8 levels were similar between these cells as well. These
levels were identical when normalized to actin, and demonstrated a
similar kinetics when measured at 0,8,24, and 48 hours. However,
IFIT3/P60 was robustly expressed in ARPE-19 cells in comparison to
Muller cells and exhibited markedly different kinetics. ARPE IFIT3
levels rapidly peaked at 24 hours and became attenuated at 48 hours,
whereas Muller cell IFIT3 protein peaked at 8 hours and sustained
this level at 48 hours.
Conclusions: Both Muller cells and ARPE-19 demonstrate cell
specific responses to extracellular virus manifested by differential
IFIT3 induction. This finding may reflect an IRF3 independent
pathway. Differential responses to identical dsRNA stimulus is
therefore cell specific and demonstrates the feasibility of using
this approach to further understand the molecular mechanism that
underlies regulation between chronic inflammation and programmed
cell death.
Commercial Relationships: Nathaniel Sears, None; Steffen
Christoffersen, None; George Hoppe, None; Jonathan E. Sears,
None
Support: Research to Prevent Blindness
Downregulating p22phox ameliorates inflammation in
Angiotensin II induced oxidative stress by regulating MAPK and
NF-κB pathways in ARPE-19 cells
Yiguo Qiu1, Lifei Tao1, Peizeng Yang1, Qiuhong Li2, Bo Lei1.
1
Department of Ophthalmology, the First Affiliated Hospital of
Chongqing Medical University, Chongqing Key Laboratory of
Ophthalmology, Chongqing Eye Institute, Chongqing, China;
2
University of Florida, Gainesville, FL.
Purpose: Oxidative stress and inflammation are interrelated
biological events and both have been identified to play important
roles in the pathological process of age-related macular degeneration
(AMD). The purpose of this study is to investigate the antiinflammation effect and the mechanism of downregulating p22phox
in Angiotensin II (Ang II) induced oxidative stress in ARPE-19 cells.
Methods: ARPE-19 cells were transfected with p22phox siRNA (P)
followed by stimulation with Ang II for 48 hours. The mRNA levels
of p22phox, inflammatory cytokines, IκBα and NOX1, 2, 4 were
analyzed by real time PCR. The protein expression of p22phox, p65
and the phosphorylation of p38 mitogen-activated protein kinase
(MAPK), extracellular signal–regulated kinase (ERK1/2) and c-Jun
N terminal kinase (JNK) were detected by Western Blotting. The
reactive oxygen species (ROS) was investigated by DCFH-DA assay
with flow cytometry. Protein concentrations of IL-6, IL-8 and MCP-1
in the supernatant were measured by ELISA. The signal transduction
mechanisms involved in cytokine production were evaluated using
inhibitors for MAPK and NF-κB pathways.
Results: The expressions of p22phox, IL-6, IL-8 and MCP-1
remarkably decreased in p22phox siRNA transfected and Ang II
treated (P plus Ang II) group at both mRNA and protein levels. The
mRNA expressions of NOX1, 2, 4 were reduced in P plus Ang II
group than Ang II group (p<0.05, p<0.01). The ROS production was
decreased in P plus Ang II group than Ang II group (p<0.01). The
mRNA level of IκBα was significantly increased and the protein
level of p65 decreased in P plus Ang II group (p<0.05, p<0.001).
Downregulating p22phox decreased the phosphorylation of p38,
ERK1/2 and JNK stimulated by Ang II. The inhibitory experiments
showed that MAPK and NF-κB pathways were involved in the
inhibitory effect of downregulating p22phox on cytokine production.
Conclusions: Downregulating p22phox shows anti-inflammation
effect in Ang II induced oxidative stress in ARPE-19 cells by
regulating MAPK and NF-κB pathways. These results support
the notions that p22phox plays an important role in inflammation
accompanied by oxidative stress and p22phox may provide a novel
therapeutic target for oxidative induced inflammation.
Commercial Relationships: Yiguo Qiu, None; Lifei Tao, None;
Peizeng Yang, None; Qiuhong Li, None; Bo Lei, None
Support: NNSF of China grants (81470621)
Minocycline abrogates glycated albumin induced MCP-1 and
IL-8 cytokine release from ARPE-19 cells
Joanna DaCosta. Cranfield University, Bedfordshire, United
Kingdom.
Purpose: Reduced choroidal blood flow may create hypoxia in the
retina, triggering formation of new vessels through the activation
of vascular endothelial growth factor. This may initiate neovascular
age-related macular degeneration.Glycated human serum albumin
(GHSA) has been shown to stimulate RPE cells to produce IL-8 and
MCP-1. Early glycated albumin may have a direct impact on cell
function during ageing. Minocycline is a semisynthetic derivative of
tetracycline with a longer half life and improved penetration through
the blood brain barrier. Apart from antimicrobial effects it also has
potent anti-inflammatory and immunomodulatory effects. It
was postulated that minocyline may suppress the induced expresion
of inflammatory cytokines from retinal pigment epithelial cells in
culture.
Methods: Research was conducted on human ARPE-19 cells in
culture. Cells were exposed to hypoxia and GHSA. Minocycline
was investigated for the effect on cell growth and cell viability. Cell
counts were obtained with a Scepter cell counter (Millipore). Cell
viability and apoptosis rates were investigated with annexin V and
propidium iodide staining by flow cytometry. The effects of hypoxia
and different concentrations of GHSA, IL-1â, TNF-á on cell viability
and with treatment with minocycline were investigated. ELISA
for IL-8 and MCP-1 was conducted with appropriate negative and
positive controls. Standards and samples were analysed in duplicate.
Intraplate and interplate reproducibility tests were performed.
Results: Cell viability decreased at minocycline doses above
5mM. Hypoxia increased the proportion of non viable cells and
cells undergoing apoptosis and minocycline treated cells had a
lower proportion in the early stage of apoptosis. ELISA results
demonstrated that hypoxia and GHSA treated cells showed a
significantly increased MCP-1 production. Minocycline significantly
reduced MCP-1 production in normoxic conditions (p< 0.01)and
also decreased MCP-1 production in hypoxia (p<0.05). For IL-8,
minocycline significantly reduced production in normoxia (p<0.01)
but not statistically significantly under hypoxia.
Conclusions: The results suggest that minocycline abrogates the
production of inflammatory cytokines IL-8 and MCP-1 in cell culture
and minocycline may have a therapeutic role in the treatment of
inflammatory changes in age related macular degeneration.
Commercial Relationships: Joanna DaCosta, None
Interleukin 33/ST2 signaling regulates inflammatory response
in choroidal stroma: implications for age-related macular
degeneration
Sofia Theodoropoulou1, Jian Liu1, Dong Li2, Damo Xu2, Iain
McInnes2, David A. Copland1, Andrew D. Dick1. 1Academic Unit of
Ophthalmology, University of Bristol, School of Clinical Sciences,
Bristol, United Kingdom; 2Institute of Infection, Immunity and
Inflammation, University of Glasgow, Glasgow, United Kingdom.
Purpose: Age-related macular degeneration (AMD) is a leading
cause of irreversible blindness. Altered immune responses are integral
in progression of disease, and in part, in response to oxidative
stress and hypoxia-induced regulation of metabolism. This includes
activation of innate immunity and complement activation, including
activation of cellular inammasome through pattern recognition
receptors (TLRs). We wished to elaborate mechanisms that regulate
RPE-choroidal microenvironment in AMD. One hypothesis is that,
via TLR stimulation, up-regulation of interleukin-33 (IL-33) in retinal
pigment epithelial cells (RPE) activates in a ST2 (Il-33 receptor)dependent manner both choroidal stromal fibroblasts and mast cells.
Through such mechanisms, change in choroidal architecture may
occur, contributing to the insidious degeneration observed clinically.
Methods: RPE cells (ARPE-19 and B6-RPE07) were stimulated
with TLR ligands and expression profile and secretion of IL-33 was
determined by RT-PCR, Western blots and immunostaining. Function
and expression profile of bone-marrow-derived mast cells (BMMC)
and human choroidal fibroblasts was also assessed.
Results: IL-33 was highly expressed in the retina of naïve mice,
and its receptor ST2 was expressed in RPE, choroidal mast cells
and choroidal fibroblasts in mouse and man. Treatment of RPE
with TLR ligands (LPS or poly(I:C)) resulted in significant upregulation of IL-33 expression and secretion, which was enhanced
upon TLR-stimulation under hypoxic conditions. ST2+ bone marrow
derived mast cells (BMMC) generated a spectrum of inflammatory
cytokines and prostaglandin synthase 2 (PGS2) when cultured
with IL-33 rich RPE supernatant. Pretreatment with soluble ST2
markedly inhibited the ability of BMMC to produce proinflammatory
mediators. Importantly, activation of inflammatory cascade upon
RPE supernatant treatment was abrogated in the absence of ST2 in
BMMC from ST2-/- mice. Furthermore, recombinant IL-33 treatment
of human choroidal fibroblasts impaired their ability to migrate
and contract collagen gel, while expression of MMP-2 and -9 was
reduced.
Conclusions: Our data illuminate an endogenous IL33/ST2 pathway
between RPE function and choroidal stroma, influencing tissue
remodeling. Our findings support IL-33/ST2 axis as a therapeutic
target in atrophic AMD.
Commercial Relationships: Sofia Theodoropoulou, None; Jian
Liu, None; Dong Li, None; Damo Xu, None; Iain McInnes, None;
David A. Copland, None; Andrew D. Dick, None
Support: NIHR (National Institute for Health Research); NERC
(National Eye Research Centre)
ROCK regulates CD163, a specific biomarker for choroidal
neovascularization
Souska Zandi1, 2, Shintaro Nakao3, 1, Sonja Frimmel1, Dawei Sun1,
Justus G. Garweg2, Tatsuro Ishibashi3, Ali Hafezi-Moghadam1.
1
Radiology, Brigham and Women’s Hospital, Boston, MA;
2
Ophthalmology, Swiss Eye Institute, Rotkreuz and Bern,
Switzerland; 3Ophthalmology, Kyushu University, Fukuoka, Japan.
Purpose: Biomarkers for age-related macular degeneration (AMD)
are urgently needed. Previously we reported a key role for Rho kinase
(ROCK) signaling in choroidal neovascularization (CNV). CD163 is
a M2- macrophage surface marker, the expression of which in CNV
has not been known. We investigate the regulatory role of ROCK in
CD163 expression and their potential as biomarkers in AMD.
Methods: CNV was induced in C57BL/6J mice using a 532-nm laser
(100mW, 50mm, 100ms).
Western Blot was performed for CD163, CCR7, CD80 and β-tubulin
. Choroids were harvested at different time points (4 h, 1, 3, 7 and 14
days) after CNV induction.
On day 7 mice were treated with dual ROCK1/2- and a ROCK 2
selective inhibitor.
For histology frozen sections of the posterior segment, including the
central portion of CNV lesions (10 lesions per eye), were prepared.
The number of CD163 positive macrophages was counted.
Results: The time course of protein expression showed significantly
elevated CD163 levels in CNV through day 14. CCR7 was
unchanged while CD80 was moderately higher in the first three
days. CD 163 was only found in CNV lesions, but not in unlasered
mice. ROCK inhibition significantly reduced CD163 in CNV lesions
compared with vehicle treated animals. Immunohistochemistry
confirmed that CD163(+) macrophages were increased in CNV
lesions, while normal unlasered retinas did not show CD163(+)
staining.
Conclusions: CD163 expression was exclusive to CNV, while it was
not found in the normal eyes. ROCK inhibition suppressed CD163
expression. The current results indicate that CD163 could become a
biomarker for exudative AMD. CD163 expression in CNV lesions is
effectively reduced by ROCK inhibition, making CD163 a sensitive
marker for evaluation of therapeutic success.
Commercial Relationships: Souska Zandi, None; Shintaro Nakao,
None; Sonja Frimmel, None; Dawei Sun, None; Justus G. Garweg,
None; Tatsuro Ishibashi, None; Ali Hafezi-Moghadam, None
Support: NH Grant 25732-30, the Bright Focus Foundation, the
Malaysian Palm Oil Board, Grants from JSPS KAKENHI, Grant-inAid for Young Scientists
Selective ROCK2 inhibition causes an M1-macrophage shift in
choroidal neovascularization
Ali Hafezi-Moghadam1, Souska Zandi1, Shintaro Nakao1, 2, Dawei
Sun1, Sonja Frimmel1, Zhongyu Zhang1, Tatsuro Ishibashi2.
1
Radiology, Harvard Medical School, Boston, MA; 2Kyushu
University, Fukuoka, Japan.
Purpose: Plasticity and diversity are fundamental characteristics
of macrophages. Undifferentiated M0 macrophages polarize into
the classical pro-inflammatory M1-like and the alternative antiinflammatory M2-like macrophages. Macrophage plasticity in agerelated macular degeneration (AMD) is not well understood. Here,
we introduce a novel role for ROCK2 in macrophage plasticity and
its impact on ocular immune balance.
Methods: CNV was induced in C57BL/6J mice using a 532-nm laser
(100mW, 50mm, 100ms). Western Blot was performed for CD206,
CCR7, CD80 and β-tubulin . Choroids were harvested at different
time points (4 h, 1, 3, 7 and 14 days) after CNV induction. On day
7 mice were treated with dual ROCK1/2- and a ROCK 2 selective
inhibitor. For flow cytometry, retinal and choroidal macrophages
were prepared from mouse eyes for flow cytometry. After laser injury,
eyes were enucleated at different time points (1, 2, 3, 5, and 7days)
and stained for CD11b-PE, CD80-FITC, CD206-FITC, or isotype
control.
Results: The number of CD11b(+)CD80(+) M1-like macrophages
increased on day 1 after laser injury and remained high through day
7 (n=6 animals, P<0.01). We found a peak of CD11b(-)CD206(+)
cells on day 2 post laser injury, which preceded the reported start
of angiogenesis. On days 3 through 7 the percentage of CD11b(+)
CD206(+) cells increased with a peak on day 7, coinciding with
the maximum angiogenic response in the laser-injury model (n=6
animals, P<0.01). Dual ROCK and selective ROCK2 inhibition
substantially decreased the CD11b(+)CD206(+) M2 population,
when examined on day 7, while ROCK2 inhibition increased the
percentage of CD80(+) cells. Western blot showed that ROCK2
inhibition but not dual ROCK1/2 inhibition increased CD80 and
CCR7 in lasered eyes.
Conclusions: Selective ROCK2 inhibition increases M1-like
macrophage markers, CD80 and CCR7. Targeting macrophage
plasticity through ROCK signaling preserves the beneficial
macrophages that are essential for retinal health and simultaneously
restores the balance between pro-angiogenic macrophages and their
angiostatic counterparts.
Commercial Relationships: Ali Hafezi-Moghadam, None; Souska
Zandi, None; Shintaro Nakao, None; Dawei Sun, None; Sonja
Frimmel, None; Zhongyu Zhang, None; Tatsuro Ishibashi, None
Support: National Institutes of Health (NIH)/National Institute
of Diabetes and Digestive and Kidney Diseases through Diabetes
Complications Consortium award 25732-30 (A.H.-M.), the
BrightFocus Foundation, the Malaysian Palm Oil Board. Grants from
JSPS KAKENHI, Grant-in-Aid for Young Scientists (A) (#25713057
to SN).
Intravitreal injection of a P2RX7 antagonist reduces
photoreceptor cell death in a model of subretinal inflammation
Xavier P. Guillonneau, shulong hu, Bertrand Calippe, Sophie
Lavalette, Jose A. Sahel, Florian Sennlaub. U968 Institut de la
Vision, INSERM, Paris, France.
Purpose: Inflammatory mononuclear phagocytes (MP) accumulate
in geographic atrophy form of age related macular degeneration
(AMD). We have previously shown that Cx3cr1-deficient mice
develop age- and light-induced subretinal accumulation of MP that
is responsible for photoreceptor degeneration. The mechanisms by
which MP accumulation leads to degeneration remains unknown.
Cx3cr1-deficient MP have been shown to lead to increased neuronal
apoptosis through IL-1beta (IL-1β) secretion in the brain. To test
whether reducing IL1 B secretion by antagonizing P2XR7 reduces
photoreceptor toxicity, we injected brilliant blue G (BBG) in the
vitreous of mice subjected to a light stress.
Methods: Mice were submitted to 4 days of light challenge to allow
for subretinal inflammation and returned to normal cyclic light
conditions. Mice received an intravitreal injection of PBS or BBG
at day 3 and day 7. Mice were sacrified at day 5 and 10 and retinal
and choroidal flatmounts were evaluated for P2RX7 expression, MP
accumulation and TUNEL staining. Photoreceptor degeneration was
evaluated at day 21 on historesin section.
Results: Immunohistochemistry of P2XR7 on Cx3cr1GFP/GFP
mice light-challenged retinal flatmounts showed that P2RX7
expression was restricted to MP that had infiltrated the subretinal
space while IBA1+. Microglial cells of the inner retina were not
P2RX7 positive. Quantification of subretinal MP on IBA1 stained
choiroidal and retinal flamounts stained at d10 did not reveal any
differences in subretinal MP accumulation after BBG injections. In
contrast, TUNEL staining of apoptotic nuclei in the ONL of retinal
flatmounts showed a two fold decreased in the number of apoptotic
photoreceptor cells in animal treated with BBG correlated with an
increased in the ONL thickness at d21.
Conclusions: Our results demonstrate P2RX7 inhibition reduces
subretinal MP toxicity in a model of light-induced subretinal
inflammation and suggest that intravitreal injections of P2RX7
antagonists might help at preventing photoreceptor cell loss in AMD.
Commercial Relationships: Xavier P. Guillonneau, None; shulong
hu, None; Bertrand Calippe, None; Sophie Lavalette, None; Jose
A. Sahel, None; Florian Sennlaub, None
Up-regulation of P2RX7 in Cx3cr1-deficient mononuclear
phagocytes leads to increased interleukin-1β secretion and
photoreceptor neurodegeneration
shulong hu, Bertrand Calippe, Sophie Lavalette, José Sahel, Florian
Sennlaub, Xavier P. Guillonneau. U968, Institut de la vision, inserm,
Paris, France.
Purpose: Inflammatory mononuclear phagocytes (MP) accumulate in
geographic atrophy form of age related macular degeneration (AMD)
which are associated with photoreceptor degeneration. We have
previously shown that Cx3cr1-deficient mice develop age-related
subretinal accumulation of MP with photoreceptor degeneration.
Cx3cr1-deficient MP have been shown to lead to increased neuronal
apoptosis through IL-1beta (IL-1β) in the brain. However the
mechanisms for this increased IL-1β secretion from Cx3cr1-deficient
MP is still unknown.
Methods: Bone marrow monocytes (BMM) were isolated from
wildtype and Cx3cr1-deficient mice and analyzed by flow cytometry
for P2RX7 expression. Equal numbers of wildtype and Cx3cr1deficient BMM were co-cultured with wildtype retinal explants or
purified photoreceptor outer segment (POS). After 18 hours, BMMderived MP mRNA expression of specific pro- or anti-infammmatory
markers were assayed by qPCR. ATP and IL-1β were assayed in
BMM-derived supernatants. In specific experiments, TUNEL staining
was performed on retinal explants to assay the neurotoxicity of
wildtype and Cx3cr1-deficient BMM.
Results: Cx3cr1-deficient BMM have an increased surface
expression of P2RX7 and secreted increased amount of IL-1β upon
TLR stimulation in an P2RX7-dependent mechanism when compared
to wildtype BMM. Culturing Cx3cr1-deficient BMM in the presence
of POS enhanced the expression of inflammatory genes such as IL1β and P2RX7 while in similar conditions wildtype BMM express
higher levels of CD206 and IL-1Ra. Cx3cr1-deficient BMM have
an increased photoreceptor toxicity in a BMM/retina co-culture
model. Addition of IL-1Ra or Brilliant blue G efficiently reduce
photoreceptor cell loss in the BMM/retina co-culture.
Conclusions: We here show that spontaneous P2RX7 activation and
enhanced IL-1β secretion are responsible for subretinal CX3CR1deficient MP photoreceptor toxicity. Our results suggest that
inhibition of the inflammasome may reduce photoreceptor cell loss in
AMD
Commercial Relationships: shulong hu, None; Bertrand Calippe,
None; Sophie Lavalette, None; José Sahel, None; Florian
Sennlaub, None; Xavier P. Guillonneau, None
Apolipoprotein E promotes subretinal mononuclear phagocyte
survival
Florian Sennlaub1, 2, Olivier Levy1, 2, Bertrand Calippe1, 2, Sophie
Lavalette1, 2, Shulong J. Hu1, 2, Elisa Dominguez1, 2, Michael Housset1,
2
, Michel Paques1, 2, José-Alain Sahel1, 2, Xavier P. Guillonneau1,
2 1
. Univ Pierre et Marie Curie, Paris, France; 2Institut de la Vision,
Paris, France.
Purpose: Physiologically, the retinal pigment epithelium (RPE)
expresses immunosuppressive signals such as FAS ligand (FASL),
which prevents the accumulation of leukocytes in the subretinal
space. Age related macular degeneration (AMD) is associated with
a breakdown of the subretinal immunosuppressive environment
and chronic accumulation of mononuclear phagocytes (MPs). MPs
are known to express high levels of Apolipoprotein E (APOE)
and the APOE isoform 2, which is associated with higher APOE
concentrations, increases the risk of AMD. Here we investigated the
influence of APOE on subretinal MP accumulation.
Methods: This study used wildtype-, Cx3cr1G/G-, ApoE-/--, Cx3cr1G/
G
ApoE-/--, Faslpr-, and FasLgld-mice, recombinant APOE, IL6, IL-6
blocking antibody and a Fas agonist. The subretinal MP accumulation
was studied in age-, light-, and-laser-induced subretinal inflammation
and subretinal adaptive transfer of MPs of the different genotypes to
wildtype mice.
Results: We show that subretinal MPs in AMD patients accumulate
on the RPE and express high levels of APOE. MPs of Cx3cr1-/mice that develop MP accumulation on the RPE, photoreceptor
degeneration, and increased choroidal neovascularization, similarly
express high levels of APOE. ApoE deletion in Cx3cr1-/-mice prevents
pathogenic age- and stress-induced subretinal MP accumulation. We
demonstrate that increased APOE levels induce IL-6 in MPs via the
activation of the TLR2-CD14-dependent innate immunity receptor
cluster. IL-6 in turn represses RPE FasL expression and prolongs
subretinal MP survival. This mechanism may account, in part, for the
MP accumulation observed in Cx3cr1-/-mice.
Conclusions: Our results underline the inflammatory role of APOE in
sterile inflammation in the immunosuppressive subretinal space. They
provide rationale for the implication of the APOE2 isoform and IL-6
in AMD, and open avenues toward therapies inhibiting pathogenic
chronic inflammation in late AMD.
Commercial Relationships: Florian Sennlaub, None; Olivier
Levy, None; Bertrand Calippe, None; Sophie Lavalette, None;
Shulong J. Hu, None; Elisa Dominguez, None; Michael Housset,
None; Michel Paques, None; José-Alain Sahel, None; Xavier P.
Guillonneau, None
Support: This work was supported by grants from INSERM, ANR
Maladies Neurologiques et Psychiatriques (ANR- 08-MNPS -003),
ANR Geno 2009 (R09099DS), Labex Lifesenses, Carnot, and ERC
starting Grant (ERC-2007 St.G. 210345) and HUMANIS.
Elevated levels of helper T cell-related cytokines in the aqueous
humor in age-related macular degeneration and retinal vein
occlusion
Tomohito Sato1, Masaru Takeuchi1, Yoko Karasawa1, Masataka Ito2,
Toshio Enoki3. 1Ophthalmology, National Defense Medical College,
Tokorozawa, Japan; 2Developmental Anatomy and Regenerative
Biology, National Defense Medical College, Tokorozawa, Japan;
3
ENOKI EYE CLINIC, Sayama, Japan.
Purpose: To investigate the involvement of helper T (Th)-related
cytokines at the onset of exudative age-related macular degeneration
(wet AMD) and retinal vein occlusion (RVO) with macular edema
patients.
Methods: Seventeen eyes of 17 patients with wet AMD, 6 eyes of
6 patients with RVO who had undergone intravitreal injection of
anti-VEGF antibody, and 8 eyes of 7 patients with cataract (Cat)
who underwent Cat surgery were studied. Undiluted aqueous humor
samples were collected at the first injection of anti-VEGF antibody
in wet AMD and RVO, and at the initiation of Cat surgery in Cat.
PDGF-BB, IL-1β, IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-12, IL-13, IL-15, IL-17A, Eotaxin, bFGF, G-CSF, GMCSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNFα
and VEGF in the aqueous humor were measured by the Bio-Plex
kit® (Bio-Rad Laboratories, Inc.). The cytokines with values below
limits of detection were assigned a numerical value of 0 pg/ml for
statistical analysis. Levels of cytokines and correlations between
VEGF and other cytokines were evaluated by Wilcoxon signed-rank
test and Spearman’s correlation, respectively. The level of statistical
significance was set under 0.05.
Results: In levels of cytokines, levels of IL-12, IP-10, MIP-1β
and VEGF in wet AMD, and of IL-7, IL-8, IL-12, IL-13, bFGF,
IP-10, MCP-1 and VEGF in RVO were significantly higher than
those in Cat. In correlations between VEGF and other cytokines,
there were positive correlation with IL-12 (rs =0.88, p0.0019) and
negative correlation with IL-7 (rs=0.74, p0.018) in Cat, positive
correlation with IL-12 (rs=0.93, p3.44x10 -8) in wet AMD, and
positive correlations with IL-6 (rs=0.89, p0.0094), IL-9 (rs =0.84,
p0.017), IL-12 (rs =0.94, p0.0024, G-CSF (rs =0.84, p0.017), MCP-1
(rs =0.77, p0.036) and negative correlation with MIP-1β (rs =0.77,
p0.036) in RVO, respectively.
Conclusions: The present study indicated that Th1-related cytokines
in wet AMD, Th1- and Th2-related cytokines in RVO were elevated
simultaneously with increase of macrophage-related cytokines. The
findings suggest that Th cells may be involved in the development of
wet AMD and RVO.
Commercial Relationships: Tomohito Sato, None; Masaru
Takeuchi, None; Yoko Karasawa, None; Masataka Ito, None;
Toshio Enoki, None
The impact of systemic inflammation on age-related macular
degeneration
Paul L. Ibbett1, Andrew J. Lotery2, V. Hugh Perry1, Jessica Teeling1.
1
Centre for Biological Sciences, University of Southampton,
Southampton, United Kingdom; 2Clinical Neurosciences, University
of Southampton, Southampton, United Kingdom.
Purpose: Systemic inflammation exacerbates neurodegenerative
diseases e.g. Alzheimer’s disease, but the impact of systemic
inflammation on age-related macular degeneration (AMD) is
unknown. We hypothesise that a) systemic inflammation can activate
and ‘prime’ retinal microglia to respond in a greatly exaggerated
manner to subsequent immune activation and b) that this contributes
to AMD pathogenesis and/or progression. We tested this hypothesis
by modelling inflammatory drusen in AMD using immune complexes
and chronic systemic inflammation using bacterial infection.
Methods: Mice received an i.p. injection of Salmonella Typhimurium
(S. Typhimurium) or saline and were perfused with heparinised saline
7 and 28 days post infection (n=5 per group). Retinal tissue was snap
frozen and stored at -80°C until qPCR analysis of GADPH and IL-1β
mRNA levels. Whole eyes were embedded in OCT and stored at
-20°C until cryosectioned and assessed for DAB immunoreactivity
to microglial markers CD11b, FcγRI and MHCII. To test for priming,
mice received an i.p. injection of S. Typhimurium followed 28 days
later by i.p. injection of LPS (0.5mg/kg) or saline (n=5 per group) and
tissue was collected 24 hours later and examined as before. Immune
complexes were formed in the retinae of mice as previously described
(Murinello et al. 2014) followed 28 days later by i.p. injection of S.
Typhimurium (n=3 per group). Tissue was collected 7 days later and
examined as before.
Results: 7 days of S. Typhimurium infection induced MHCII
expression associated with retinal blood vessels. Retinal IL-1β
mRNA expression was undetectable in saline controls and detectable
after 28 days of S. Typhimurium infection. LPS injection, induced
a threefold increase in IL-1β mRNA in the retina after 24hrs in S.
Typhimurium infected animals (p<0.05; Two-tailed T test), but not
saline or LPS only controls. CD11b, FcγRI and MHCII expression in
the retina after 7 days of S. Typhimurium infection was not increased
by previous immune complex induced retinal inflammation.
Conclusions: Chronic systemic inflammation induced blood-retinalbarrier activation after 7 days and microglial activation and IL-1β
expression in the retina after 28 days. These microglia are primed
to respond in an exaggerated manner to subsequent inflammatory
stimuli. This may contribute to the pathogenesis of AMD, if microglia
are primed by systemic inflammation to respond to inflammatory
drusen formation in AMD.
Commercial Relationships: Paul L. Ibbett, None; Andrew J.
Lotery, None; V. Hugh Perry, None; Jessica Teeling, None
Support: Fight for Sight PhD Studentship
The TGFβ co-receptor betaglycan (TGFBR3) is not implicated in
TGFβ-dependent T-cell responses
Robert J. Barry1, 2, David Withers3, Graham R. Wallace1, 2, Peter
Lane3, Philip I. Murray1, 2, John Curnow1, 2. 1Academic Unit of
Ophthalmology, University of Birmingham, Birmingham, United
Kingdom; 2Centre for Translational Inflammation Research,
University of Birmingham, Birmingham, United Kingdom; 3Institute
of Biomedical Research, University of Birmingham, Birmingham,
United Kingdom.
Purpose: Transforming Growth Factor-β (TGFβ) is considered an
important immunoregulatory cytokine. In experimental models of
uveitis, TGFβ, in the presence of pro-inflammatory co-stimulatory
molecules, drives differentiation of naïve CD4+ T-cells to a
pathogenic Th17 phenotype. TGFβ exists in three isoforms and
signals through a receptor comprising three subunits. The role of
TGFBR1 and 2 is well characterized but the function of TGFBR3
(betaglycan) is unclear. Betaglycan is implicated in embryogenesis,
carcinogenesis, and intrathymic T-cell development, but its role in
peripheral immune responses is unknown. Betgalycan is considered
necessary to present TGFβ2, but not the other isoforms, to the
TGFBR1/2 receptor complex and may be important in environments
where TGFβ2 predominates, such as the eye. Our aim was to
examine the role of betaglycan in controlling T-cell responses to
TGFβ.
Methods: Homozygous betaglycan deficiency is embryonic lethal;
we therefore generated fetal liver chimeras in rag-deficient mice
to create a model in which all T and B-cells lack betaglycan. We
phenotyped this model, performed controlled antigenic challenge
in vivo to assess Th1, Treg and Th17 responses, and created naive
T-cell differentiation assays in vitro to assess FoxP3 induction by all
isoforms of TGFβ.
Results: Betaglycan-deficient mice showed reconstitution of
peripheral lymphocyte populations within 6 weeks of cell transfer.
We observed sporadic immune activation in betaglycan-deficient
mice, demonstrated by increased proportions of effector CD4+ and
CD8+ T-cells in circulating, lymph node and splenocyte populations.
We have found no evidence of circulating serum autoantibodies. No
significant difference was observed between betaglycan-deficient and
wild-type chimeras in response to antigenic challenge by attenuated
Listeria monocytogenes, incomplete Freund’s adjuvant or bacterial
porins. All isoforms of TGFβ induced FoxP3 expression in naïve
CD4+ T-cells with no significant difference between cells derived
from betaglycan-deficient and wild-type chimeras.
Conclusions: We have created a novel experimental model in
which to assess the role of betaglycan on mature T-cell responses.
In contrast to its role in other areas, betaglycan appears unnecessary
for TGFβ-dependent T-cell responses in our assays. Further work
is required to assess betaglycan function in environments where
TGFβ2-driven T-cell responses predominate.
Commercial Relationships: Robert J. Barry, None; David
Withers, None; Graham R. Wallace, None; Peter Lane, None;
Philip I. Murray, None; John Curnow, None
Support: Fight for Sight (UK) Clinical Fellowship
Paradoxically Decreased Uveitis Susceptibility in PD-L1/L2
Double Knockout Mice
Negin Ashki, Ann Chan, Yu Qin, Lynn K. Gordon. Ophthalmology,
University of Los Angeles California, Los Angeles, CA.
Purpose: Uveitis is a potentially blinding, immune mediated
intraocular inflammatory disease. Programmed death-1 (PD-1), an
immune molecule with two known ligands, is believed to downregulate autoimmunity. Mice that lack PD-1 or its ligands are
typically more susceptible to specific types of spontaneous or induced
autoimmune disease. We tested the hypothesis that susceptibility to
uveitis increases in animals that lack PD-ligands using an animal
model of experimental autoimmune uveitis (EAU).
Methods: Uveitis was induced in C57Bl/6 (WT) and PD-L1/L2
double knockout (DKO) mice on the C57Bl/6 background using
IRBP (1-20) according to published protocols. All experiments
were carried out in strict accordance with ARVO guidelines and the
Guide for the Care and Use of Laboratory Animals. Animals that
did not receive IRBP immunization served as controls for the uveitis
studies. A masked clinical assessment by funduscopic examinations
of the retinal inflammation was done every day for 3 weeks after the
immunization. The severity of the retinal inflammation was graded on
a four-point scale. At 3 weeks following immunization, the eyes were
enucleated, fixed tissues were stained, and the histological severity
was graded by multiple masked observers using a published scale.
Results: Surprisingly, we found significant abrogation of uveitis in
the DKO animals. This experiment was repeated with a total number
of 20 animals per group. The percent of WT mice that developed
uveitis was 70%, whereas only 30% of the DKO developed
intraocular inflammation (P<0.05). Histologic activity score by
immunohistochemistry was also reduced in the DKO animals as
compared to WT (P<0.001).
Conclusions: The decrease in uveitis susceptibility in the DKO mice
was an unanticipated result. First, this observation may lead to a
new understanding of uveitis pathogenesis. Second, the availability
of blocking antibodies for PD-1 and PD-ligands, recently approved
for use in cancer immunotherapy allows us to explore the feasibility
blocking the PD-1 system as a possible therapeutic target in ocular
inflammation
Commercial Relationships: Negin Ashki, None; Ann Chan, None;
Yu Qin, None; Lynn K. Gordon, None
TMP778 Inhibits Induction of Experimental Autoimmune Uveitis
and related immune responses
Samuel Hinshaw1, Wambul (Sylvia) W. Wandu1, Guangpu Shi1, Jianfei
Yang2, Igal Gery1. 1National Eye Institute, NIH, Bethesda, MD;
2
Tempero Pharmaceuticals, GSK, Boston, MA.
Purpose: TMP778, an inverse agonist of RORγt, was recently
demonstrated to inhibit induction of experimental autoimmune
disease (Xiao, et al., Immunity, 2014). Here, we determined the
immunosuppressive property of TMP778 on the development
of experimental autoimmune uveitis (EAU) and related immune
responses.
Methods: Female B10.A mice immunized with an emulsion of
interphotoreceptor retinoid-binding protein (IRBP) in complete
Freund’s adjuvant (CFA) were treated twice daily with TMP778
(provided by Tempero Pharmaceuticals) by subcutaneous injection.
Pathological changes in the eyes were determined by fundoscopy,
while spleen cells were tested in vitro for cytokine production in
response to IRBP. Separately, splenocytes from mice immunized with
IRBP were cultured with TMP778 to test its inhibitory effect on the
response to IRBP by cytokine release.
Results: Fundoscopic analysis of the eyes showed that treatment of
mice with TMP778 significantly inhibited the development of EAU
(p ≤ 0.01). Furthermore, splenocytes from mice treated with TMP778
produced significantly less IFN-γ (p ≤ 0.001) and IL-17 (p ≤ 0.01)
in response to IRBP than their controls. When added to cultures of
splenocytes sensitized against IRBP, TMP778 strongly inhibited the
release of IL-17. Additional data of ongoing studies are to be reported
at the meeting.
Conclusions: Treatment of mice with TMP778 inhibits the
development of EAU and cellular immune responses to IRBP.
TMP778 and other inverse agonists of RORγt are a promising class
of compounds for inhibition of immune-mediated disease.
Commercial Relationships: Samuel Hinshaw, None; Wambul
(Sylvia) W. Wandu, None; Guangpu Shi, None; Jianfei Yang,
Tempero Pharmaceuticals, GlaxoSmithKline (E); Igal Gery, None
Intermediate (CD14++CD16+) monocytes are expanded in noninfectious uveitis patients and have a reduced capacity to drive
Th17 differentiation
Emily L. Williams1, 2, Baoying Liu3, Ashwin Dhanda1, 5, Philippa
J. Lait1, 2, Lauren P. Schewitz-Bowers1, 2, Peter Collins6, Andrew
D. Dick1, 2, Robert B. Nussenblatt3, 4, Richard W. Lee1, 2. 1School of
Clinical Sciences, University of Bristol, Bristol, United Kingdom;
2
National Institute for Health Research (NIHR) Biomedical Research
Centre at Moorfields Eye Hospital and University College London
Institute of Ophthalmology, London, United Kingdom; 3Laboratory
of Immunology, National Eye Institute, National Institutes of Health,
Bethesda, MD; 4Centre for Human Immunology, Autoimmunity
and Inflammation, National Institutes of Health, Bethesda, MD;
5
Plymouth University Peninsula Schools of Medicine and Dentistry,
Plymouth, United Kingdom; 6University Hospitals Bristol NHS
Foundation Trust, Bristol, United Kingdom.
Purpose: Human monocyte subsets are phenotypically distinct
and differ in their ability to stimulate T cells. It is unclear whether
the proportion and function of monocyte subsets is skewed under
inflammatory conditions or influenced by glucocorticoid treatment,
thereby promoting or regulating tissue inflammation. The purpose of
this project was therefore to characterise human monocyte subsets in
the context of non-infectious uveitis and to interrogate their effect on
CD4+ T cell phenotype and response to glucocorticoids.
Methods: The proportion of classical (CD14++CD16-), non-classical
(CD14dimCD16++) and intermediate monocyte (CD14++CD16+)
subsets in the peripheral blood (PB) of patients with non-infectious
uveitis was determined by flow cytometry (n=98). As these patients
were receiving immunosuppressive therapy, monocyte subsets were
similarly assessed in treatment naïve alcoholic hepatitis patients as
comparators (n=19). Classical and intermediate monocytes were
isolated from the PB of healthy volunteers by fluorescent activated
cell sorting (n=14). These subsets were co-cultured for 5 days
with autologous memory CD4+CD45RO+ T cells, in the presence
or absence of the synthetic glucocorticoid Dexamethasone (Dex).
T cell proliferation and intracellular cytokine expression was then
quantified.
Results: Analysis of monocyte subsets in the PB of patients showed
that intermediate monocytes were enriched in both conditions.
Following glucocorticoid treatment, intermediate monocytes were
further expanded in non-infectious uveitis patients. During co-culture,
intermediate monocytes induced less memory T cell proliferation
(29% v 41%; p=0.003), less IL-17 expression (8% v 12%; p<0.001)
but similar IFNγ production (27% v 26%) than classical monocytes.
Dex-mediated suppression of T cell proliferation and IFNγ production
was abrogated to a greater degree in intermediate monocyte cocultures than in classical monocyte co-cultures. T cell proliferation
and IL-17 production was also reduced in co-cultures containing both
monocyte subsets compared with co-cultures containing classical
monocytes alone.
Conclusions: Intermediate monocytes are expanded in the PB of
patients with non-infectious uveitis and have a reduced capacity
to drive memory T cell proliferation and IL-17 production. This is
associated with enhanced T cell suppression by glucocorticoids.
Commercial Relationships: Emily L. Williams, None; Baoying
Liu, None; Ashwin Dhanda, None; Philippa J. Lait, None; Lauren
P. Schewitz-Bowers, None; Peter Collins, None; Andrew D. Dick,
None; Robert B. Nussenblatt, None; Richard W. Lee, None
Support: BRC NIHR Grant
Expression kinetics of Glucocorticoid Receptor Isoforms Predicts
Treatment Response to Glucocorticoids in Uveitis patients
Cristhian A. Urzua1, 2, Han Si2, Baoying Liu2, Philippa Lait3, Richard
W. Lee3, Annelise Goecke4, Robert B. Nussenblatt2. 1Ophthalmology,
Universidad de Chile, Santiago, Chile; 2Laboratory of Immunology,
National Eye Institute, Bethesda, MD; 3School of clinical sciences,
University of Bristol, Bristol, United Kingdom, Bristol, United
Kingdom; 4Universidad de Chile, Santiago, Chile.
Purpose: Glucocorticoids (GC) have been the mainstay therapy for
autoimmue uveitis for decades, but up to one third of patients are
unable to achieve disease control at tolerable GC doses, developing
vision-threatening complications and requiring immunosuppressive
therapy (IMT). Glucocorticoid receptor (GR)-isoforms - including
two classical post-transcriptional GR alpha isoform (GRα) and
beta isoform (GRβ)- have been implicated in the mechanism of
GC resistance. However, the utility of using GR isoforms in uveitis
patients, to identify GC resistance, have not been fully investigated.
In this study, we evaluate the expression kinetics of GRα and GRβ in
peripheral blood mononuclear cells (PBMCs) in uveitis patients, and
test whether it can be used to identify GC resistance at an early point.
Methods: Twenty-one systemic treatment naïve autoimmune uveitis
patients were recruited. GC resistance was defined as a persistence
of active intraocular inflammation, despite of treatment with 1 mg /
kg/day of oral prednisone for at least one month. Otherwise, patients
were categorized as GC-sensitive. Real-Time qPCR were performed
to measure mRNA levels of GR α/β in PBMCs, at baseline and two
weeks after prednisone initiation.
Results: There is no significant difference on the expression levels
of GRα and GRβ between GC-sensitive and GC-resistant patients at
baseline. After two weeks of prednisone treatment, the expression of
GRα increased in GC-sensitive patients, while there was a decrease
of this isoform in GC-resistant patients (5.5 fold vs 0.7 fold, p=0.01).
GRβ expression increased in both groups with a significant higher
level in GC-sensitive patients (6.6 fold vs 4.6 fold, p=0.03). The
expression levels of GR isoforms were independent of disease
activity.
Conclusions: The evaluation of expression kinetics of GR isoforms
could potentially serve as a biomarker to early identify GC-resistant
uveitis patients. These results contribute to our knowledge in
understanding the complex mechanism of GR resistance and may
facilitate clinical decision-making in the management of autoimmune
uveitis.
Commercial Relationships: Cristhian A. Urzua, None; Han Si,
None; Baoying Liu, None; Philippa Lait, None; Richard W. Lee,
None; Annelise Goecke, None; Robert B. Nussenblatt, None
Expression of MSR-1—a Class-A Macrophage Scavenger
Receptor—May Be Altered in Ocular Sarcoidosis
Amol Sura1, 2, Baoying Liu1, Zhiyu Li1, Susan Hannes1, Siamon
Gordon3, Robert B. Nussenblatt1. 1National Eye Institute, National
Institutes of Health, Bethesda, MD; 2Louisiana State University
Health Sciences Center, New Orleans, LA; 3Sir William Dunn School
of Pathology, University of Oxford, Oxford, United Kingdom.
Purpose: Ocular sarcoidosis is a multisystem, idiopathic disorder
characterized by noncaseating granulomas in affected tissues
including the eye. Granulomas arise from the transformation and
reorganization of macrophages into epithelioid cells. The role that
macrophages play in ocular inflammation, however, is less clear. In
this present study we aimed to investigate the relationship between a
macrophage scavenger receptor, MSR-1, and ocular sarcoidosis.
Methods: Human peripheral blood mononuclear cells were isolated
from sarcoid patients and healthy controls using a Ficoll gradient
centrifugation protocol. Monocytes were isolated from PBMCs using
CD14+ magnetic separation beads (Miltenyi). RNA sequencing
(Illumina) was used to study the transcriptome profiling of monoctyes
from sarcoid patients, when compared to age and race matched
controls. Quantitative real-time PCR (qPCR) was used to confirm
RNAseq findings. For animal studies, Class-A scavenger receptor
(SR-A) knockout mice and their wild type controls on a C57BL/6
background were immunized with LPS (100 μg) to induce endotoxininduced uveitis (EIU). Anterior chamber taps for leukocyte cell count
were performed at 24 h and 48 h after immunization.
Results: RNA Sequencing (n=3 controls, n=4 patients) revealed
a 1.49 ± .370 fold increase in MSR-1 RNA expression in sarcoid
patients compared to healthy controls (p=.007). Quantitative RT-PCR
(n=8 controls, n=11 patients) of MSR-1 RNA expression showed a
non-significant increase in MSR-1 expression (95% CI: .821 ± 2.00)
in sarcoid patients compared to controls. Mouse studies revealed that
at 48 h, mice without EIU (n=5) had no cell filtration, SR-A (+) mice
with EIU (n=5) had an average infiltration of 49 cells/μl, and SR-A
(-) mice with EIU (n=5) had an average infiltration of 28 cells/μl.
Conclusions: Our data to date suggest that changes in MSR-1
expression may affect ocular inflammation. In affected patients, we
found altered levels of MSR-1—a Class-A macrophage scavenger
receptor (SR-A) known to play an important role in atherosclerotic
inflammation and murine choroidal neovascularization. The
decreased cell infiltration in SR-A knockout mice with uveitis—as
well as the central role of macrophages in inflammatory eye diseases
and granuloma formation—provide plausible connections between
MSR-1 and the macrophage-mediated inflammatory response.
Commercial Relationships: Amol Sura, None; Baoying Liu, None;
Zhiyu Li, None; Susan Hannes, None; Siamon Gordon, None;
Robert B. Nussenblatt, None
Tau oligomers trigger inflammation in the eyes of the Alzhiemer’s
disease mouse models
Praveena Gupta1, Diana Castillo-Carranza3, Barton Whittle2, Nick
Crain2, Adriana Paulucci4, Bernard F. Godley1, Julia Gerson3,
Urmi Sengupta2, Rakez Kayed3. 1Ophthalmology & Visual Sciences,
Univ of Texas Medical Branch, Galveston, TX; 2University of
Texas Medical Branch, Galveston, TX; 3Mitchell Center for
Neurodegenerative Diseases, University of Texas Medical Branch,
Galveston, TX; 4Optical Microscopy Core, University of Texas
Medical Branch, Galveston, TX.
Purpose: Neurofibrillary tangles (NFT’s) are a pathological hallmark
of neurodegenerative tauopathies. Recent findings suggest that tau
oligomers, a precursor to NFT’s, are the true neurotoxic tau entities
in these diseases. This study is undertaken to determine if the
accumulation of tau oligomers initiate inflammatory response in the
eyes of Alzheimer’s disease mouse models.
Methods: Eyes from wild type, htau (overexpressing human tau) and
P301L (mutant tau) mouse models were dissected and processed for
cryosections. Immunohistochemistry was performed using anti-T22
oligomer specific antibody, anti-tau 5 (for total tau) and GFAP (Glial
cell) and Iba1 (microglia) antibodies. Images were then captured
using a confocal fluorescence microscope. In addition, Western
blot of eye homogenates were performed using antibodies for tau
oligomers (T22) and tau 5.
Results: Tau oligomers and total tau proteins were detected in the
ganglion cells and upto outer nuclear layers of the retina in the AD
mouse models and were also confirmed by Western blots. GFAP
positive Muller cell processes were seen extending from the ganglion
cell layer to the inner nuclear layer. A few of their cell bodies
colocalized with the tau oligomers suggesting the engulfment of the
oligomers by the Muller cells. Increased number of hyperactivated
astrocytes were noted in both the transgenic mouse models mostly at
the vicinity of the oligomer deposits in all the cell layers of the retina.
In addition, extensive staining of the Iba1 antibody was prominent
in the plexiform and nuclear layers of the tau transgenic models
indicating microglia activation. Age-matched wild type did not show
any signs of inflammation.
Conclusions: Our findings provide evidence of inflammation
associated with tau-oligomers in the retina of the tau-transgenic
mouse models and that tau oligomers play a vital role in the
pathogenesis of Alzheimer’s disease.
Commercial Relationships: Praveena Gupta, None; Diana
Castillo-Carranza, None; Barton Whittle, None; Nick Crain,
None; Adriana Paulucci, None; Bernard F. Godley, None; Julia
Gerson, None; Urmi Sengupta, None; Rakez Kayed, None
Support: RPB to the department
In vivo confocal microscopy of inflammatory cells in the central
cornea in patients with different subtypes of anterior uveitis
Friederike Mackensen1, 2, Alexandra B. Knoll1, 2, Gerd Auffarth2,
Silvia Postole1. 1Ophthalmology, Interdisciplinary Uveitis Center,
Heidelberg, Germany; 2Ophthalmology, University Hospital
Heidelberg, Heidelberg, Germany.
Purpose: Previously we could show increased numbers and densities
of dendritic-like cells (DLC) in the subbasal nerve plexus of the
central cornea in patients with Herpetic Anterior Uveitis (HAU). Now
we aimed to explore these and other inflammatory cells seen in this
layer in different subtypes of anterior uveitis using in vivo confocal
microscopy.
Methods: Eyes of patients with different types of anterior uveitis,
Herpetic Anterior Uveitis (HAU), Fuchs Uveitis Syndrome (FUS),
Juvenile Idiopathic Arthritis (JIA), Sarcoid Uveitis and HLA-B27
related anterior Uveitis, were examined in vivo with the Heidelberg
Retina Tomograph II/III and Rostock Cornea Module (HRT RCM).
The contralateral eye and published data on healthy eyes was used
as control. Inflammatory cells were defined on the basis of their
morphology: type 1 (dendritic-like cells (DLC) and type 2 (cell
bodies lacking dendrites). Frequencies were determined with the
in-built cell counting software and means of 3 images evaluated
statistically.
Results: So far 89 eyes of 48 patients were included. The difference
between means of type 1 cells density of affected eyes in all four
groups was significant (One-way ANOVA p=0.039). The difference
between means of type 1 cell densities of affected eyes in patients
with HAU (96.8 ± 44.2 cells/mm2 n=10) and that of FUS patients
(46.4 ± 38.7 cells/mm2 n=17) was significant (Tukey’s Post-hoc
p=0.025), whereas the difference between HAU and JIA (53.3 ± 34.5
cells/mm2 n=7) and HAU and HLA-B27 patients (63.1 ± 59.2 cells/
mm2 n=10) was not significant (Tukey’s Post-hoc; p=0.181 and
p=0.300). A cut-off value of 61 DLC/mm2 was calculated for HAU.
The difference between means of affected eyes for type 2 cells was
not significant (One-way ANOVA p=0.185)
Fellow eyes, even though clinically unaffected, showed an
inflammatory response although to a lesser extent. In two of the
groups we noticed a significant difference between affected and
contralateral eye (HAU p=0.0010; FUS p=0.0199) whereas in the
other groups both eyes showed similar snumbers.
Conclusions: The high density and morphology of DLC in the
central cornea of patients with HAU assessed by confocal microscopy
supports the clinical diagnosis of HAU especially when compared
to FUS patients. Interestingly these cells are also found in eyes with
other anterior uveitis subtypes and unaffected eyes, although to a
lesser extent.
Commercial Relationships: Friederike Mackensen, Heidelberg
Engineering (R); Alexandra B. Knoll, None; Gerd Auffarth, None;
Silvia Postole, None
Infliximab for the treatment of ocular cicatricial pemphigoid, a
Preliminary Report
Joan J. Lee1, Charles S. Foster1, 2. 1Uveitis, Massachusetts Eye
Research and Surgery Institution, Cambridge, MA; 2Ophthalmology,
Harvard Medical School, Cambridge, MA.
Purpose: The purpose of this study is to evaluate the efficacy of antiTNF drug, Infliximab, in ocular cicatricial pemphigoid.
Methods: Retrospective chart review of a specialty practice.
Results: Nine patients with the diagnosis of ocular cicatricial
pemphigoid who were treated with infliximab infusion were
identified. Age at diagnosis ranged from 29-88 years old, six were
female and 3 were male, and all patients had stage 1-3 disease. The
follow up following diagnosis ranged from 1 consultation visit to 111
months. The dosage of infliximab used ranged from 4.76mg/kg to
7.5mg/kg. Four patients achieved remission with Infliximab alone,
1 patient achieved remission with infliximab and one other immunemodulatory drug and the remaining 4 patients failed to achieve
remission with Infliximab.
Conclusions: Ocular cicatricial pemphigoid (OCP) is
an inflammatory condition that is potentially blinding.
Immunosuppressive chemotherapy is required to control this disease.
Several chemotherapy medications have been used successfully in
the treatment of OCP. However, treatment resistance and side effects
to medication may limit a patient’s options for treatment. Elevated
levels of tumor necrosis factor alpha in the serum and conjunctiva
have been previously documented in patients with OCP. We report
a preliminary set of data that show some promise in the efficacy of
infused infliximab at doses of 4.76-7.5mg/kg in ocular cicatricial
pemphigoid.
Commercial Relationships: Joan J. Lee, None; Charles S. Foster,
None
Long-term Ocular Analysis in Murine Model of Anterior Scleritis
Hiroko Taniguchi, Yuki Kitahara, Junko Hori. Nippon Medical
School, Tokyo, Japan.
Purpose: We have previously established a model of anterior scleritis
by modifying a collagen-induced autoimmune arthritis model. In
the present study, the long-term observation of the clinical and the
immunological findings were performed.
Methods: Male DBA/1J mice (8-week-old) received primary
immunization in the back of the neck with 200ug of bovine type
II collagen (CII) emulsified using equal volume of complete
Freund’s adjuvant (CFA, containing 100ug H37Ra Mycobacterium
tuberculosis). After 3 weeks, CFA-emulsified CII was injected
intradermally around the eye for secondary immunization, then
the arthritis and eyes were examined. Eyeballs were excised at 3,
5, 8, 12 and 24 weeks after secondary immunization and analyzed
histologically and immunohistologically.
Results: Clinical findings comprised severe arthritis and dilation of
scleral blood vessels from 3 weeks after secondary immunization.
Histological findings revealed anterior scleral thickening, with
significantly large number of infiltrating cells as compared to
untreated mice. The number of infiltrating cells in anterior sclera
peaked at 8 weeks and remained for 24 weeks. The inflammation of
anterior sclera peaked at a later time point compared with arthritis.
Infiltration of CD4+, CD11b+ cells were present in the Tenon’s
layer, while deposition of plasma cells (CD138), complement (C3),
immunoglobulin (Ig)G and IgM were seen in the anterior sclera
in contact with the ciliary body and blood and lymphatic growth
(CD31 and LYVE-1) expression was increased in the corneal limbus
compared to untreated mice, throughout all observation periods.
Conclusions: T cells, macrophages, plasma cells, complement,
immunoglobulins, the outgrowth of blood and lymphatic vessels
were persistently found in the sclera of the collagen-induced
anterior scleritis model. It is suggested that the involvement of
immunocomplex deposition, and blood and lymphatic growth in the
sclera is one of immunopathology of this scleritis model.
Commercial Relationships: Hiroko Taniguchi, None; Yuki
Kitahara, None; Junko Hori, None
Support: Grant-in-Aid for Scientific Research (C) from the Japan
Society for the Promotion of Science
Increased tear IL-17 levels are related with HLAB27 associated
uveitis patient
Miriam Gómez1, Maria Valdez1, Milton Maldonado1, Francisco
Martínez-Castro1, David De La O-Altamirano2, Atzin RoblesContreras2, Stephanie Voorduin1. 1Ocular Inflammatory Disease,
Fundación Hospital “Nuestra Señora de la Luz” IAP, Mexico City,
Mexico; 2Centro de Investigación Biomédica, Fundación Hospital
“Nuestra Señora de la Luz” IAP, Mexico City, Mexico.
Purpose: It is known that IL-17 has an important role in autoimmune
processes. We did not found studies that relate levels of IL-17 in
HLA-B27 associated uveitis patients. So the purpose of this study
was to measure IL-17 levels in tear and serum of patients with
HLA-B27 associated uveitis and compare them with healthy donors.
Methods: An observational, analytical, transversal and comparative
study was performed. Male and female adults with HLA-B27
associated uveitis and healthy donors were included. Patients were
divided in groups according to presence/absence of inflammatory
activity and type of treatment (no treatment, topical treatment and
systemic treatment independet of topical tratment). IL-17 was
measured in tears and serum of patients and healthy donors by
ELISA (R&D systems). IL-17 levels obtained from each group were
compared with control group. A non paired t test was used to compare
the groups (GraphpadPrism v.5.0 software). A value of p<0.05 was
considered statistically significant.
Results: Twenty-three tear and 21 serum samples of 22 patients
were obtained from the HLA-B27 associated uveitis group, and 9
tear and 9 serum samples from the control group. In the HLA-B27
associated uveitis group, the average age was 42±13years, 13
patients (59%) were female; 8eyes (35%) with no activity, 15eyes
(65%) with activity, 1(4.3%) with no treatment, 12(52%) with
topical treatment and 10eyes (43.5%) with systemic treatment were
included. In the control group, the average age was 28±3 years,
6 patients(67%) were female. There was a statistical significant
difference between the control (20.89±1.01pg/mL) and active patients
(25.07±1.35pg/mL) (p<0.05). Also, there was a difference between
the control (20.89±1.01pg/mL) and patients with topical treatment
(25.27±1.56pg/mL) (p<0.05). There was no difference between
control group vs non active neither patients with systemic treatment.
It wasn’t possible to compare the no treatment group. The IL17 levels
in serum were lower than limit detection in all participants.
Conclusions: IL-17 levels are increased during inflammatory
activity. Systemic treatment reduces tear IL-17 to normal levels, but
topical treatment does not. HLA-B27 associated uveitis is probably
mediated by Th17 response, larger studies are needed in order to
confirm this hypothesis.
Commercial Relationships: Miriam Gómez, None; Maria Valdez,
None; Milton Maldonado, None; Francisco Martínez-Castro,
None; David De La O-Altamirano, None; Atzin Robles-Contreras,
None; Stephanie Voorduin, None
An Open-Label Trial to Assess the Efficacy and Safety of
Tocilizumab in the Management of Juvenile Idiopathic ArthritisAssociated Uveitis: Preliminary Results
Eric B. Suhler3, 1, Tracy R. Giles1, Phoebe Lin1, Amde S. Shifera1,
James T. Rosenbaum1, 2. 1Ophthalmology/Casey Eye Institute, Oregon
Health & Science University, Portland, OR; 2Ophthalmology, Devers
Eye Institute, Portland, OR; 3Ophthalmology, VA Portland Health
Care System, Portland, OR.
Purpose: To assess the efficacy and safety of tocilizumab, a
monoclonal antibody against the IL-6 receptor, in the treatment
of JIA-associated uveitis refractory to other modes of systemic
immunosuppression.
Methods: We received IRB and FDA approval to recruit 5 patients
into a 16 month open-label study of the effectiveness of monthly
tocilizumab 8-10 mg/kg infusions for the treatment of refractory
juvenile idiopathic arthritis (JIA)-associated uveitis. Patients
characterized as refractory had failed treatment with corticosteroids
and at least one standard immunosuppressive. Initial treatment
outcome was ascertained at 16 weeks after study initiation by ability
to control anterior chamber intraocular inflammation and without
need to increase adjunctive systemic immunosuppressive therapy
and utilizing no more than two drops of prednisolone acetate daily.
Secondary outcome measures included visual acuity, macular edema
as measured by ocular coherence tomography (OCT), and counts
of swollen or painful joints. Patients who meet criteria for clinical
success at 16 weeks will be allowed to complete up to 52 additional
weeks in the study with final treatment outcome ascertained at week
68.
Results: Two patients have enrolled in this study at the time of this
abstract submission. One met the primary endpoint of control of
inflammation with steroid tapering by week 16, and has continued
in the study. The second patient failed to gain control of bilateral
anterior chamber inflammation with high dose corticosteroids two
tocilizumab infusion, and was withdrawn from the study at week 8.
Interestingly, both patients had OCT-documented macular edema at
study outset and had resolution of macular edema on study therapy.
Conclusions: Our preliminary results suggest that tocilizumab
may be effective for the treatment of uveitic macular edema in
patients with refractory JIA-associated uveitis, and was beneficial
in achieving and maintaining control of inflammation in one of two
early study enrollees. No treatment-limiting toxicity was noted.
Enrollment is ongoing and further study is required to define patient
populations who may derive the most benefit from tocilizumab
therapy for JIA-associated uveitis.
Commercial Relationships: Eric B. Suhler, AbbVie (C), AbbVie
(F), Bristol-Meyers-Squibb (F), EyeGate (F), Genentech (F), XOMA
(C); Tracy R. Giles, Genentech (F); Phoebe Lin, Genentech (F);
Amde S. Shifera, Genentech (F); James T. Rosenbaum, Genentech
(F)
Support: Genentech
Epidemiology of cicatricial conjunctivitis in a reference
ophthalmological center of Mexico City
Arriozola Rodriguez Karen Janeth, Monica Almanza Monterrubio,
Miguel Pedroza-Seres. Conde de Valenciana, Distrito Federal,
Mexico.
Purpose: To analyze the characteristics of cicatricial conjunctivitis in
a tertiary eye center in Mexico.
Methods: We review electronic charts of patients with diagnosis of
cicatricial conjunctivitis between 2001 and 2014. Infectious etiology
of conjunctival scarring were ruled out. Information on demographic
characteristics, ophthalmologic examination, systemic illness,
medications and results of conjunctival biopsies were used for this
analysis. Patients with mucous membrane pemphigoid with ocular
involvement (ocular cicatricial pemphigoid) were classified according
to Foster’s classification.
Results: From a total of 847 patients with diagnosis of cicatricial
conjunctivitis, 104 were included in the study. 71 (68%) were female
and 34 (32%) male. The mean age at presentation was 61.7 years. 41
patients (39%) were diagnosed with ocular cicatricial pemphigoid,
16 (16%) pseudopemphigoid, 16 (15%) Steven’s Johnson syndrome
and 15 (14%) chronic unspecific conjunctivitis. Other diagnosis were
pemphigus, Sjogren syndrome, squamous cell carcinoma, linear IgA
bullous dermatosis. Conjunctival biopsies were made in 40 patients
(38%), 14 (35%) were positive to ocular cicatricial pemphigoid, 13
(32%) to chronic unspecific conjunctivitis, 5 (12.5%) to pemphigus
vulgar. Most of the patients with cicatricial conjunctivitis had ocular
cicatricial pemphigoid and 20 (50%) were stage 3, 13 (32%) stage
2 and 6 (15%) stage 1 at initial visit and at the end 5 (12%) were
stage 1, 7 (17%) stage 2, 21 (52%) stage 3 and 4 (1%) progress
to stage 4. The mean visual acuity in this group was 0.78 at first
visit and 0.79 LogMar at last one. Follow-up time varies from 1
month to 8 years. A total of 65% of patients with ocular cicatricial
phemphigoid showed clinical activity of the disease and required
systemic immunosupressor medication for control: 20 (74%) patients
received oral steroid, 14 (51%) diamino-diphenyl-sulfone, 11 (40%)
methotrexate, 9 (33%) azathioprine, 7 (27%) cyclophosphamide
and 1 (3%) mycophenolate mophetil. Of all these patients 20 (74%)
required two or more systemic medication due to poor control or
recurrent reactivation.
Conclusions: The main diagnosis of cicatricial conjunctivitis in our
study was mucous membrane pemphigoid with ocular involvement.
Patients with ocular cicatricial pemphigoid are seen in an advanced
clinical stage. This is the first study of the causes of cicatricial
conjunctivitis in our media.
Commercial Relationships: Arriozola Rodriguez Karen Janeth,
None; Monica Almanza Monterrubio, None; Miguel PedrozaSeres, None
Efficacy and safety of infliximab and adalimumab in treating
ocular autoimmune diseases
Miriam Allende, Lucia Ibares-Frias, Sonia C. Labrador, Cristina
Wong, Lidia Cocho, Jose M. Herreras. IOBA, Valladolid, Spain.
Purpose: To establish the efficacy and safety of the biological
response modifiers (BRMs) infliximab and adalimumab in treating
refractory ocular autoimmune disease.
Methods: Retrospective observational clinical analysis of case
series of resistant autoimmune diseases from a single University
Hospital which were treated with adalimumab or infliximab. All
patients with intermediate uveitis were required to undergo magnetic
resonance imaging of the brain to rule out demyelinating disease.
Epidemiological data, data related to treatment with BRMs, other
immunomodulatory adjuvant medications and other treatments for
the control of the ocular pathology were collected from the beginning
of the treatment with BRMs to the last visit closest to the 1st of July
2014. The main outcomes were: visual acuity, degree of anterior
and posterior chamber inflammation (Standardization of Uveitis
Nomenclature Working Group criteria), immunosuppression load (as
defined by Nussenblat et al) and presence of complications. Student’s
dependent, independent t test or non-parametric test of Wilcoxon was
used to make comparisons between means. Statistically significant
differences were considered when p<0.05. Data were analyzed with
SPSS 20 statistics software.
Results: 31 patients, 13 men and 18 women were included. All
patients had bilateral ocular involvement. The most common
site of inflammation was the panuveitis (42.40%). Six patients
were classified as having idiopathic forms of uveitis (19.35%).
The most common diagnoses were Behçets disease (16.12%),
HLA-B27 associated anterior uveitis (16.12%), Juvenile idiopathic
arthritis (12.90%) and Vogt-Koyanagi-Harada (12.90%). There
were improvements in the parameters analyzed in most patients
with statistically significant results in the reduction of the
immunomodulatory load with infliximab, reduction of cells in the
anterior chamber and reduction of immunomodulatory load with
adalimumab. Infliximab outperformed adalimumab in the first 24
months of use and adalimumab until the end of the study, in certain
specific uveitis. Complications were observed in 14.70% of patients.
Conclusions: Infliximab and adalimumab are both effective and
safe drugs for resistant uveitis and may reduce immunomodulation
requirement. However, there are slight differences between the two
treatments that should be considered.
Commercial Relationships: Miriam Allende, None; Lucia IbaresFrias, None; Sonia C. Labrador, None; Cristina Wong, None;
Lidia Cocho, None; Jose M. Herreras, None
Intermediate uveitis: Pattern of etiology, complications,
treatment and outcome in a tertiary academic center
Thomas Ness, Daniel Boehringer, Sonja Heinzelmann. Eye Center,
University of Freiburg, Freiburg, Germany.
Purpose: Patients with intermediate uveitis (IU) represent a
heterogenous group characterized by a wide spectrum of etiologies
and regional differences. The aim of the study was to analyze the
characteristics of patients with IU seen in an academic center in
Germany.
Methods: We conducted a retrospective analysis of the clinical
records of all patients with intermediate uveitis referred to the Eye
Center, University of Freiburg from 2007 to 2014. Diagnosis was
established according to the SUN criteria. Data analyzed were
etiology, demographics, complications, treatment and final visual
acuity.
Results: In the period 159 patients with intermediate uveitis were
identified. The median age at diagnosis was 35 years. The majority
was female (64%) and the mean duration of IU was 6.1 years (range
1 month – 35 years). Etiology of IU was idiopathic in 59%. Multiple
sclerosis (MS) (20%) and sarcoidosis (10%) were frequent systemic
causes of IU. In 11 % several other etiologies including infectious
diseases (tuberculosis, borreliosis) or immune mediated conditions
(e.g. after vaccination) were found. The pattern of complications
included macular edema (36%), cataract (24%), secundary glaucoma
(7%), and epiretinal membrane formation (19%). Periphlebitis was
more frequent with multiple sclerosis. Treatment comprised local and
systemic steroids, immunosupressive agents, biologics, and surgery.
Last visual acuity was better than 20/25 in 75 % of the eyes.
Conclusions: In a german acedemic center most cases of IU were
idiopathic or associated with MS or sarcoidosis. In contrast to other
countries infectious cases were rare. Even with a long duration and
despite of numerous complications the overall visual prognosis is
favorable.
Commercial Relationships: Thomas Ness, Abbvie (C), Allergan
(C), Novartis (C), Sanofi (C), Santen (C); Daniel Boehringer, None;
Sonja Heinzelmann, Abbvie (C), Allergan (C), Sanofi (C), Santen
(C)
Severity of Experimental Autoimmune Uveoretinitis (EAU) is
Reduced in Germ-Free Mice
Aneta Klimova1, Petra Seidler Stangova1, Petra Svozilkova1, Tomas
Hrncir2, Renata Stepankova2, Miloslav Kverka2, Helena TlaskalovaHogenova2, John V. Forrester3, 4, Jarmila Heissigerova1. 1Department
of Ophthalmology, 1st Faculty of Medicine and General University
Hospital, Prague 2, Czech Republic; 2Department of Immunology and
Gnotobiology, Institute of Microbiology, v.v.i., Academy of Sciences
of the Czech Republic, Prague and Novy Hradek, Czech Republic;
3
Section of Immunology and Infection, Institute of Medical Sciences,
University of Aberdeen, Aberdeen, United Kingdom; 4Immunology
and Virology Program, Centre for Ophthalmology and Visual
Science, The University of Western Australia, Crawley, Austria.
Purpose: The gut microbiome is now recognized as an important
regulatory element in immunological homeostasis and autoimmunity.
The aim of our study is to evaluate the impact of a reduced microbial
environment in the model of IRBP-peptide induced EAU in
gnotobiotic mice i.e. mice bred in germ free conditions.
Methods: EAU was induced in C57BL/6 mice by subcutaneous
inoculation of 500 ug IRBP (interphotoreceptor retinoid binding
protein) in complete Freund’s adjuvant and intraperitoneal application
of pertussis toxin. Severity of disease was compared in 4 study
groups: (a) germ-free mice (14 eyes) (b) conventionally- housed (c-h)
mice in which EAU was induced using sterile reagents (38 eyes) (c)
c-h EAU mice treated with antibiotics (metronidazole and cetirizine)
in the drinking water, (32 eyes) and (d) c-h EAU mice induced
using standard reagents (50 eyes). Histological grading of EAU was
performed at day 35 post induction on a scale of 0 (no inflammation)
to 4 (severe inflammation).
Results: When compared to control mice, in germ-free mice the
inflammation was significantly lower (p = 0,007). The median of
inflammation intensity reaches in germ-free mice grade 1 compared
to grade 2 in control group. The mice induced with sterile chemicals
did not show a significant difference in inflammation intensity when
compared to the control group (p = 0,564) nor was there a statistically
significant difference in inflammation intensity between control mice
and mice treated with antibiotics in drinking water.
Conclusions: In this study we have confirmed that germ-free mice
developed less severe intensity of EAU. Our results also show that
exposure to microbial material, in addition to the mycobacteria and
CFA and the pertussis toxin as used in the standard induction protocol
does not influence the severity of EAU. This study demonstrates a
close link between the microbiome and experimental autoimmune
uveoretinitis.
Commercial Relationships: Aneta Klimova, None; Petra Seidler
Stangova, None; Petra Svozilkova, None; Tomas Hrncir, None;
Renata Stepankova, None; Miloslav Kverka, None; Helena
Tlaskalova-Hogenova, None; John V. Forrester, None; Jarmila
Heissigerova, None
Support: IGA MZ NT/14017-3/2013, SVV UK 260022/2014, SVV
UK 254260023/2014
Loss of Natural Immune Tolerance Toward Recoverin in Patients
with Autoimmune Retinopathy
Steven Lundy1, John R. Heckenlively2. 1Internal MedicineRheumatology, University of Michigan Medical School, Ann Arbor,
MI; 2Ophthalmology and Visual Sciences, W.K. Kellogg Eye Center,
Ann Arbor, MI.
Purpose: Autoimmune retinopathy (AIR) leads to a rapid
degeneration of visual fields that is distinct from retinitis pigmentosa
and correlates with increases in anti-retinal autoantibodies. When
properly diagnosed, a majority of patients with active AIR respond
to immune suppression with stabilized or improved vision. The
pathogenesis of AIR is under investigation to better define the
immune response toward retinal antigens, facilitate diagnosis, and
help tailor treatments.
Methods: The cellular and humoral immune response toward the
retinal protein recoverin was measured in AIR patients and compared
to control subjects. Peripheral blood mononuclear cells (PBMC)
from patients and controls were stimulated with recombinant human
recoverin and analyzed for the release of interferon gamma (IFNγ)
and interleukin 10 (IL-10). Plasma was screened for anti-recoverin
antibodies of IgM and IgG subclasses by ELISA. Lymphocyte
subsets in peripheral blood were analyzed by flow cytometry.
Results: Control subject PBMC responded to recoverin by producing
a high level of the immune suppressive cytokine IL-10, suggesting
a normal role of recoverin in maintaining retinal tolerance. AIR
patient PBMC responded to recoverin with significantly lower levels
of IL-10 but elevated levels of TH1-associated IFNγ when compared
to controls. AIR patients were frequently positive for anti-recoverin
IgG with IgG1 being the most dominant subtype. In contrast, controls
frequently had measurable titers of circulating anti-recoverin IgM
with low or no presence of anti-recoverin IgG antibodies. AIR
patients also consistently had a reduced level of CD24highCD38high
regulatory B lymphocytes in comparison to controls.
Conclusions: The high levels of anti-recoverin IgM and IL-10
producing lymphocytes specific toward recoverin in control subjects
suggest that this protein is an immune tolerizing retinal antigen.
These data support our working hypothesis that the switch from
immune tolerance toward recoverin to a more inflammatory TH1driven response plays an important role in the pathogenesis of AIR.
Further studies are in progress to determine correlations between
these parameters, disease activity and response to therapy. This
information should help improve the diagnosis of AIR and will
hopefully lead to significant advances in treatment.
Numerical values are P values from Student’s t test for controls (n =
12) versus AIR patients (n = 16).
Commercial Relationships: Steven Lundy, None; John R.
Heckenlively, None
Support: Grant from Research to Prevent Blindness Foundation
IL-10 expression by Th17 cells correlates with their response to
glucocorticoids
Philippa J. Lait1, 2, Lauren P. Schewitz-Bowers1, 2, Emily L. Williams1,
2
, Ester Carreno3, Andrew D. Dick1, 2, Richard W. Lee1, 2. 1School of
Clinical Sciences, University of Bristol, Bristol, United Kingdom;
2
National Institute for Health Research (NIHR) Biomedical Research
Centre at Moorfields Eye Hospital and University College London
Institute of Ophthalmology, Bristol, United Kingdom; 3University
Hospitals Bristol NHS Foundation Trust, Bristol, United Kingdom.
Purpose: We have previously shown that CD4+ T cells from
steroid refractory (SR) uveitis patients demonstrate both increased
IL-17 expression and a failure to up-regulate IL-10 in response to
glucocorticoids; leading to a significantly reduced ratio of IL10 to IL-17 in CD4+ cells from these patients. Hence, due to the
relationship between these two cytokines and the clinical SR uveitis
phenotype, we sought to determine the capacity of the synthetic
glucocorticoid dexamethasone (Dex) to up-regulate IL-10 in Th17
cells and how production of IL-10 from these cells affects their
response to Dex.
Methods: All experiments used blood from healthy donors (N=4).
CD4+ T cells were isolated and cultured with anti CD3/CD28
microbeads and 1x10-6M Dex for 4 days. Post culture, Dex induced
IL-10 cells were isolated using capture antibodies (Miltenyi Biotech)
and sorted (BD influx). Their suppression of CD4+CD25- effector
cells was functionally assessed in a standard proliferation assay.
Similarly, Th1 and Th17 CD4+ T cells were isolated on the basis of
differential expression of CXCR3, CCR4, CCR6 and CD161 and
cultured as described. On day 4, intracellular IL-17, IFNγ and IL-10
expression was determined by flow cytometry (BD, LSRII). To assess
the effects of IL-10 on suppression by Dex, sorted Th1 and Th17
cells were cultured with IL-2 and autologous irradiated APCs for 14
days, after which intracellular cytokine expression was determined
by flow cytometry. Cells were then cultured with 1x10-6M Dex for
24hours and proliferation was quantified using tritiated thymidine
incorporation.
Results: Dex induced IL-10+ CD4+ T cells exhibited functional
suppression of CD4+CD25- cells (1 Treg:15 effector cell ratio). Both
Th17 and Th1 cells were able to up-regulate IL-10 expression after
culture with Dex. However, the suppression of proliferation by Dex
in Th17 cells correlated positively with the level of IL-10 in these
cells.
Conclusions: IL-10 and IL-17 are key cytokines involved in the
clinical SR phenotype. In normal volunteers Dex up regulates IL-10
in CD4+ T cells and these cells are functionally suppressive. Further,
in Th17 cells from normal volunteers, Dex induces increased IL-10
expression and expression of IL-10 in this cell subset correlates
with Dex mediated suppression of proliferation suggesting this
may function as a negative feedback mechanism to curb immune
responses.
Commercial Relationships: Philippa J. Lait, None; Lauren P.
Schewitz-Bowers, None; Emily L. Williams, None; Ester Carreno,
None; Andrew D. Dick, None; Richard W. Lee, None
Support: NIHR Moorfields BRC Grant
IL-21 induces Expansion of IL-35-producing B cells (i35-Bregs)
Chengrong Yu, Lin Sun, Chang He, Rashid M. Mahdi, CHRALES
E EGWUAGU. Laboratory Immunology, National Eye Inst/NIH,
Bethesda, MD.
Purpose: IL-35-producing B cells (i35-Bregs) are critical regulators
of immunity during autoimmune and infectious diseases and recent
studies suggest that IL-35 plus LPS can induce the conversion of
naive or IL-10-producing B cells (Bregs) into i35-Breg. Interestingly,
while LPS or IL-21 plus CD40 signaling induces naïve B cells to
produce IL-10, co-stimulation with LPS plus anti-CD40 inhibits
Bregs and promotes i35-Bregs expansion. In this study, we
investigated whether similar to IL-35, if IL-21 plus LPS can also
induce the expansion of i35-breg population.
Methods: CD19+ B cells were isolated by sorting from the
spleen of C57BL/6 mice and are activated with LPS or aCD40-/
aIgM antibodies in absence or presence of rIL-35 or IL-21.
Immunophynotype of the cells was characterized by FACS using
labeled mAbs specific to CD19, B220, CD1d, CD5, CD21, CD23,
CD27, CD38, CD40, CD138, GL-7, IgM, IgD, IL-10, TNF-a,
IL-12p35, EBI3, WSX-1 or IL-12Rb2. The B cells were also
characterized by ELISA, PCR and Western blotting.
Results: IL-21 plus CD40 signaling induced IL-10 while IL-21
inhibited LPS-induced IL-10 production. On the other hand, IL-21
plus CD40 or IL-21 plus LPS induced expression of IL-35 (IL-12p35
and Ebi3). Interestingly, co-stimulation of B cells by IL-21 and
aCD40/aIgM abs induced more IL-35 compared to stimulation with
IL-21 plus LPS.
Conclusions: We show here for the first time that IL-21 induces the
expansion of IL-35-producing B cells and suggests that IL-21 and IL35 may form an axis that promotes the expansion of IL-35+Bregs.
Commercial Relationships: Chengrong Yu, None; Lin Sun,
None; Chang He, None; Rashid M. Mahdi, None; CHRALES E
EGWUAGU, None
Interface between Mesenchymal Stem Cells and Regulatory T
Cell
Sunil Chauhan1, Masahiro Omoto1, 2. 1Schepens Eye Research
Institute, Harvard Medical School, Boston, MA; 2Ophthalmology,
Okayama University Hospital, Okayama, Japan.
Purpose: While mesenchymal stem cells (MSC) and regulatory T
cells (Tregs) are immunomodulatory in their own right, MSCs can
also enhance the suppressive function of Tregs to efficiently regulate
the host immune response. However, the mechanisms how MSCs
and Tregs interact are unclear. The purpose of this study was to
investigate the mechanisms of MSC–Treg interaction, and its effect
on Treg function.
Methods: Naïve Balb/c mice were used to generate bone marrowderived MSCs (CD45–CD34–SCA1+ CD29+), and to isolate
CD4+CD25+ Tregs (purity: >95% Foxp3+) from spleen and lymph
nodes. MSCs and purified Tregs were co-cultured with or without
transwells (1 mm pore size). After 72h, Tregs (CD4+CD25+Foxp3+)
were assessed by flow cytometry, or live Tregs were sorted to
quantify Foxp3 protein level using ELISA. MSC expression of CD80
was investigated using flow cytometry. The effect of blocking MSCexpressed CD80 on Treg suppressive function was analyzed using
CD80 blocking antibodies.
<!—[endif]—>
Results: Flow cytometry and ELISA analyses showed that Tregs
co-cultured with MSCs either in direct cell-to-cell contact or in
transwells expressed significantly increased (2–3 fold) levels of
Foxp3 compared with Tregs cultured without MSCs (p<0.01).
However, MSCs were more effective in amplifying Treg- expression
of Foxp3 when co-cultured in direct contact with Tregs than in
transwells (p<0.02). All MSCs expressed CD80, and addition of
anti-CD80 antibodies in a MSC-Treg co-culture assay significantly
reduced the suppressive function of Tregs (~35%, p<0.02).
Conclusions: Our results demonstrate that direct cell-to-cell
interaction between MSCs and Tregs is essential for maximal
enhancement of Treg function, which in part is mediated by MSCexpressed CD80 molecule.
Commercial Relationships: Sunil Chauhan, None; Masahiro
Omoto, None
Support: Massachusetts Lions Foundation, Harvard Scholar in
Medicine Fellowship
Th1 and Th17 autoimmune responses differ in responding to
adenosine receptor (AR) agonists
Deming Sun1, 2, Aijun Zuo1, 2, Hui Shao3, Henry Kaplan3, Dongchun
Liang1. 1DVRC-411, Doheny Eye Institute, Los Angeles, CA;
2
Ophthalmology, UCLA, Los Angeles, CA; 3Ophthalmology,
University of Louisville, Louisville, KY.
Purpose: ATP release together with increased adenosine generation
is a general phenomenon in inflammation. Control activation of
adenosine receptors has been shown to be beneficial in treating a
series of inflammatory and autoimmune disorders. While previous
studies mostly examined the effects of AR agonists on Th1-type
immune responses, in this study, we compared the effect of AR
agonists on Th17 and Th1 autoimmune responses in experimental
autoimmune uveitis (EAU).
Methods: EAU was induced in B6 mice by subcutaneous injection of
200 μl of emulsion containing 200 μg of human IRBP1-20 in CFA.
aβ and γδ T cells were purified from immunized mice. For NECA
treatment, immunized B6 mice received a single i.p. injection of
NECA (5 mg/kg) on different days after immunization. The intensity
of Th1 and Th17 response and the pathogenic activity of isolated
autoreactive T cells were compared between in vivo treated and nontreated mice and between cells with or without AR agonist treatment
in vitro.
Results: The effects of NECA on Th1 and Th17 responses were
completely dissociated. The enhancing effect of NECA on Th17
responses was modulated by γδ T cells. Activated γδ T cells
expressed the highest levels of A2AR receptors. A2AR ligation
inhibits αβ T cell activation, but enhances γδ T cell activation. AR
agonist promotes the differentiation of a DC subset co-expressing
CD11c and Gr-1, which possess a strong stimulating effect on Th17
autoreactive T cells.
Conclusions: AR agonists had a consistent inhibitory effect on
the Th1 autoimmune response; however, their effect on the Th17
autoimmune response could be either inhibitory or enhancing,
depending on both environmental conditions and the activation status
of the T cells. Our results show that the inflammatory environment
has a strong impact on converting the effect of AR agonist on the
Th17 autoimmune response from anti- to pro-inflammatory. Given
that both Th1 and Th17 autoreactive T cells are pathogenic for
autoimmune diseases, identification of the mechanisms by which
adenosine enhances or inhibits should allow us to minimize the
enhancing effect of AR agonists on the Th17 response and improve
the therapeutic goal of controlling both Th1 and Th17 responses in
autoimmune diseases.
Commercial Relationships: Deming Sun, None; Aijun Zuo, None;
Hui Shao, None; Henry Kaplan, None; Dongchun Liang, None
Support: This work was supported in part by NIH grants EY
0022403, EY018827, and EY003040.
The role of the gut microbiota in immune-mediated uveitis
Phoebe Lin1, Christina Metea1, Mark Asquith1, Henry Gruner1, James
T. Rosenbaum1, 2, Yukiko K. Nakamura1. 1Ophthalmology, Casey Eye
Institute, OHSU, Portland, OR; 2Devers Eye Institute, Portland, OR.
Purpose: In both germ free and broad-spectrum oral antibiotic
experiments, investigators have demonstrated an important role for
the gut microbiota in extraintestinal immune-mediated diseases such
as multiple sclerosis. We found that alteration of the gut microbiota
using oral broad-spectrum antibiotics ameliorated experimental
autoimmune uveitis (EAU). The objective of this study was to
investigate the mechanism of this effect on regulatory and effector T
cells.
Methods: Interphotoreceptor binding protein peptide 161-180 was
used to induce uveitis in antibiotic-treated B10.RIII mice. Eyes,
spleen, cervical, mesenteric lymph nodes (CLN, MLN), and lamina
propia lymphocytes (LPL) were collected on days 7, 14, 21, and 28
after immunization. We performed flow cytometry for Foxp3, CD4,
IL-17, and IFN-gamma staining to quantify regulatory T lymphocytes
(Tregs), Th1, and Th17 cells.
Results: A smaller proportion of antibiotic-fed animals had clinical
EAU scores ≥ 3 compared with water-fed animals (7.7% vs 52%,
p<0.001). There were higher proportions of Tregs (CD4+, FoxP3+) in
the LPL of oral antibiotic-fed mice on day 7, 14, and 28, compared to
water-fed mice (day 7: 41.66% vs. 25.05%, p<0.05; day 14: 45.76%
vs. 31.61%, p<0.05; day 28: 32.73% vs. 14.03%, p<0.01; Figure 1A).
Higher frequencies of Tregs were observed in MLN of antibiotic-fed
mice on day 21 and day 28, compared to water-fed mice (day 21:
17.73% vs. 13.18%, p=0.05; day 28: 12.22% vs. 9.93 %, p<0.05,
Figure 1B). Th1 and Th17 proportions were reduced early on, prior to
onset of inflammation, in oral antibiotic-fed mice in both MLN and
CLN (p<0.01 for both), but not spleen (p>0.05). We found a higher
number of Tregs among infiltrating CD4+ T lymphocytes in the lessinflamed antibiotic-treated animal eyes at 2 weeks after immunization
compared to water-fed EAU mice.
Conclusions: Alteration of the gut microbiota by oral antibiotic
administration modulates changes in CD4+ effector and regulatory T
cell populations in lymphoid organs and eyes, all tissues distant from
the gut, which may explain the reduction in severity of EAU. These
findings may lead to a better understanding of how uveitis can be
treated or prevented by modulating the gut microbiota.
Commercial Relationships: Phoebe Lin, None; Christina Metea,
None; Mark Asquith, None; Henry Gruner, None; James T.
Rosenbaum, None; Yukiko K. Nakamura, None
Support: NIH K08EY022948
Searching for phenotypic differences between microglia and
monocyte-derived macrophages in the neuroretina
Emily O’Koren1, Rose Mathew1, Daniel Saban1, 2. 1Ophthalmology,
Duke University School of Medicine, Durham, NC; 2Immunology,
Duke University School of Medicine, Durham, NC.
Purpose: It is now established that monocyte-derived macrophages
seen in neuroinflammation and normal tissue resident microglia
have distinct developmental origins (bone marrow and yolk
sac, respectively). Lack of discriminatory markers for these two
populations has thwarted efforts to understand their respective
contributions in pathological states. The current study sought to
identify differentially expressed cell surface markers of monocytederived macrophages versus microglia in the neuroretina.
Methods: C57BL/6 mice were lethally irradiated and reconstituted
with GFP+ bone marrow cells in order to discriminate microglia
(GFP-) from monocyte-derived macrophages (GFP+). One cohort of
host mice was given lead helmet shielding against irradiation-induced
neuroretinal injury, whereas the other cohort was left unshielded
(n=4 to 5 mice/group). Non-irradiated/naïve mice (n=5) were
also included. 4 months post-irradiation, flow cytometric analysis
of neuroretinal immune cells was performed. Discrimination of
intravascular cells was enabled by infusion of CD45/BV650 Ab five
minutes prior to euthanasia. Discrimination of extravascular cells
was enabled via additional ex vivo staining with CD45/APCCy7 Ab.
Samples were additionally stained for CD11b, Ly6C, Ly6G, F4/80,
CD64, I-A/I-E.
Results: Microglia and monocyte-derived macrophages in
neuroretina were identified as BV650/CD45- APCCy7/CD45+
CD11b+ Ly6C- Ly6G- F4/80+ CD64+. Greater than 60% of these
cells were GFP+ in unshielded mice, whereas GFP+ cells were
completely undetectable in shielded counterparts. GFP+ cells from
unshielded mice were analyzed for mean fluorescence intensity
(MFI) levels of CD45, CD11b, F4/80, CD64, I-A/E expression, and
compared to GFP- cells from unshielded, shielded, and naïve mice.
While the MFI of CD45 and I-A/E were not different (p>0.05), the
MFI of CD11b was slightly higher (p=0.002) and CD64 was slightly
lower (p=0.02) in GFP+ cells. Strikingly, MFI of F4/80 showed a
4-fold increase in GFP+ cells (p=0.001).
Conclusions: Our data suggest that the two populations express
certain markers at different levels. Surprisingly, CD45 was not
differentially expressed, which may suggest that differentiation of
monocytes into macrophages in the neuroretina is associated with
down-regulation of CD45. Most striking difference was in F4/80,
possibly suggesting that it can help distinguish the 2 populations in
neuroinflammation.
Commercial Relationships: Emily O’Koren, None; Rose Mathew,
None; Daniel Saban, None
Support: NEI-R01EY021798 and Research to Prevent Blindness
[RPB] Career Development Award
Molecular mechanisms of retinal cell death in Staphylococcus
aureus endophthalmitis.
Pawan Kumar Singh1, 2, Ajay Kumar1, Ashok Kumar1, 2. 1Kresge Eye
Institute, Wayne State University School of Medicine, Detroit, MI;
2
Anatomy and Cell Biology, Wayne State University, Detroit, MI.
Purpose: Severe vision loss in bacterial endophthalmitis is associated
with the death of retinal cells including photoreceptors. In the present
study, we investigated the molecular mechanisms of cell death
triggered by Staphylococcus aureus using both in vivo and in vitro
experimental models.
Methods: For in vivo studies, endophthalmitis was induced in
C57BL/6 mice by intravitreal injection of 5000 cfu of S. aureus
(strain RN6390). TUNEL and Annexin V & PI staining were used
to determine cell death using microscopy and flowcytometry,
respectively. qRT-PCR was used to determine the induced expression
of pathway-specific genes and death domain receptors. Western blot
analysis was performed to assess the activation of caspases and other
executioner proteins (cytochrome C, AIF, PARP). Human Müller glia
(MIO-M1 cells) and mouse cone photoreceptor cells (661W) were
used for in vitro studies. The mitochondrial membrane potential was
studied by JC1 staining using fluorescence microscopy, as well as by
flowcytometry. The caspase and PARP1 inhibitors were used for the
inhibition study.
Results: S. aureus caused significant cell death in the mouse retina
and cultured MIO-M1 and 661W cells as evidenced by increased
number of TUNEL positive cells. S. aureus-challenged retina
and retinal cells exhibited increased generation of ROS, reduced
mitochondrial membrane potential (JC1 staining), the release of
cytochrome C into the cytoplasm and the activation of caspase 3. We
also observed the cleavage of PARP-1 and the release of Apoptosis
Inducing Factor (AIF) from mitochondria in S. aureus-infected cells.
Our inhibition studies showed that pancaspase inhibitor Q-VD-OPH
and PARP-1 inhibitor DPQ reduced retinal cell death.
Conclusions: Our data demonstrate that in bacterial endophthalmitis,
retinal cells undergo apoptosis in both caspase-dependent and
caspase-independent manner and mitochondria seems to play a
central role in this process. These insights prompt further study to
assess potential therapeutic candidates for retinal protection against
bystander damage induced by microbial pathogens.
Commercial Relationships: Pawan Kumar Singh, None; Ajay
Kumar, None; Ashok Kumar, None
Support: NIH Grant EY019888, Fight For Sight
TLR4-dependent retinal gene expression in Bacillus cereus
endophthalmitis
Phillip S. Coburn, Salai Madhumathi Arasi Parkunan, Blake Randall,
Michelle C. Callegan. Ophthalmology, The Univ of Oklahoma Hlth
Sci Ctr, Oklahoma City, OK.
Purpose: To evaluate the retinal transcriptional response to B.
cereus infection early during the course of experimental murine
endophthalmitis and to identify Toll-like receptor (TLR) 4-dependent
genes.
Methods: The right eyes of male C57BL/6J or TLR4-/- mice were
intravitreally injected with 100 CFU of B. cereus ATCC 14579. Left
eyes served as uninfected controls. At 4 h post-infection, RNA was
isolated from retinas, converted to cDNA, and hybridized to the
Affymetrix GeneChip Mouse Genome 430 2.0 array. Data analysis
was performed using Partek’s Genomics Suite software to obtain
differential gene expression data. A 5-fold change in gene expression
and p < 0.05 threshold were selected as the criteria for comparative
array analyses. Arrays were performed in duplicate and the results
were confirmed by qPCR and ELISA.
Results: Genes related to the acute inflammatory response and
inflammatory cell recruitment were significantly upregulated
5-fold or greater at 4 h post-infection with B. cereus in C57BL/6J
retinas [CXCL1 (KC), CXCL2 (MIP2-α) CXCL10 (IP-10), CCL2
(MCP1), and CCL3 (MIP1-α)]. The pro- and anti-inflammatory
mediator IL6, the intercellular adhesion molecular ICAM-1, and
the inhibitor of cytokine signal transduction, SOCS3, were also
significantly upregulated 5-fold or greater. Leukemia inhibitory
factor (LIF), crucial for the survival of photoreceptor cells, was also
highly induced in response to B. cereus infection. Prostaglandinendoperoxide synthase 2 (PTGS2/COX-2), which converts
arachidonic acid to prostaglandin endoperoxide H2, was upregulated,
as was pentraxin 3 (PTX3), which is produced in response to
TLR engagement, activates the classical complement pathway,
and facilitates pathogen recognition and clearance. Among the
genes related to inflammation and infection that are upregulated in
wild type retinas at 4h post-infection, only CCL3 was found to be
significantly upregulated in B. cereus-infected TLR4-/- retinas.
Conclusions: These studies demonstrate that TLR4 regulates
cytokine and chemokine genes important for the acute inflammatory
response and neutrophil recruitment, as well as genes related to
photoreceptor survival and pathogen recognition and clearance.
Future studies will evaluate the retinal and global ocular
inflammatory responses over the course of B. cereus endophthalmitis
to identify pathway-based anti-inflammatory targets, specifically,
those that are regulated by TLR4.
Commercial Relationships: Phillip S. Coburn, None; Salai
Madhumathi Arasi Parkunan, None; Blake Randall, None;
Michelle C. Callegan, None
Support: NIH/NEI R01 EY024140
Induction of T Cell Anergy in Response to Endogenous Retinal
Self-Antigens
Scott W. McPherson, Neal D. Heuss, Mark J. Pierson, Dale S.
Gregerson. Department of Ophthalmology and Visual Neurosciences,
University of Minnesota, Minneapolis, MN.
Purpose: Previously, we demonstrated that peripheral regulatory T
cells (pTregs) can be generated within the retina and are protective
against spontaneous and induced experimental autoimmune
uveoretinitis (EAU). In this study we investigated the role of T cell
anergy induced by endogenous retinal self-antigens as a mechanism
retinal immune privilege.
Methods: Transgenic (Tg) mice expressing beta-galactosidase
(bgal mice) as a retinal neo-self antigen were used in conjunction
with Tg mice expressing diphtheria toxin receptor (DTR) and green
fluorescent protein (GFP) under control of the FoxP3 promoter (FDG
mice) and bgal-specific T cell receptor Tg mice (BG2 mice). Tg
mice expressing DTR and GFP under control of the CD11c promoter
(CDG mice) were used assess the role of dendritic cells (DC) in the
T cell response. Backcrossing was done to generate mice expressing
two or more of these transgenes. Antigen specific retinal T cells
responses were induced by posterior segment injection of bgal. At
day three post-injection, retinal T cells were analyzed and sorted by
FACS into three populations; Tregs (GFP+CD25+), candidate anergic
T cells (GFP-CD25-), and activated effector T cells (GFP-CD25+).
Analysis for expression of specific genes associated with each
population was done by FACS and RT-PCR.
Results: In response to bgal injection, retinal Treg numbers were
reduced in bgal-FDG-BG2 mice compared to FDG-BG2 mice.
Retinal effector T cells numbers were also reduced in bgal-FDG-BG2
mice even when existing Tregs were deleted by diphtheria toxin. The
candidate anergic T cell population was increased in mice expressing
retinal bgal. Analysis of gene expression in Tregs from bgal+ and
bgal- retinas showed similar elevated levels of TGF-b, IL-10, and
Helios compared to effector T cells. Analysis of candidate anergic T
cells revealed a lack of markers associated with activated T cells (Ki67, IL-2) and a lack of markers associated with Tregs (FoxP3, IL-10).
In all cases the response to bgal was dependent on the presence of
dendritic cells within the retina.
Conclusions: Local expression of a retinal self-antigen alters the
response to that antigen in the retina. The most significant difference
is the appearance of cells that bear characteristics of anergic T
cells. No evidence for local induction of anergic T cells by antigen
presenting cells other than DC was found.
Commercial Relationships: Scott W. McPherson, None; Neal D.
Heuss, None; Mark J. Pierson, None; Dale S. Gregerson, None
Support: Support by NIH grants RO1-EY0210996, RO1-EY016376,
P30-EY011373, Research to Prevent Blindness, Minnesota Lion’s
Club, and Wallin Neuroscience Discovery Fund
Capability of vitreous fluid to enhance TGF-β-induced Foxp3+
regulatory T cell conversion
Hiroshi Keino, Takayo Watanabe, Annabelle A. Okada.
Ophthalmology, Kyorin University School of Medicine, Mitaka,
Japan.
Purpose: To determine whether vitreous fluid can promote the
generation of Foxp3+ CD4+ T regulatory (Treg) cells in vitro.
Methods: Vitreous fluid was collected from fresh porcine
eyes, sterilized by filtration. Vitreous, used for culture at a final
concentration of 25%. Mouse CD4+ T cells purified from pooled
splenocytes were stimulated with plate-bound anti-CD3 for 72 h in
the presence or absence of vitreous fluid. CD4+ T cells were analyzed
for Foxp3 expression using flow cytometry. In some experiments,
CD4+ T cells were cultured in the presence of vitreous fluid with
or without TGF-β2. For evaluation of T cell activation, culture
supernatant was harvested for ELISA assay of interferon (IFN)-γ.
Results: Vitreous fluid alone did not increase the frequency of
Foxp3+ Tregs. Although TGF-β2 induced Foxp3+ Tregs in vitro, the
frequency of TGF-β2 induced Foxp3+ Tregs increased significantly
in the presence of vitreous fluid. IFN-γ production increased in the
presence of vitreous fluid, but decreased significantly in the presence
of vitreous fluid plus TGF-β2.
Conclusions: These findings suggest that vitreous fluid may enhance
TGF-β-induced Foxp3+ Treg conversion.
Commercial Relationships: Hiroshi Keino, None; Takayo
Watanabe, None; Annabelle A. Okada, None
Support: Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science, and Technology, Japan
26462696
CXCL 12 is a chemoattract induced by HMGB1 in T cell induced
uveitis (tEAU)
Guomin Jiang1, Yunsong Wang2, Yuan Zhao4, Amir Reza
Hajrasouliha1, Jinhui Wu3, Deming Sun5, Henry J. Kaplan1, Hui
Shao1. 1Department of Ophthalmology, University of Louisville,
Louisville, KY; 2Department of Ophthalmology, Gongren Hospital,
Tangshan, China; 3Ophthalmology, Changhai Hospital, Shanghai,
China; 4Department of Pharmaceutical Sciences, Sullivan University
College of Pharmacy, Louisville, KY; 5Doheny Eye Institute, Los
Angeles, CA.
Purpose: In EAU induced by injection of uveitogenic,
interphotoreceptor retinoid-binding protein (IRBP)-specific T cells
(tEAU) in mice, we have previously reported that high mobility
group box 1 (HMGB1), an important endogenous danger signal, was
an early and critical mediator for induction of disease. Our current
study explores the role of HMGB1 in intraocular inflammation,
focusing on its role in recruiting infiltrating cells into the eye.
Methods: Supernatants collected from the co-culture of retinal
explants with HMGB1 or IRBP-specific T cells in the presence or
absence of anti-HMGB1 Ab were tested for their chemoattractive
effect on leukocytes. Mice receiving IRBP-specific T cells were
treated with CXCR4 antagonist (AMD3100), and intraocular
inflammation was examined by funduscopy and histology.
Results: HMGB1 alone or culture supernatants from retinal explants
did not attract leukocytes. However, supernatants from retinal
explants treated with HMGB1 or co-cultured with IRBP-specific T
cells did attract lymphocytes; co-culture in the presence of antiHMGB1 Ab abrogated chemoattraction, implying molecules released
by HMGB1 treated retinal cells are chemoattractive. Moreover,
supernatants neutralized by anti-CXCL12 Ab or leukocytes pretreated with AMD3100 (an antagonist of CXCR4, a receptor for
CXCL12) resulted in decreased chemoattraction. Finally, tEAU was
significantly inhibited by AMD3100.
Conclusions: CXCL12, a ligand for CXCR4, is produced by
retinal cells after exposure to HMGB1 and is a chemoattract for
lymphocytes in tEAU.
Commercial Relationships: Guomin Jiang, None; Yunsong Wang,
None; Yuan Zhao, None; Amir Reza Hajrasouliha, None; Jinhui
Wu, None; Deming Sun, None; Henry J. Kaplan, None; Hui Shao,
None
Support: Supported in part by an RPB Lew R Wasserman Merit
Award (HS), the Commonwealth of Kentucky Research Challenge
Trust Fund (HK), and grants from the University of Louisville School
of Medicine, Fight for Sight (GJ), Kentucky Science & Engineering
foundation and Sullivan University College of Pharmacy (YZ).
Graft versus Host Disease of the retina and recognition of selfantigens after allogeneic hematopoietic cell transplantation
Manfred Zierhut1, Nora Roth2, Andreas Korn3, Antje Bornemann4,
Christoph Simon2, Annika Böhm1, Wolfgang Bethge1, Lothar
Kanz2, Hans-Georg Rammensee5, Sebastian Haen2. 1Centre for
Ophthalmology, University of Tuebingen, Tuebingen, Germany;
2
Hematology, University Hospital, Tuebingen, Germany;
3
Neuroradiology, University of Tuebingen, Tuebingen, Germany;
4
Neuropathology, University of Tuebingen, Tuebingen, Germany;
5
Immunology, University of Tuebingen, Tuebingen, Germany.
Purpose: Graft versus Host Disease (GvHD) is believed to
be mediated by T cells recognizing major (MHC) or minor
histocompatibility antigen mismatches. The exact T cell epitopes
recognized on recipient cells remain unknown. Here, we evaluated
the GvHD in the posterior eye segment (PS) and the antigens
recognized by allogeneic T cells.
Methods: Six patients suffered from retinal diseases after HCT, 22
further patients were recruited before HCT irrespective of ocular
symptoms. As controls, T cells from 5 family donors and 8 healthy
individuals per epitope were evaluated.Autologous DNA was isolated
from blood before or oral mucosa cells after HCT. Allogeneic DNA
was obtained from blood after HCT (complete donor chimerism).
DNA sequencing was used to identify donor-recipient single
nucleotide polymorphisms (SNP). Retina specific candidate epitopes
derived from the retinal guanylate cyclase 2D (GUCY2D), the
retinoid binding protein (RBP) and the guanylate cyclase activating
proteins A1 und B1 (GUCA1A/GUCA1B) were predicted based
on known SNP and individual gene sequences using the database
EpiToolKit. Reactivity of T cells was determined in ELISpot and
intracellular cytokine staining.
Results: PS diagnoses were optic nerve atrophy (n=2), in 1 case
combined with a selective dysfunction of the cones, optic neuritis
(n=2), ischemic retinopathy (n=1) and VZV retinitis (n=1). In 2 of
6 patients specific T cells against GUCY2D epitopes were detected
24 and 40 months after HCT. DNA sequencing did not reveal a SNP
indicating recognition of self-antigens. In 6/22 patients without
PS symptoms, retina-specific T cells could be detected directed
against epitopes derived from GUCA1A (n=3), GUCA1B (n=3)
and GUCY2D (n=3) between 4 and 14 months after HCT. Again,
no SNP could be observed. Hence, reactivity was directed against
self-epitopes. Transplantation of retina-antigen specific cells and
reactivity against naturally occurring epitopes were excluded in
donors and healthy individuals.
Conclusions: GvHD manifestations of the retina can be detected
after allogeneic HCT and mediated by antigen-specific T cells. The
antigens recognized hereby can be self-antigens and do not need to
be based on SNP between donor and recipient. Development of PS
GvHD may be triggered by viral infections and should be considered
in case of atypical ophthalmologic findings.
Commercial Relationships: Manfred Zierhut, None; Nora Roth,
None; Andreas Korn, None; Antje Bornemann, None; Christoph
Simon, None; Annika Böhm, None; Wolfgang Bethge, None;
Lothar Kanz, None; Hans-Georg Rammensee, None; Sebastian
Haen, None
Support: Deutsche José Carreara Leukämie Stiftung Grant DJS
08/04; Fortüne project of University of TuebingenGrant 1832-0-1;
Deutsche Krebshilfe Grant 110465
Development of autoimmune type lacrimal gland GVHD in
adoptively transferred mice with recipient derived T cells
Yoko Ogawa, Shigeto Shimmura, Kazuo Tsubota. Department of
Ophthalmology, Keio Univ School of Medicine, Shinjuku-Ku, Japan.
Purpose: Lacrimal gland chronic graft-versus-host disease
(cGVHD) is a complication following bone marrow
transplantation, which resembles autoimmune disease such as
Sjogren’s syndrome. T cells are believed to play a possible role
in immunomodulation. However, the precise source and role of T
cells in lacrimal gland cGVHD are unknown. Here we show using
a MHC compatible, minor antigen mismatched cGVHD model
that donor mesenchymal stem cells (MSC), and recipient T cells
play a role in the pathogenesis of lacrimal gland cGVHD. This
study was undertaken to investigate whether recipient derived
T cells are capable of inducing autoimmune lesions in cGVHD
lacrimal gland.
Methods: We obtained freshly isolated MSCs and hematopoietic
stem cells (HSCs) by established methods by Morikawa S, and
Matsuzaki Y, et al. To confirm the function of mismatched MSCtransplanted recipient derived T cells, we adoptively transferred
splenic T cells from mismatched MSC-transplanted recipient into
naïve nude mice (BALB/c background) and compared with that
of the wild type control. The animals were analyzed 45 days after
transfer. Analyses of tissue samples from lacrimal glands including
other target organs were performed by using immunohistochemistry
and immunofluorescence. Splenic cells were collected and stained
with anti-CD4 and anti-IL-17 antibody for flowcytometry. Purified
donor-derived and mismatched B10.D2 MSCs were co-cultured with
T cells isolated from mismatched MSC-transplanted recipient mice.
Cells were treated with either blocking anti-MHC class II antibody or
isotype control.
Results: We found that cGVHD-like inflammation and fibrosis
occurred in the target organs including lacrimal glands as well as
systemic organs. The number of HSP47+ fibroblasts per field in the
target organs was significantly increased in the adoptively transferred
nude mice compared with that of the wild type control. FACS
analysis of splenic PBMC of the adoptively transferred recipients
revealed that CD4+Th 17 cells were markedly elevated compared
with wild type BALB/c background nude mice. Effector T cells
proliferation co-cultured with mismatched MSC was blocked by anti
MHC Class II antibody in vitro.
Conclusions: Our results show that donor MSCs express MHC
Class II following transplantation, and trigger immune responses
in residual host T cells in the pathogenesis of autoimmune
pathology in lacrimal gland cGVHD.
Commercial Relationships: Yoko Ogawa, None; Shigeto
Shimmura, None; Kazuo Tsubota, None
Support: the Japanese Ministry of Education, Science, Sports, and
Culture #26462668 and Japan Medical Association 2014.
Identification of the vital role of the innate receptor Mincle in the
pathogenesis of autoimmune uveitis
Ellen J. Lee1, 2, Brieanna Brown2, 1, Emily Vance2, 1, Riley Hazard2,
Phyllis Silver3, Rachel R. Caspi3, Holly L. Rosenzweig1, 2. 1Oregon
Health & Science University, Portland, OR; 2Veterans Affairs
Medical Center, Portland, OR; 3Laboratory of Immunology, National
Eye Institute/NIH, Bethesda, MD.
Purpose: We previously demonstrated a role for the Syk/CARD9
signaling axis, a pathway for innate C-type lectin receptors
(CLRs), in the pathogenesis of autoimmune uveitis as modeled
in experimental autoimmune uveitis, EAU. Here, we investigated
further the source and timing of Syk/CARD9-associated disease
susceptibility, and delineated the role of individual upstream CLRs
(e.g. Dectin-1, Dectin-2, Mincle), which are involved in fungal and/or
mycobacterial host defense responses.
Methods: EAU was induced by immunization with
interphotoreceptor retinoid-binding protein (IRBP) and uveitis was
evaluated longitudinally by fundus imaging (TEFI) and histology.
The impact of the Syk inhibitor, piceatannol, versus vehicle control,
on the effector phase of disease was evaluated in wild-type (WT)
mice by initiating treatment (n=10 mice/treatment) after uveitis onset
(5mg/kg given on d14,17,21,24 post-immunization) and compared
to prophylactic administration of piceatannol (d0,4, 8,12,16) to
target the induction phase. To assess the contribution of individual
CLRs, onset and severity of uveitis were evaluated in mice deficient
in Dectin-1, Dectin-2, or Mincle versus C57BL/6J controls (n=1020 mice/genotype). Flow cytometry was used to evaluate the
composition of ocular leukocytes.
Results: While protection of CARD9 KO mice to EAU was
reproduced in WT mice by prophylactic inhibition of the upstream
kinase Syk, indicating a role for CARD9 in the disease induction
phase, piceatannol did not offer sustained protection when
administered therapeutically. By d21 post-immunization, Dectin-1
and Dectin-2 KO mice exhibited similar or exacerbated uveitis
compared to WT controls, while Mincle KO mice had significantly
reduced uveitis (TEFI p=0.0017, histology p=0.0001). Interestingly,
Dectin-1 and Dectin-2 KO mice had increased numbers of leukocytes
(particularly macrophages, neutrophils, and CD4+ T cells) compared
to WT, while Mincle KO mice had similar composition but few
numbers compared to WT.
Conclusions: Syk inhibition studies support a role for the Syk/
CARD9 pathway in the induction rather than effector phase of
disease. The proximal CLR Mincle, but not Dectin-1 or Dectin-2, is
implicated in the initiation of this innate pathway for autoimmune
disease in the eye. These findings provide insight into the role of
CLRs and the Syk/CARD9 signaling pathway in ocular inflammatory
disease.
Commercial Relationships: Ellen J. Lee, None; Brieanna Brown,
None; Emily Vance, None; Riley Hazard, None; Phyllis Silver,
None; Rachel R. Caspi, None; Holly L. Rosenzweig, None
Support: NIH/NEI (grant EY019020) and the Research to
Prevent Blindness Foundation, the Department of Veterans Affairs
Biomedical Laboratory, and NEI Intramural support (project #
EY000184)
Experimental autoimmune uveitis in rabbits: clinical and
functional evaluation
Gabriela L. Ioshimoto1, 2, Andre Liber1, 2, Thais Z. igami3, Francisco
M. Damico3, Dora F. Ventura1, 2. 1Neuroscience and Behavior,
Universidade de São Paulo, São Paulo, Brazil; 2Dept. of Experimental
Psychology, Universidade de São Paulo, São Paulo, Brazil; 3Dept. of
Ophthalmology, Universidade de São Paulo, São Paulo, Brazil.
Purpose: Autoimmune uveitis is an ocular inflammatory disease of
unknown etiology in which the eye can be the only organ involved
or can be a part of a syndrome involving multiple tissues. Several
studies have been using models of experimental autoimmune uveitis
(EAU), a disease model that affects specifically the eye and is
mediated by T lymphocytes. However, EAU is well established in
mice and rats while data in rabbits are scarce. The aim of this study
is to evaluate the clinical and functional findings in the rabbit eye
during the course of EAU.
Methods: EAU was induced by intravitreal injections of M.
tuberculosis H37Ra antigen (Difco Lab) in 10 rabbits preimmunized
with a subcutaneous injection of 10 mg of the same antigen. Fellow
eye was used as control. ERG was recorded 1 day before and
sequentially on the following 48 days after induction (total of 12
sessions). The ERGs were recorded according to a ISCEV protocol,
which used five scotopic stimulus intensities after 30 min of dark
adaptation: 0.0009; 0.009; 0.09; 0.9 and 9.0 cd.s/m2 and one photopic
stimuli after 2 min of light adaption: 9.0 cd.s/m2 in a background of
25 cd/m2. The biomicroscopy was performed in 8 animals with slit
lamp Topcon SL-3G model after each ERG session.
Results: ERG showed significant functional retinal impairments. In
scotopic condition there was increase in a-wave latencies (p<0.03)
and decrease in a-wave amplitudes (p<0.04). There was an increase
in b-wave latency (p<0.01) and a decrease in b-wave amplitude
(p<0.04). Photopic ERG showed no change in a-wave but increased
implicit time 20 days after uveitis induction (p<0.02). Biomicroscopy
showed that 66% of the animals had progressive moderate conjuntiva
hyperemia that started on day 7 and recovered by day 35. Anterior
chamber cells and flare presented the same profile but more severe.
Subcapsular cataract was present in 83% of the eyes.
Conclusions: The uveitis induction promoted alterations in both
a- and b-waves suggesting pre- and post-receptoral permanent
retinal damage. Clinical findings showed improvement over time
and indicated that the presence of cataracts is a consequence of
the inflammation. Our results are in agreement with literature and
indicate that this EAU model produces a limited spectrum of the
disease and can be used as a self-limited model of autoimmune
uveitis.
Commercial Relationships: Gabriela L. Ioshimoto, None; Andre
Liber, None; Thais Z. igami, None; Francisco M. Damico, None;
Dora F. Ventura, None
Altered micro-RNA expression contributes to the inflammatory
environment observed in patients with primary Sjögren’s
syndrome.
Joan Ní Gabhann1, 2, Qistina Pilson2, 3, Caroline A. Jefferies1, Conor
C. Murphy2, 3. 1Molecular and Cellular Therapeutics, Royal College of
Surgeons in Ireland, Dublin, Ireland; 2Department of Ophthalmology,
Royal College of Surgeons in Ireland, Dublin, Ireland; 3Department
of Ophthalmology, Royal Victoria Eye and Ear Hospital, Dublin 2,
Ireland.
Purpose: Sjögren’s syndrome (SS) is a systemic autoimmune
disorder characterized by inflammation that affects mucous
membranes particularly those of the exocrine glands, causing dry
eyes and dry mouth. The exocrine glands become infiltrated with
lymphocytes resulting in severe damage to both the salivary glands
and the lacrimal glands. Previous investigations have suggested that
dysregulated systemic inflammation contributes to the development
and pathogenesis of SS. This study aimed to investigate potential
mechanisms responsible for over production of pathogenic cytokines
in SS patients.
Methods: Peripheral blood mononuclear cells and serum were
prepared from whole blood taken from both healthy controls and
pSS patients. Gene induction and micro-RNA expression were
analysed by real-time PCR. Cytokine levels were determined by
ELISA. Differences in miR expression, cytokine levels and gene
induction between patients and controls were examined using the
non-parametric Mann–Whitney test. Spearman’s rank correlation was
used to assess the interrelationship between miR expression, gene
induction and peripheral cytokine levels.
Results: We observed significantly enhanced expression of the
pro-inflammatory micro-RNA, miR-155 (p≤0.05) and significantly
reduced levels of the IL-10 promoting miR, miRNA-21 (p≤0.05)
compared to healthy controls. In keeping with this altered pattern of
miR expression we observed significantly increased serum levels of
pro-inflammatory cytokines (IL-6, IL-8 and TNF-a) as well as the
Th17 promoting cytokine, IL-23p19 (p ≤0.05). Altered expression of
previously identified targets of miR-155 (SHIP-1, SOCS1 – potent
anti-inflammatory regulators) and miR-21 (IL12p35 and PDCD4,
positive regulators of inflammation) was also observed. Significantly
a moderate negative correlation (r =-0.657, p≤0.05) was observed
between reduced miR-21 expression and increased peripheral IL23p19 levels in pSS patients, suggesting a link between altered miR
expression and disease pathogenesis.
Conclusions: Our data suggest that abnormal expression or
regulation of miRs and consequently miR regulated genes in innate
immunity may contribute to the initiation and progression of SS via
overproduction of pathogenic cytokines.
Commercial Relationships: Joan Ní Gabhann, None; Qistina
Pilson, None; Caroline A. Jefferies, None; Conor C. Murphy,
None
Support: This work was supported by the Health Research Board
and the Royal Victoria Eye and Ear Hospital Research Foundation
through the Medical Research Charities Group (grant code 1409)
Effect of HDACi Givinostat in Treating Experimental Ocular
Autoimmunity
Baoying Liu, Zhiyu Li, Phyllis Silver, Cuiyan Tan, Ping Chen, ChiChao Chan, Robert B. Nussenblatt. National Eye Insitute, Bethesda,
MD.
Purpose: Histone deacetylase inhibitor (HDACi) Givinostat, also
called ITF2357, binds the zinc-containing catalytic domain of the
HDAC and potentially has anti-inflammatory, anti-angiogenic and
anti-neoplastic activities. Givinostat has been shown to inhibit
expression of tumor necrosis factor-α, interferon γ, interleukin 1α
and β and interleukin 6. It is currently in 11 phase II clinical trials
and has been designated an orphan drug for the treatment of juvenile
idiopathic arthritis in the European Union. In this study, we aim
to examine the effect of Givinostat in treating experimental ocular
autoimmunity.
Methods: Experimental autoimmune uveitis (EAU) was induced
in B10.A mice by immunization with interphotoreceptor retinoidbinding protein (IRBP; 100 μg) in CFA. Treatment with Givinostat
was by twice a week intraperitoneal injection of 0.2 mg. Disease
severity was assessed by both fundoscopy and histological
examination. Draining lymph node cells were tested for proliferation
by thymidine uptake. In addition, the intracellular expression of
cytokines and Foxp3 was determined by flow cytometry.
Results: Treatment with Givinostat efficiently inhibited the
development of EAU in mice, as well as the cellular proliferation
immune responses against IRBP. Givinostat treatment moderately
increased the proportion of Foxp3 expressing T-regulatory cells.
However, preliminary results indicated that the treatment exhibited
little effect on the expression of the signature cytokines of Th1 and
Th17 subpopulations, IFN-gamma and IL-17.
Conclusions: Givinostat, a histone deacetylase inhibitor, efficiently
inhibits EAU development and related cellular immune responses.
Givinostat may be considered for treatment of pathogenic immunity
in humans.
Commercial Relationships: Baoying Liu, None; Zhiyu Li, None;
Phyllis Silver, None; Cuiyan Tan, None; Ping Chen, None; ChiChao Chan, None; Robert B. Nussenblatt, None
Support: NEI intramural research grant
The Effect of HDACi Givinostat on the Expression of Immune
Markers Related to Human Ocular Inflammation
Timothy P. Quinn1, 2, Robert B. Nussenblatt2, Baoying Liu2. 1Wayne
State University School of Medicine, Grosse Pointe Park, MI;
2
Clinical Immunology, National Institutes of Health National Eye
Purpose: Uveitis involves swelling of the uvea, is responsible for
10-20% of blindness in the United States and is often attributable
to an autoimmune disorder. Histone deacetylase inhibitor (HDACi)
Givinostat, also known as ITF2357, prevents HDAC from altering
a histone lysine residue leading to a condensed chromatin and
has been shown to have an anti-inflammatory, anti-neoplastic and
anti-angiogenic effect. It is currently in 11 phase II clinical trials
and has been designated an orphan drug in the European Union for
the treatment of juvenile idiopathic arthritis. Givinostat has been
shown to inhibit expression of tumor necrosis factor α, interferon
γ, interleukin 1α and β, and interleukin 6 and it as well has been
shown to decrease inflammation in an experimental autoimmune
uveoretinitis (EAU) murine model. We aimed to investigate the
effects of Givinostat on the expression of immune markers related to
ocular inflammation.
Methods: Human peripheral blood mononuclear cells (PBMCs)
were isolated from whole blood samples and activated with either
anti-CD3/CD28 or lippolysaccaride (LPS) and cultured with varying
concentrations of Givinostat. Cultures activated with anti-CD3/CD28
were labeled with CD4, CD3 and either CD25 or CD28 and those
activated with LPS labeled with CD16, CD14 and either CD163,
CD80, CD86, or HLA-DR. Populations were then analyzed using
flow cytometry FACScalibur and mean fluorescence intensity was
assessed. Further, isolated CD4+ T cells were isolated and cultured
with varying concentrations of Givinostat and with or without the
presence of monocytes and labeled with CD4, CD3 and either CD25
or CD28 and analyzed as above.
Results: A dose response curve effect was observed that decreased
the expression of CD25, CD28, CD163, HLA-DR and CD80.
Increasing Givinostat concentrations did not, however, decrease the
expression of CD86. The observed dose response effect on CD25
and CD28 is independent of the presence of monocytes. Further,
monocytes showed a greater degree of sensitivity to Givinostat as
compared to T cells.
Conclusions: These results reveal an anti-inflammatory effect of
Givinostat and as a potential treatment for autoimmune uveitis. This
reveals a possibility to treat the disease at the level of epigenetics
rather than symptomatically and further investigation into Givinostat
is warranted.
Commercial Relationships: Timothy P. Quinn, None; Robert B.
Nussenblatt, None; Baoying Liu, None
Support: NEI Intramural Training Award
Peripheral Monocyte Subset-Derived Macrophages Show Distinct
Expression Kinetics of Immune Biomarkers Associated with
Ocular Autoimmunity
Susan Hannes, Baoying Liu, Zhiyu Li, Diamond Ling, H Nida Sen,
Robert B. Nussenblatt. Clinical Immunology, National Eye Institute,
Bethesda, MD.
Purpose: Human peripheral blood monocytes are precursors of
some tissue-resident macrophages and have been implicated in the
pathogenesis of non-infectious uveitis. They can be categorized
into three subgroups: CD14++CD16- (classical), CD14++CD16+
(intermediate), and CD14+CD16++ (non-classical). Previously, we
observed that monocytes isolated from the circulating blood of
non-infectious uveitis patients exhibit a skewed phenotype toward
an elevated level of the CD14++CD16+ subset. However, the destiny
of these monocyte subsets after differentiation has not been fully
investigated. Here, we sought to evaluate the expression kinetics of
immune biomarkers associated with non-infectious uveitis in both
M1 (pro-inflammatory) and M2 (alternative) macrophages in vitro.
Methods: Human peripheral blood mononuclear cells (PBMCs)
were isolated from the peripheral blood of healthy donors and uveitis
patients using a Ficoll gradient centrifugation protocol. Subsets of
monocytes were purified by flow cytometry (BDFACSAria II) based
on CD14 and CD16 staining. The subsets were differentiated over
a time course using GM-CSF and M-CSF to produce M1 and M2
macrophages, respectively. An Annexin V Apoptosis Assay was used
to monitor viability. The following cell surface markers were assayed
with flow cytometry: CD1a, CD11b, CD11c, CD69, CD80, CD86,
and CD163.
Results: We observed a time dependent effect in biomarker changes
during polarization procedures using either GM-CSF or M-CSF
by monitoring cell-surface marker changes. During M1 and M2
differentiation conditions, marker expression levels varied, with
perceptible increases in CD80, CD86, and CD163 for GM-CSF
differentiated
CD14++CD16- and CD14++CD16+ subpopulations. Regarding GMCSF versus M-CSF conditions, CD80 exhibited increased expression
with M-CSF polarization in the CD14+CD16++ subpopulation. CD86
levels are higher with M-CSF polarization in CD14++CD16+ and
CD14+CD16++ subpopulations. During either polarization condition,
the CD14+CD16++ population was found to die more quickly than the
other two.
Conclusions: Our study showed that under both M1 and M2
polarization conditions, peripheral monocyte subset-derived
macrophages exhibit distinctive expression kinetics of immune
biomarkers associated with ocular autoimmunity. Our results
contribute to knowledge of the innate immune mechanisms involved
in non-infectious uveitis.
Commercial Relationships: Susan Hannes, None; Baoying Liu,
None; Zhiyu Li, None; Diamond Ling, None; H Nida Sen, None;
Robert B. Nussenblatt, None
Support: NEI Intramural Research Program
Polysialic acid attenuates alternative complement activation,
inhibits microglial reactivity and reduces vascular leakage after
retinal laser-damage
Marcus Karlstetter1, Janine Claude2, Albert Caramoy1, Bettina
Linnartz-Gerlach2, Jens Kopatz2, Anika Lückoff1, Yiner Wang2,
Christine Skerka3, Harald Neumann2, Thomas Langmann1.
1
Department of Ophthalmology, University of Cologne, Cologne,
Germany; 2Institute of Reconstructive Neurobiology, University of
Bonn, Bonn, Germany; 3Department of Infection Biology, Leibniz
Institute for Natural Product Research and Infection Biology, Jena,
Germany.
Purpose: Unbalanced activation of the complement system and
radical production by activated microglia/macrophages are key
features of age-related macular degeneration (AMD). Polysialic acids
constitute the outermost part of the neuronal glycocalyx and serve as
ligands for the inhibitory human-specific Siglec-11 receptor which
regulates microglial activation. Sialic acid loss induces complement
activation and complement receptor mediated superoxide production
by microglia. Here, we hypothesized that low molecular polysialic
acid (PSA) may interfere with complement activation and could
reduce microglial reactivity via interaction with the Siglec-11
receptor and thus may attenuate immune mechanisms during AMD
pathology.
Methods: Retinal polysialic acid expression was studied by
immunohistochemistry and Siglec-11 receptor transcripts were
analyzed by RT-PCR. To determine the immune modulatory
potential of PSA on human induced pluripotent stem (iPS)cell derived microglia, we analyzed pro-inflammatory cytokine
production, phagocytosis of RPE cell debris and superoxide
release after PSA treatment. We further studied the effect of PSA
on membrane attack complex formation and subsequent cell lysis
by immunohistochemistry. To study the in vivo effects of PSA, we
applied PSA intravitreally in humanized Siglec-11 transgenic mice
directly after retinal laser-coagulation. Fourty eight hours later
microglial reactivity was analyzed in the retina and the RPE/choroid
and vessel leakage was assessed by fluorescein angiography.
Results: We found that polysialic acids and the Siglec-11 receptor
are expressed in the human retina. Application of PSA on human
microglial cells inhibited production of the pro-inflammatory
cytokine tumor necrosis factor-alpha, reduced inflammatory
phagocytosis and blocked superoxide release after cell debris
stimulation. PSA inhibited formation of the membrane attack
complex and complement mediated lysis. Intravitreally injected PSA
led to decreased accumulation of reactive microglia/macrophages in
the laser spots of Siglec-11 transgenic mice and significantly reduced
retinal vessel leakage.
Conclusions: We conclude that PSA attenuates neurotoxicity of
human microglia in vitro and that intravitreal application of PSA
decreases pathological features of AMD. Our findings suggest that
PSA may provide a novel therapeutic strategy for AMD.
Commercial Relationships: Marcus Karlstetter, EP2783691 (P);
Janine Claude, None; Albert Caramoy, None; Bettina LinnartzGerlach, None; Jens Kopatz, EP2783691 (P); Anika Lückoff,
None; Yiner Wang, None; Christine Skerka, None; Harald
Neumann, EP2783691 (P); Thomas Langmann, EP2783691 (P)
Immunotherapy of Non-Infectious Uveitis using Collagen IIspecific regulatory Type 1 (Col-Treg) cells
Hélène Asnagli1, Nathalie Belmonte2, Julie Gertner-Dardenne2,
Marie Jacquin1, Papa Babacar Fall1, Irène Marchetti2, MarieFrançoise Hubert3, André Sales4, Arnaud Foussat5. 1Preclinical &
Safety Pharmacology, TxCell, Valbonne-Sophia Antipolis, France;
2
Cell Processing & New Products, TxCell, Valbonne, France; 3COLA,
Jozerand, France; 4Vetopath, Antibes, France; 5Research & New
Products, TxCell, Valbonne, France.
Purpose: Non-infectious uveitis(NIU) is a condition characterized
by dysregulation of the immune system in the eye for which only
steroids are currently approved. Col-Treg is a T-cell immunotherapy
composed of autologous type-1 regulatory T (Treg) cells specific for
collagen-II. The use of Col-Treg was evaluated for NIU in mice as
Collagen-II is naturally present in the eye and so triggers the activity
of the Col-Treg cells in situ.
Methods: Col-Treg cells were produced from blood of healthy
volunteers or from splenocytes of transgenic mice for Collagen-IIspecific TCR. Cells were characterized for marker expression by
FACS and for in vitro immuno-modulatory functions. NIU model was
developed by IRBP immunization. In vivo efficacy was evaluated
using ophtalmoscopy and histology. In vivo tracking was performed
using a Col-Treg TCR Vβ chain-specific quantitative PCR.
Results: Col-Treg cells secrete IL-10 and IL-13 and express GITR,
CD39 and Granzyme B, molecules known to be involved in the
control of inflammation. In vitro assays confirmed the capacity of
Col-Treg cells to hydrolyse ATP, kill myeloid cells in a contactdependent manner and inhibit T effector cell IL-17 and IFNγ
secretion using soluble factor dependent pathways. Intravenous
administration of Col-Treg inhibited ocular inflammation in NIU
mice. Moreover, Col-Treg injection decreased cell infiltration in the
ocular tissues, vasculitis and retinal folds. Tracking experiments
demonstrated a tropism of Col-Treg for inflammatory eyes. An in
vivo GLP toxicity study performed in healthy mice did not reveal
Col-Treg related adverse events. Characterization of human Col-Treg
GMP batches demonstrated comparability with mouse Col-Treg for
marker expression and in vitro function. Human Col-Treg cells did
not show evidence of tumorigenicity or uncontrolled proliferation
in vitro as witnessed by a limited survival capacity upon chronic
stimulation and a strict dependence to exogenous stimulation for their
exponential proliferation.
Conclusions: These results demonstrate the safety and efficacy of
Col-Treg administration for the treatment of NIU in mice and suggest
that Col-Treg could be used as a therapeutic tool for patients with
non-infectious uveitis refractory to approved medications. These
results will be taken as a basis for a First in Man clinical study with
Col-Treg in NIU patients that is expected to start in 1H 2015.
Commercial Relationships: Hélène Asnagli, TxCell (E); Nathalie
Belmonte, TxCell (E); Julie Gertner-Dardenne, TxCell (E);
Marie Jacquin, TxCell (E); Papa Babacar Fall, TxCell (E); Irène
Marchetti, TxCell (E); Marie-Françoise Hubert, TxCell (C);
André Sales, TxCell (C); Arnaud Foussat, TxCell (E)
Glucocorticoid Receptor Role in the Mouse Retina
Mahita Kadmiel, Sivapriya Ramamoorthy, John Cidlowski. NIH/
NIEHS, Durham, NC.
Purpose: Glucocorticoids are widely used in the treatment of
ocular disorders. However, the physiological function of retinal
glucocorticoid receptor (GR) is poorly understood. Glucocorticoid
receptor signaling activates anti-inflammatory responses in various
organs in the body. Therefore, we hypothesize that the glucocorticoid
receptor in the retina functions to regulate the inflammatory response
and thereby maintain retinal homeostasis.
Methods: Mice with conditional deletion of the glucocorticoid
receptor in the retina (RetinaGRKO mice) were generated by
intercrossing GRflox/flox mice (Oakley RH, 2013) and RxCre mice
(Swindell E.C, 2006). At 2 months of age, control and knockout male
mice were euthanized and their eyes were harvested for downstream
analysis. Reduction in GR mRNA and protein level in the retinas
from RetinaGRKO mice was determined by RT-PCR, western blotting
and immunohistochemistry. For histological and immunofluorescence
studies, paraformaldehyde-fixed eyes were cryoprotected in sucrose
and embedded in OCT compound before cryosectioning. For gene
expression studies, NanoString technology was used to measure the
expression of 248 genes involved in inflammation.
Results: Immunofluorescence in adult wild type mice revealed
that glucocorticoid receptor is expressed predominantly by the
Muller glial cells of the neural retina and by the retinal pigmented
epithelium. Compared to the control animals, Retina GRKO mice
exhibited a 60% reduction in glucocorticoid receptor, both at the
mRNA level and the protein level. Loss of GR in the retina results
in the thinning of the inner nuclear layer of the peripheral retina.
Furthermore, NanoString analysis revealed that glucocorticoid
receptor is important for retinal homeostasis, as the expression level
of 26 inflammatory genes was significantly altered in RetinaGRKO
mice (ANOVA p value <0.05). Members of the toll-like receptor
family and of the complement system were among the genes
whose expression was significantly altered in the absence of the
glucocorticoid receptor.
Conclusions: Our results suggest that retinal glucocorticoid
receptor plays a critical role in maintaining retinal homeostasis
by regulating the inflammatory response. Future studies involving
electroretinograms would reveal the functionality of retinas in the
absence of glucocorticoid receptor. Findings from this study may lead
to improved glucocorticoid-based therapies for inflammatory diseases
of the retina.
Commercial Relationships: Mahita Kadmiel, None; Sivapriya
Ramamoorthy, None; John Cidlowski, None
Support: NIEHS Intramural Research Program
Expression of The Regulatory Melanocortin-Adenosinergic
Pathway in Human Uveitis Patients
Darren J. Lee. Ophthalmology, Boston University School of Med,
Boston, MA.
Purpose: Mice that have recovered from experimental autoimmune
uveoretinitis (EAU) have regulatory immunity in their spleen. This
post-EAU regulatory immunity involves an APC that activates
antigen-specific inducible Treg cells. It requires expression of
melanocortin 5 receptor (MC5r) on the APC, and adenosine 2A
receptor (A2Ar) on the T cells. This melanocortin-adenosinergic
pathway induced regulation provides long-term protection from
recurrence of EAU. Therefore, this conserved pathway is a potential
therapeutic approach for uveitis. PBMC were assayed to determine
whether MC5r and A2Ar are expressed on immune cells in uveitis
patients.
Methods: PBMC were collected from healthy volunteers and
uveitis patients being treated at Massachusetts Eye Research and
Surgery Institute. Patients were grouped as suppressed (US) and
active (UA), based on the location of uveitis, and by the type of
immunosuppressive therapy. US patients had no uveitis within a
year from the time of collection. PBMC were analyzed by FACS
for CD14, CD16, CD4, MC5r, and A2Ar expression. The CD4 T
cells were sorted out, and the monocytes were sorted into classical
(CD14+CD16lo) and non-classical (CD14-CD16hi) subsets. The
monocytes were pulsed with tetanus toxin and treated with the
neuropeptide α-MSH to stimulate the melanocortin pathway. The T
cells were added to the cultures, and the supernatants were assayed
for TGF-β.
Results: Analysis of PBMC from US (n = 11) and UA (n = 21)
patients and healthy volunteers (n = 9) revealed a similar percentage
of classical and non-classical monocytes. Further analysis of MC5r
expression on each of the subsets showed no significant difference.
The expression of A2Ar on CD4+ T cells was also similar. There was
no difference in MC5r or A2Ar expression with age, type of therapy,
or location of uveitis. Both monocyte subsets produced slightly more
TGF-β from US (n = 5) and UA (n = 6) patients compared to healthy
volunteers (n = 3), and treatment with α-MSH had no effect. When T
cells were added to the monocyte cultures a similar pattern of TGF-β
production was observed.
Conclusions: While our initial functional analysis saw no effect
on TGF-β production, the potential to activate the melanocortinadenosinergic pathway to suppress effector T cells should be possible
in uveitic patients.
Commercial Relationships: Darren J. Lee, None
Dynamic quantitative proteomic analysis of AqH from
experimental recurrent autoimmune uveitis
Hui Shao1, Guomin Jiang1, Yunsong Wang1, 2, Xiaomin Zhang3,
Deming Sun4, Henry J. Kaplan1, Lei Zhou5, 6. 1Ophthalmology
& Visual Sciences, University of Louisville, Louisville, KY;
2
Ophthalmology, Tangshan Gongren Hospital, Tangshan, China;
3
Uveitis & Ocular immunology, Tianjin Medical University Eye
Hospital, Eye Institute & School of Optometry and Ophthalmology,
Tianjin, China; 4Doheny Eye Institute, Los Angeles, CA; 5Singapore
Eye Research Institute, Singapore, Singapore; 6Ophthalmology, Yong
Loo Lin School of Medicine, National University of Singapore,
Singapore, Singapore.
Purpose: The mechanism of recurrent autoimmune uveitis is
unknown. We hypothesized that quantitative proteomic analysis of
AqH would identify proteins that correlate with recurrent episodes of
EAU induced by interphotoreceptor retinoid-binding protein (IRBP)specific T cells (tEAU) in rats.
Methods: tEAU was induced in Lewis rats by injection of IRBP
peptide (R16)-specific T cells collected from R16 immunized
Lewis rats. Intraocular inflammation was clinically evaluated. AqH
samples were collected on days 0 (naïve), 1, 4 (onset), 7 (peak), 11
(remission), 14 (second attack), 24 (second peak) and 30 (second
remission), and analyzed by iTRAQ Quantitative Proteomics. AqH
samples were pooled from six eyes at each time point and assessed
by Spearman correlation coefficient by analyzing the samples in
replicates from two independent experiments.
Results: Among ~200 identified AqH proteins (<1% false discovery
rate, FDR), 34 were found to correlate with clinical recurrence.
Pathway analysis using MetaCore revealed that proteins in all three
complement pathways (classical, lectin and alternative) showed
the strongest association with intraocular inflammation, with blood
coagulation and HDL-mediated reverse cholesterol transport being
second and third, respectively. C-reactive protein (CRP), a protein
that binds to lysophosphatidylcholine and activates the complement
system via the C1Q complex, was increased during inflammatory
episodes and subsided during remission.
Conclusions: This study using proteomic analysis in AqH during
recurrent EAU induced by uveitogenic T cells in Lewis rats
demonstrated the involvement of complement pathways and CRP.
However, it is unknown whether complement activation is the
initiator or consequence of recurrent intraocular inflammation.
Commercial Relationships: Hui Shao, None; Guomin Jiang,
None; Yunsong Wang, None; Xiaomin Zhang, None; Deming Sun,
None; Henry J. Kaplan, None; Lei Zhou, None
Support: Supported in part by a RPB Lew R Wasserman Merit
Award (HS), a RPB unrestricted grant to UofL, the Commonwealth
of Kentucky Research Challenge Trust Fund (HK), and the
University of Louisville School of Medicine (GJ).
156 Corneal immunology and infections
Sunday, May 03, 2015 3:15 PM–5:00 PM
601/603 Paper Session
Program #/Board # Range: 938–944
Program Number: 938
Mechanisms of enhanced corneal graft rejection on clinically
uninflamed herpes simplex virus type 1 infected recipient corneal
beds
Robert L. Hendricks1, Alexander Rowe2, HONGMIN YUN2.
1
Ophthalmology, Immunology, Microbiology and Molecular
Genetics, University of Pittsburgh, Pittsburgh, PA; 2Ophthalmology,
University of Pittsburgh, Pittsburgh, PA.
Purpose: The frequency of corneal graft rejection is significantly
increased on herpes simplex virus type 1 (HSV-1) infected recipient
corneal beds, even when recipient corneas lack clinically detectable
inflammation at the time of grafting. The purpose of this study was to
define mechanisms that promote allograft rejection on these clinically
quiet HSV-1 infected corneal beds.
Methods: Wild type (WT) C57BL/6 corneas or those expressing
chicken ovalbumin (Ova) as a surrogate alloantigen were transplanted
orthotopically to recipient WT C57BL/6 mice. Prior to grafting,
recipient mice were mock depleted or depleted of CD4+ T cells
systemically or locally in the cornea (subconjunctival). Thirty days
prior to grafting corneas were mock infected or infected with a 1
x 105 pfu of KOS HSV-1, resulting in corneal epithelial lesions,
establishment of latent infections of the trigeminal ganglion, but
no corneal inflammation that was clinically detectable at the time
of grafting. Quantification of corneal chemokine transcripts by
qRT-PCR and leukocytic infiltrates by flow cytometry on dispersed
corneal cells was performed before and after grafting.
Results: Prior to grafting, transcripts for chemokines including
CXCL10, CXCL9, CCL5, and CCL2 were significantly elevated
in infected, but uninflamed recipient corneal beds, and were
dramatically reduced by local (corneal) or systemic CD4+ T cell
depletion. Leukocytic infiltrates were significantly elevated in
infected corneal beds prior to grafting and in corneal beds and
corneal allografts after grafting, but prior to rejection. The frequency
of corneal allograft rejection was significantly increased in infected
cornea. Non-immunologic rejection of syngeneic corneal grafts was
not significantly increased on HSV-1 infected beds (20% infected
vs. 0% non-infected, p > 0.05), but those on infected beds showed
significantly increased pathology that did not reach the criterion for
rejection.
Conclusions: HSV-1 infected corneas that never develop clinically
detectable inflammation nonetheless maintain an inflammatory
environment comprised of elevated chemokines and leukocytes for
at least 30 days that is regulated by CD4+ T cells. This preexisting
inflammation predisposes to corneal graft rejection.
Commercial Relationships: Robert L. Hendricks, None;
Alexander Rowe, None; HONGMIN YUN, None
Support: NIH EY10359, EY05945, P30-EY08098, Unrestricted
grants from Research to prevent Blindnes (New York, NY) and The
Eye and Ear Foundation of Pittsburbh
Program Number: 939
A STING-dependent innate sensing pathway mediates resistance
to corneal HSV-1 infection
Derek J. Royer1, Daniel J. Carr1, 2. 1Microbiology & Immunology,
Univ of Oklahoma Health Sci Ctr, Oklahoma City, OK;
2
Ophthalmology, Univ of Oklahoma Health Sci Ctr, Oklahoma City,
OK.
Purpose: Type 1 interferons (IFN-α/β) mediate host resistance to
herpes simplex virus type 1 (HSV-1) infection in the cornea. Our
lab has previously shown that the loss of toll-like receptor (TLR)
signaling does not affect host resistance to HSV-1 in the cornea;
rather, a TLR-independent pathway drives the production of IFN-α/β
at day 2 post infection (pi) through the cytosolic innate sensor adaptor
protein, the stimulator of interferon genes (STING). We hypothesized
that STING also mediates host resistance to corneal HSV-1 infection
and preserves the corneal integrity through day 5 pi via the induction
of IFN-α/β.
Methods: In the current study, wildtype (WT), STING-deficient
(STING-/-), and IFN-α/β receptor deficient (CD118-/-) mice were
infected with HSV-1. Corneal viral burden at day 5 pi was assessed
by plaque assay, edema monitored with a pachymeter, and infiltrating
cells characterized via flow cytometry. Corneal whole mounts were
prepared to visualize lymphatic vessels and mast cells by confocal
microscopy.
Results: STING-/- and CD118-/- mice had comparable corneal
HSV-1 titers that were both significantly higher than WT at day
5 pi. However, the increase in viral titer did not directly correlate
with tissue edema or infiltrating leukocytes. Corneal edema was
unremarkable in WT and STING-/- corneas through day 5 pi despite
the difference in viral burden, while CD118-/- corneas exhibited
substantial edema beginning on day 3 pi. Edema was independent
of leukocyte infiltration, as similar numbers of total leukocytes
and specifically neutrophils were observed in STING-/- and CD118/corneas at day 5 pi. Preliminary evidence supports a role for the
lymphatic vasculature surrounding the cornea in regulating corneal
edema, as the lymphatic vessels and limbal mast cells are lost by day
5 pi in CD118-/- corneas but not in WT or STING-/- corneas.
Conclusions: Consistent with the hypothesis, our results support
a role for STING in innate host resistance to HSV-1 infection with
respect to viral surveillance and containment within the corneal
microenvironment. However, auxiliary STING-independent,
IFN-α/β-dependent pathways are necessary to limit corneal edema
attributed here to the loss of lymphatic vessels surrounding the tissue.
Furthermore, our results suggest that limbal mast cells preserve and
protect the lymphatic vasculature via auxiliary IFN-α/β-dependent
pathways.
Commercial Relationships: Derek J. Royer, None; Daniel J. Carr,
None
Support: NIH Grants EY021238 and T32EY023202
Program Number: 940
Fine-tuning of Autophagy Is Required for a Productive Herpes
Simplex Virus Ocular Infection
Deepak Shukla, Abraam Yakoub. Ophthal/Visual Sciences, University
of Illinois at Chicago, Chicago, IL.
Purpose: Autophagy is an important catabolic process of the cell,
and also an essential defense mechanism. Our study demonstrates a
unique role of autophagy in regulating herpes simplex virus type-1
(HSV-1) infection, which has been debatable and poorly defined for
ocular infection.
Methods: Multiple methods including starvation, physical stress,
and pharmacological agents including proteosomal inhibitors,
Bafilomycin A1, and Chloroquine were used to modulate autophagy
in human corneal epithelial cells. Flow cytometry, fluorescence
microscopy, and confocal fluorescence microscopy were used for
monitoring and quantitative assessment of LC3-GFP or HcRedLC3 autophagosomal punctae. Quantitative PCR was used for HSV
genome quantification and immunoblotting to assess autophagy
proteins. Wild-type (WT) or ATG5-/- MEFs were also used to study
infection and autophagy.
Results: Utilizing a comprehensive approach, we show that
inducing autophagy of host cells suppresses HSV-1 infection in
ocular cells that represent natural targets of HSV infection in vivo.
Measuring the autophagic response of cells to HSV infection, we
found that HSV requires a “normo-autophagic” state in host cells
for productive infection. Suppression of this physiologically normal
level of autophagy dramatically diminished the viral infection and
resulted in a measureable loss of viral DNA copy numbers. Along
these lines, ATG5-/- mouse embryo fibroblasts showed a clear loss of
productive infection. Inducing autophagy, on the other hand, also had
detrimental effects on viral growth and copy numbers.
Conclusions: Overall, our findings suggest a model, whereby HSV1, in order to support its own replication, fine-tunes the autophagic
activity of the host, preventing induction of autophagy that suppresses
the infection, while maintaining an intermediate level of autophagy
required for virus replication. Our work establishes the existence
of various tints of autophagy with distinct biological functions, and
provides a druggable pathway for ocular herpes management.
Commercial Relationships: Deepak Shukla, None; Abraam
Yakoub, None
Support: NIH grant R21 EY023058 and an unrestricted support from
Research to Prevent Blindness
Program Number: 941
Herpes Zoster Vaccination: Impact on the Incidence and
Complications of Herpes Zoster Ophthalmicus
Shruti Aggarwal, Rodrigo Müller, Deborah Pavan-Langston.
Ophthalmology, Massachusetts Eye & Ear Infirmary, Cambridge,
MA.
Purpose: The incidence of Herpes zoster (HZ) infection in the US is
about 1 million/year, of which about 20% have ocular involvement,
Herpes zoster Ophthalmicus (HZO). Zostavax™, HZ vaccine, is
currently approved for people > 60 years of age to prevent and/or
minimize HZ. However, the impact of vaccination is not well known.
The purpose of this study is to evaluate the effect of Zostavax on the
incidence and complications of HZO.
Methods: Patients were enrolled from the Cornea Service at
Massachusetts Eye and Ear Infirmary. The patients’ previous history
of shingles, vaccination and clinical courses were recorded. Outcome
measures were the age of incidence of HZ, Zostavax™ vaccination,
clinical signs, symptoms and complications of HZO.
Results: 341 patients (114 males, 227 females) were enrolled, mean
age being 56.0 ±17.6. 165 patients developed HZ; the mean age at
the first episode being 59.0±15.6. The mean age of HZ vaccination
was significantly higher at 66.7±12.5 (p=0.008). The current cut off
of eligibility for Zostavax™ is 60 years; number of patients >60
was 148. Out of the eligible patients, 112 (75.7%) did not get the
vaccine and 47.3% of this group developed shingles, while out of
the 36 (24.3%) eligible patients who did get the vaccine, only 4%
developed the disease. Relative risk of developing HZ was 3.21 in
non-vaccinated group compared to the vaccinated group (p = 0.0008).
Ocular involvement was seen in 85% of HZ; with complications
such as residual corneal opacities, secondary glaucoma and cataracts
seen in 70%, 8.7% and 5%, respectively in 3.7 years follow up. Out
of the 4 patients who developed HZ after vaccination, mean age of
incidence was 67±14.
Conclusions: Zostavax™ can decrease the risk of development
of Herpes zoster by 3 times. In current practice, the mean age of
vaccination occurs 8 years later compared to the peak incidence of
disease. Patients should be encouraged to be vaccinated about ten
years earlier to minimize the surge in incidence that occurs in the 5th
decade of life. Based on our data as a large tertiary care ophthalmic
institute, it is indicative that zoster vaccine delays the age of onset
of the first episode of HZO and may result in fewer long term
complications even in patients who develop disease after vaccination.
Large population studies based on systemic data are required to
further elucidate vaccine efficacy.
Commercial Relationships: Shruti Aggarwal, None; Rodrigo
Müller, None; Deborah Pavan-Langston, None
Program Number: 942
Bacterial induced corneal inflammation is inhibited by
Mesenchymal Stem Cells (MSCs)
Rui Zhang, Yan Sun, Eric Pearlman, Suber S. Huang. Department of
Ophthlamology, University Hospitals Eye Institute, Cleveland, OH.
Purpose: Numerous studies have demonstrated inflammation to
play a key role in the development of pathologic ocular scarring.
Mesenchymal stem cells (MSCs) have been shown in recent research
to help with the healing response in various diseases. This study
evaluates whether the targeted delivery of MSCs decreases the
amount of pathologic and inflammatory response to ocular injury
using a bacterial infection model.
Methods: C57BL/6 mice corneas were abraded, and infection was
induced by Pseudomonas aeruginosa. The mice were divided into
3 groups. One group received topical antibiotic (moxifloxacin) 2
hrs post procedure, then every 12 hrs for 24 hrs; one group received
Human-derived Mesenchymal Stem Cells (MSCs) and the third
group received topical moxifloxacin and MSCs. The human derived
MSCs were injected into the subconjunctival space immediately
after bacterial innoculation. Progression of corneal changes was
followed with clinical photos. The mice were sacrificed on day 7, and
the degree of corneal infiltration and inflammation was studied via
confoscan and histological sections, and neutrophil recruitment to the
corneal stroma was detected by immunohistochemistry.
Results: The corneas of mice treated with MSCs and antibiotic
displayed less stromal edema, infiltration and clinical disease on day
2 and 5 after treatment. Confoscan data at day 7 showed that mice
treated with MSC and antibiotic had significantly less stromal haze
and thickness with a P<0.05 when compared to mice treated with
antibiotics. Clinical photos taken on day 7 also displayed less disease,
scarring, and inflammation in mice treated with MSCs and antibiotic
versus antibiotic alone. Immunohistochemistry performed on corneal
sections also showed less neutrophil recruitment and disruption of
overall structure in mice treated with both MSCs and antibiotic.
Conclusions: These results suggest that the local delivery of
MSCs may be an effective and safe way to supplement and
immunomodulate the ocular inflammatory process driven by
the innate and adaptive immune responses. MSCs have little
immunogenicity, do not require HLA typing, and has already been
used intravenously in humans without serious consequences. These
results hold promise for future clinical trials using MSCs in ocular
keratitis and other inflammatory diseases, such as being primary
or an adjunctive treatment for neovascular age related macular
degeneration.
Figure 1
Figure 2
Commercial Relationships: Rui Zhang, None; Yan Sun, None;
Eric Pearlman, None; Suber S. Huang, None
Support: Philip F and Elizabeth G. Searle - Suber Huang MD
endowed chair, School of Medicine, CWRU
Program Number: 943
Inactivation of miR-183/96/182 Reduces Severity of Pseudomonas
aeruginosa Infection of the Cornea
Shunbin Xu1, 2, Linda D. Hazlett2, 1, Elizabeth A. Berger2, 1, Susmit
Suvas1, 2, Cui Li2, Chithra Muraleedharan1, 2, Ronald Barrett2,
Sharon A. McClellan2, Thomas Carion2, Daniel Montenegro1.
1
Ophthalmology, Wayne State University, Detroit, MI; 2Anatomy &
Cell Biology, Wayne State University, Detroit, MI.
Purpose: The miR-183/96/182 cluster (referred to as miR183/96/182) is highly expressed in all sensory organs. The cornea is
densely innervated by sensory neurons. The purpose of the current
study is to test the role of miR-183/96/182 in the murine response to
corneal infection with Pseudomonas aeruginosa (PA).
Methods: miR-183/96/182 knockout (ko), miR-183CGT/GT, and their
wild type control (wt) mice were employed. The central corneas of
anesthetized mice were scarred and 5 ml of 1X106 CFU/ml of PA
(strain 19660; ATCC) was topically applied. Corneal disease was
graded at 1, 3, and 5 days post infection (dpi).Corneal RNA was
harvested at 5 dpi. X-gal staining and immunofluorescence were
performed on corneal flat-mounts and cryosections. Peritoneal
polymorphonuclear neutrophils (PMNs) and macrophages (MΦ)
were isolated for RNA preparation and quantitative (q)RT-PCR, or
phagocytosis and intracellular bacterial killing assays.
Results: 1. miR-183/96/182 is highly expressed in corneal epithelium
and in trigeminal ganglia of wild type (wt) mice; while completely
inactivated in ko animals.
2. Compared to wt, ko animals showed decreased disease upon PA
infection, as well as decreased expression of inflammation-related
cytokines and their receptors in the cornea.
3. Inactivation of miR-183/96/182 results in decreased corneal nerve
density, branching and number of beaded nerves, and, consistently,
expression of pain receptor, TRPV1, and several key neuropeptides.
4. miR-183/96/182 is highly expressed in peritoneal MΦ. Its
inactivation resulted in significant changes in their cytokine
expression profile.
5. miR-183 and miR-182 are expressed in PMNs at levels lower than
in MΦ. In vitro assays suggested changes of PMN phagocytosis and
bacterial killing capacity in ko mice.
Conclusions: Inactivation of miR-183/96/182 resulted in
significantly reduced disease in PA-induced keratitis. Loss of
miR-183/96/182 leads to changes in corneal innervation and
innate immune responses, as well as neuro-immune interactions,
contributing to decreased corneal disease. Since sensory innervation
and innate immunity also play an important role in other forms of
microbial keratitis, corneal wound healing, diabetic neuropathy,
and dry eye syndrome, miR-183/96/182 ko mice may provide a
new opportunity to study the roles of miR-183/96/182 in various
physiological and pathological processes.
Commercial Relationships: Shunbin Xu, None; Linda D. Hazlett,
None; Elizabeth A. Berger, None; Susmit Suvas, None; Cui Li,
None; Chithra Muraleedharan, None; Ronald Barrett, None;
Sharon A. McClellan, None; Thomas Carion, None; Daniel
Montenegro, None
Support: The Research to Prevent Blindness Foundation (to
Department of Ophthalmology, KEI), NIH Grants R01EY002986,
R01EY016058 and P30EY004068 (to LDH)
Program Number: 944
Corneal burn in one eye disrupts conjunctival immunological
tolerance of the fellow eye
Jeremias G. Galletti, Irene Keitelman, Mauricio Guzmán, Florencia
G. Sabbione, Mirta N. Giordano, Analía Trevani. Immunology,
Institute of Experimental Medicine, Buenos Aires, Argentina.
Purpose: Both conjunctivas are regarded as immunologically
independent from each other, but there is anecdotal evidence that one
ocular surface might condition its fellow. The purpose of this work
was to evaluate the effect that a corneal lesion on one eye exerts
over the conjunctival immune response of the opposite eye and the
potential mechanisms involved.
Methods: An unilateral controlled chemical burn with NaOH was
induced in the cornea of 8-week-old female Balb/c mice (day 1, 3
animals/group, n=4), which were then instilled ovalbumin (OVA)
daily (days 2-5) either in the same or the fellow eye. Mice were then
immunized with OVA in adjuvant (day 8) and their T cell responses
were measured by delayed-type hypersensitivity (DTH) assays in
the footpads (day 15). Some mice underwent unilateral superior
cervical sympathectomy one week before corneal burn. To assess
lymphatic drainage pathways, fluorescent OVA was injected in the
subconjunctival space of one eye and 2 hours later each cervical
lymph node was separately harvested.
Results: OVA instillation in one eye of uninjured mice blunted the
DTH response (51±10%, p<0.05), while instillation after corneal
burn did not reduce it when done either in the injured (86±7%, ns)
or in the fellow eye (83±6%, ns). Regarding lymphatic drainage,
fluorescent OVA was detected in homolateral (p<0.001) but not in
contralateral submandibular and preauricular lymph nodes. However,
T cell activation was observed in both homo- (+42% CD69+ and
+22% CD25+ cells, p<0.05) and contralateral (+31% CD69+ and
+23% CD25+ cells, p<0.05) lymph nodes 48 h after corneal injury.
Unilateral sympathectomy did not prevent conditioning of the
conjunctival response by the contralateral corneal burn, but instead
enhanced the DTH response after ocular OVA instillation on the
operated side (p<0.05).
Conclusions: A unilateral corneal lesion is able to tip the immune
balance of the contralateral eye despite the absence of direct chemical
damage or crossed lymphatic drainage. Nevertheless, T cell activation
in the opposite lymph nodes shows that the local immune response
is indeed affected. Sympathetic nerve terminals apparently exert an
inhibitory immune influence on the conjunctiva but are not involved
in eye-to-eye immune crosstalk. These results altogether suggest that
both ocular surfaces are immunologically interdependent.
Commercial Relationships: Jeremias G. Galletti, None; Irene
Keitelman, None; Mauricio Guzmán, None; Florencia G.
Sabbione, None; Mirta N. Giordano, None; Analía Trevani, None
Support: PICT 2013-1436, FONCyT, Argentina
214 Myeloid cells in ocular health and disease - Minisymposium
Monday, May 04, 2015 8:30 AM–10:15 AM
601/603 Minisymposium
Program Number: 1341
Immune Suppressive Myeloid Cells
Suzanne Ostrand-Rosenberg. Biological Sciences, Univ. Maryland
Baltimore County, Baltimore, MD.
Presentation Description: Immune suppressive cells of myeloid
origin accumulate in individuals with a variety of conditions.
These conditions typically involve inflammation, and range from
an inflammatory tumor microenvironment to infection, stress,
and aging. The predominant cell types are myeloid-derived
suppressor cells (MDSC) and type 2 or so-called tumor-associated
macrophages (TAMs). The cells are present at low levels in healthy
and young individuals; however, when elevated, MDSC and TAMs
are profoundly immune suppressive cells that neutralize natural
immunity and impede strategies for activating the immune system
to cancer or infectious diseases. A variety of pro-inflammatory
mediators acting through multiple receptors drive the accumulation of
suppressive cells from myeloid progenitor cells. MDSC are present
in most cancer patients and TAMs infiltrate many solid tumors.
MDSC facilitate tumor progression by inhibiting both the innate
and adaptive immune systems, and also by non-immune effects that
promote angiogenesis and tumor invasion and metastasis. This talk
will review the inflammatory conditions that drive the accumulation
of immune suppressive MDSC and macrophages, describe the
mechanisms used by these cells to suppress anti-tumor immunity,
discuss how MDSC both support and dampen inflammation in the
tumor microenvironment, and review some to the strategies that have
been developed to neutralize undesirable effects of hyper-activated
myeloid cells.
Commercial Relationships: Suzanne Ostrand-Rosenberg,
MedImmune, Inc. (F), Nora Therapeutics, Inc. (F)
Support: NIH RO1 CA84232, NIH RO1 GM021248
Influence of macrophages on intraocular tumor growth
Kyle C. McKenna. 1Univ. Pittsburgh, Pittsburgh, PA; 2Biology,
Franciscan University of Steubenville, Steubenville, OH.
Presentation Description: Experimentation in a mouse model of
intraocular tumor development which indicates that tumor associated
macrophages contribute to both growth and elimination of intraocular
tumors is described and related to our current understanding of the
immunobiology of uveal melanoma.
Commercial Relationships: Kyle C. McKenna, None
Support: NEI EY018355
Influence of Myeloid Cells in Corneal Wound Healing
Lars Bellner. Pharmacology, New York Medical College, Valhalla,
NY.
Presentation Description: The heme oxygenase (HO) system has
been implicated in the resolution of inflammation. HO is the ratelimiting enzyme in heme catabolism. It cleaves heme to biliverdin,
carbon monoxide (CO), and iron; biliverdin is subsequently
converted by biliverdin reductase to bilirubin.
Our previous studies have shown that a deficiency in HO activity, as
in the HO-2 null (KO) mice, exacerbates ocular surface inflammation
allowing an acute inflammation to become chronic with the hallmarks
of neovascularization, ulceration, perforation and accumulation of
apoptotic cells.
This presentation will give a description of the impact of heme
oxygenase 2 protein deficiency on the function of infiltrating
neutrophils and macrophages in corneal wound healing from its
initial stage, the inflammatory response, and through the resolution
and repair stages.
Commercial Relationships: Lars Bellner, None
Support: NIH Grant EY06513
Role of cholesterol metabolism in retinal macrophage function
Rajendra S. Apte. Washington Univ., St. Louis, MO.
Presentation Description: Inability of macrophages to effectively
metabolize cholesterol is a cardinal event in the aging of the immune
system. It is also critical to the pathobiology of diverse diseases of
aging including atherosclerosis and macular degeneration (AMD).
Although genetic studies have been successful in associating innate
immunity and cholesterol metabolism with AMD, the link between
dysregulated cholesterol homeostasis in macrophages and initiation
of AMD is not well understood. Current concepts and data on the
regulation of genes involved in macrophage cholesterol homeostasis
during aging and disease will be presented.
Commercial Relationships: Rajendra S. Apte, Metro Midwest (I),
Washington University (P)
Support: This work was supported by NIH Grant K08EY016139
(RSA), NIH Grant R01EY019287 (RSA), NIH Vision Core Grant
P30EY02687, U.S. Civilian Research and Development Foundation
(RSA, IC), Carl Marshall Reeves and Mildred Almen Reeves
Foundation Inc. Award (RSA), Research to Prevent Blindness Inc.
Career Development Award and RPB Physician Scientist Award
(RSA), International Retina Research Foundation (RSA), American
Health Assistance Foundation (RSA), Lacy Foundation Research
Award (AS), Thome Foundation (RSA) and a Research to Prevent
Blindness Inc. Unrestricted Grant to Washington University.
Optic Nerve Regeneration and Glaucoma: the Yin and Yang of
Intraocular Inflammation
Larry Benowitz. 1Depts. Neurosurgery and Ophthalmology, Harvard
Medical School, Boston, MA; 2Neurosurgery; Kirby Neurobiology
Center, Boston Children’s Hospital, Boston, MA.
Presentation Description: In glaucoma, retinal ganglion cells
(RGCs) undergo apoptotic death due to axonal damage in the
vicinity of the lamina cribrosa. Mechanical injury to the optic
nerve similarly results in apoptotic death of RGCs and is widely
used as a model to investigate mechanisms underlying RGC loss in
glaucoma. Surprisingly, induction of a limited inflammatory reaction
in the eye enhances RGCs’ ability to survive optic nerve damage
and to regenerate axons well beyond the injury site1. The effects
of inflammation on regeneration are due primarily to the atypical
growth factor oncomodulin (Ocm). Ocm mRNA and protein increase
markedly in the eye following the induction of an inflammatory
reaction, and the role of Ocm in mediating the pro-regenerative
effects of inflammation have been established by gain- and loss-offunction studies2-5. Combining intraocular inflammation with cAMP
elevation and pten gene deletion enables some RGCs to regenerate
axons the full length of the optic nerve, reinnervate central target
areas, and restore simple visual responses6. Although activated
neutrophils and macrophages both express high levels of Ocm,
immunodepletion studies show that neutrophils represent the more
biologically relevant source5. In animal models of glaucoma, in
contrast, inflammation has a strongly negative effect. Elevation of
intraocular pressure, either by episcleral vein cauterization or lasermediated angle closure, leads to a marked increase in the chemokine
TNF-a in the eye, activation of microglia at the optic nerve head, and
delayed loss of RGCs. Deleting the gene for TNF-a or the TNFR2
receptor, immune-depletion of TNF-a, or systemic injections of
Etanercept, a soluble decoy receptor for TNF-a, greatly increase RGC
survival in animal models of glaucoma despite persistent elevation of
intraocular pressure7,8. Thus, inflammation can have either a strongly
deleterious or a strongly supportive effect on RGCs depending on the
injury model and the type of inflammatory cells that is activated.
1
Yin et al., J Neurosci 23, 2284 (2003); 2Yin et al., Nat Neurosci 9,
843 (2006); 3Yin et al., Proc Natl Acad Sci U S A 106, 19587 (2009);
4
Kurimoto et al., J Neurosci 30, 15654 (2010); 5Kurimoto et al., J
Neurosci 33, 14816 (2013); 6de Lima et al., Proc Natl Acad Sci U S
A 109, 9149 (2012); 7Nakazawa et al., J Neurosci 26, 12633 (2006);
8
Roh et al., PLoS One 7, e40065 (2012).
Commercial Relationships: Larry Benowitz, Boston Children’s
Hospital (R)
Support: NIH/NEI EY05690; Dr. Miriam and Sheldon G. Adelson
Medical Research Foundation
238 Human uveitis: Posterior
Monday, May 04, 2015 11:00 AM–12:45 PM
A Darwinian view of Behcet’s Disease
Graham R. Wallace1, Alice M. Roberts2, Rhodri L. Smith3, Robert
J. Moots3. 1Academic Ophthalmology, University of Birmingham,
Birmingham, United Kingdom; 2Biosciences, University of
Birmingham, Birmingham, United Kingdom; 3Rheumatology,
University of Liverpool, Liverpool, United Kingdom.
Purpose: Purpose - Behçet’s Disease (BD) is a multi-system
inflammatory disorder of unknown aetiology, characterised by oral
and genital ulceration, with other complications including eye, skin,
joint and CNS lesions that may cause blindness or stroke. The MHC
gene, HLA-B*51, is the strongest genetic association across the
geographic spread of disease. Many genetic studies including GWAS
have identified gene loci and we propose that BD is based on several
mutants with small effect, which together lead to disease and which
have accumulated over time. Interestingly, these mutations vary in
different parts of the world. Recent studies have catalogued archaic
human genomes, and show admixture with early humans, which
can impact on human disease. We sought to address the role of such
introgression with known genetic associations in Behcet’s Disease.
Methods: Methods – analysis of genome-wide association,
imputation and candidate gene studies, was used to determine links
between early hominid DNA, geographical differences in gene
association and pathway analysis to determine how these mutations
are linked.
Results: Results - HLA molecules associated with BD have be shown
to be derived from archaic genomes. Maintenance of these alleles
into modern day strongly supports a protective effect. Analysis of five
biological processes as examples to show that mutations mutations
specific to different geographical areas affect have a similar
functional outcome.
Conclusions: Conclusion – admixture or archaic and early human
genomes has led to particular genes that are now associated with
disease being introgressed into modern humans as protection against
infection. However, further mutations in different genes, that affect
the same cellular process have led to the association with disease.
This knowledge might allow us to target pathways rather than
individual gene products, ultimately leading to better diagnosis and
control of disease.
Commercial Relationships: Graham R. Wallace, None; Alice M.
Roberts, None; Rhodri L. Smith, None; Robert J. Moots, None
Analysis of Th cell-related cytokine production in Behçet’s
disease patients with uveitis before and after treatment with
infliximab
Masaru Takeuchi, Atsushi Tanaka, Kouzo Harimoto, Izumi Mine,
Tomohito Sato, Yoko Karasawa, Takayuki Kanda. Ophthalmology,
National Defense Medical College, Tokorozawa, Japan.
Purpose: Infliximab (Inflix), is an anti- TNFα monoclonal antibody,
resolves effectively ocular inflammation in Behçet’s disease (BD)
patients by suppressing inflammatory process via TNFα. It is
reported that treatment with Inflix inhibits Th-cell differentiation into
Th17 cells and promotes regulatory T cell production in BD patients
with uveitis.
In this study, we examined quantitative changes of cytokines
produced by various Th cells specific for ocular tissue antigens before
and after treatment with Inflix in BD patients with uveitis.
Methods: Eight BD patients with uveitis during treatment with
infliximab were recruited in this study. Peripheral blood mononuclear
cells (PBMCs) were obtained before infusion of infliximab and at 1
week after the infusion. PBMCs were also obtained from 10 healthy
subjects as the control. Each sample was cultured for 48 hrs with
IRBP, and supernatants were collected at the end of culture. IL-1β,
IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-25, IL-31, IL-33,
IFN-γ, sCD40L, and TNFα in the supernatants were measured by
Bio-Plex kit®. The study was approved by the institutional review
board of National Defense Medical College.
Results: All measured cytokines except for IL-25 and IL-33 were
higher in BD patients before Inflix infusion compared with BD
patients after Inflix infusion or healthy subjects. The levels of
IL-4, IL-10, and sCD40L in BD patients after Inflix infusion were
comparable with those of healthy subjects, however IL-1β, IL-6, IL17A, IL-17F, IL-21, IL-22, IL-31, IFN-γ, and TNFα were still higher
in BD patients than in healthy subjects.
Conclusions: It was indicated that cytokines produced by Th
cells stimulated with IRBP in BD patients were suppressed by
Inflix treatment, which was not only cytokines promoting ocular
inflammation but also the inhibitory cytokines, but that the levels of
most cytokines did not reach to those of healthy subjects.
Commercial Relationships: Masaru Takeuchi, None; Atsushi
Tanaka, None; Kouzo Harimoto, None; Izumi Mine, None;
Tomohito Sato, None; Yoko Karasawa, None; Takayuki Kanda,
None
Support: Grant-in-Aid 24592689 for Scientific Research from the
Japan Society for the Promotion of Science
develop a novel in-silico pipeline of orthogonal analyses translatable
to other diseases.
Methods: Gene expression was assayed in the OHSU Gene
Microarray Shared Resource using Affymetrix Hu U133 Plus 2.0
Microarray chips on peripheral whole blood samples from active
uveitis compared with inactive uveitis for separate etiologies and
as a combined class of heterogeneous aetiologies. We studied 35
subjects with active noninfectious (NIU) uveitis, 28 inactive NIU
in Ankylosing Spondylitis (AS), Sarcoidosis, Idiopathic Uveitis,
Behcets Disease and 25 healthy controls. We developed an in-silico
pipeline including frozen Robust Multi-array Average normalisation,
Linear Model for Microarray in ‘R’, differential gene expression
identification. False discovery rate <5% defined significance.
Methods for downstream analysis included Gene Set enrichment
Analysis, Ingenuity Pathway Analysis modules, Panther and textmining literature search.
Results: TOLL, RIG-1 and NOD, IL21 and Integrin pathways were
enriched in subjects with active uveitis. Functional distinctions
between individual uveitis aetiologies are present: IL4 pathway is
downregulated in Idiopathic uveitis; IL12, p53, IL17/23 pathways
are upregulated and wnt-catenin, integrin pathways downregulated.
The IL2 pathway is downregulated in AS Uveitis. We identified 13
(3.5 %) DEGs concordant between active uveitis disease classes, e.g.,
secretory leukocyte peptidase inhibitor & peroxisomal proliferatoractivated receptor A (PPAR) interacting complex 285.
Conclusions: Combining multiple analysis modalities enables
discovery of genes and pathways shared among heterogeneous
diseases & potentially specific to a single clinical features in these
diseases. We were able to identify both a common mRNA profile
associated with active uveitis in various diseases in peripheral blood
and show that each uveitis phenotype is associated with functional
relationships distinct from systemic disease and other uveitides.
Identification of a gene expression profile specific to non
infectious uveitis using high throughput microarray data and a
novel pipeline of in-silico methods
Srilakshmi Sharma1, 2, Sarah Wheelan3, Luigi Marchionni3, Christina
A. Harrington4, Dongseok Choi5, 4, Stephen R. Planck5, 6, James T.
Rosenbaum5, 6. 1Ophthalmology, Oxford Eye Hospital, Oxford, United
Kingdom; 2Moorfields Eye Hospital, London, United Kingdom;
3
Johns Hopkins University, Baltimore, MD; 4Oregon Health &
Sciences University, Portland, OR; 5Casey Eye Institute, Portland,
OR; 6Legacy Devers Eye Institute, Legacy Devers Eye Institute, OR.
Purpose: Microarray technology presents opportunities to study
etiological or genetic commonalities within ocular manifestations of
heterogenous diseases. Our aims are to characterise gene expression
in active uveitis of distinct etiologies and as single heterotype and to
Heatmap following Single Sample Gene Set Enrichment Analysis
showing distinctions in pathways between Uveitis and Inactive
Uveitis
Commercial Relationships: Srilakshmi Sharma, None;
Sarah Wheelan, None; Luigi Marchionni, None; Christina A.
Harrington, None; Dongseok Choi, None; Stephen R. Planck,
None; James T. Rosenbaum, None
Support: NIH/NEI EYO15858
Presentation Time: 11:45 AM–12:00 PM
Development of Choroidal Depigmentation in Birdshot
Retinochoroidopathy Patients Treated with Fluocinolone
Acetonide Implants
Blake Isernhagen1, Heena Patel1, 6, Sara Haug2, Emmett
Cunningham3, Francesco Pichi5, Careen Y. Lowder4, Sunil K.
Srivastava4, Janet L. Davis1. 1Ophthalmology, Bascom Palmer Eye
Institute, Miami Beach, FL; 2West Coast Retina, San Francisco, CA;
3
California Pacific Medical Center, San Francisco, CA; 4Cleveland
Clinic, Cleveland, OH; 5San Giuseppe Hospital, University Eye
Clinic, Milan, Italy; 6Retina Institute, Los Angeles, CA.
Purpose: To report progressive posterior choroidal
depigmentation during long-term follow-up in patients with
birdshot chorioretinopathy (BCR) undergoing implantation with a
fluocinolone acetonide containing device despite otherwise excellent
control of inflammation.
Methods: Retrospective comparative case series of BCR patients
from one university and two private uveitis clinics. Evaluation of
baseline vs. follow-up fundus color photographs after a minimum of
30 months were compared in patients treated with the fluocinolone
implant or systemic immunosuppression. Available fluorescein
angiography, indocyanine green, autofluorescence, and optical
coherence tomography were also compared. The primary outcome
measure was an “unequivocal” change in background choroidal
depigmentation on color fundus photography graded by two masked
observers, compared by chi-square. Mean group follow-up time was
compared by t-test.
Results: Group 1 included 10 patients (20 eyes) treated with
fluocinolone implants and Group 2 included 10 patients (20 eyes)
treated with immunosuppression during the study period. Both
groups had similar demographics for age, sex, and initial and
follow-up visual acuity. Follow-up for Group 1 was median 75
months and mean 69 months. Group 2 was median 57 months and
mean 64 months (P=0.29 by t-test). Ocular inflammation in both
groups improved based on clinical information and imaging studies.
Evaluable photographs demonstrated depigmentation in 10 eyes of 6
patients in Group 1 and 4 eyes of 4 patients in Group 2 (chi-square
3.96, P< 0.05).
Conclusions: Patients treated with the fluocinolone implant had a
higher frequency of choroidal depigmentation unassociated with RPE
atrophy despite good clinical control of inflammation and preserved
vision. The results may indicate a specific effect of corticosteroids on
choroidal pigment or a differential effect of intravitreal vs. systemic
treatment in BCR, a panuveitis that involves vitreous, retina, RPE,
and choroid. Additional follow-up and standardized photographs are
needed to confirm this finding and determine if similar changes occur
in other types of uveitis.
Fluocinolone patient: initial photo on left and follow-up photo on
right demonstrating progressive choroidal depigmentation.
Patient treated with immunosupression: initial photo on left
and follow-up photo on right demonstrating stable choroidal
pigmentation.
Commercial Relationships: Blake Isernhagen, None; Heena Patel,
None; Sara Haug, None; Emmett Cunningham, None; Francesco
Pichi, None; Careen Y. Lowder, Xoma (C); Sunil K. Srivastava,
None; Janet L. Davis, None
Risk Factors for Poor Visual Outcome in Birdshot
Retinochoroidopathy Patients
Sukhum Silpa-archa1, 2, Joan J. Lee1, Sutasinee Boonsopon1, Pranav
Patel1, Charles S. Foster1, 3. 1Massachusetts Eye Research & Surgery
Institution, North Quincy, MA; 2Department of Ophthalmology,
Faculty of Medicine, Rajavithi Hospital, College of Medicine,
Rangsit University, Bangkok, Thailand; 3Harvard Medical School,
Boston, MA.
Purpose: Though recognized as chronic disease with poor visual
prognosis, no study identifies risk factors of poor visual outcome
in birdshot retinochoroidopathy (BSRC) patients. We conducted a
retrospective, comparative clinical study to explore the associated
risk factors to predict poor visual outcome in BSRC patients.
Methods: We performed chart review of BSRC patients who were
evaluated at Massachusetts Eye Research & Surgery Institution
(MERSI) from 2005 to 2014 with a follow-up period at least 12
months. Ninety-eight patients (196 eyes) were identified. They were
categorized into 2 groups; poor vision and good vision. The definition
of poor visual outcome was defined as less than -6 mean deviation
on Swedish interactive threshold algorithm (SITA) short-wavelength
automated perimetry (SWAP) and abnormality (amplitude or implicit
time) in 30 Hz flickering electro-retinogram. Twenty-eight factors
were statistically analyzed by chi-square test and logistic regression
model.
Results: Of all the 98 patients, 50 patients (51%) had poor visual
outcome. There were no differences between the 2 groups in terms
of sex, age of diagnosis, race, family history of BSRC, HLA-A29
positivity. Univariate analysis revealed significant associations
between the poor visual outcomes group and initial visual acuity
more than 0.3 LogMAR, ocular hypertension or glaucoma, cystoid
macular edema, optic atrophy and duration from onset of symptoms
to evaluation at MERSI of longer than 24 months. Multivariate
logistic regression analysis identified only initial visual acuity
more than 0.3 LogMAR, and ocular hypertension or glaucoma as
independent predictors for poor visual outcome with odd ratios 4.49
(95% CI 1.49, 13.52) and 4.51 (95% CI 1.11, 18.31) respectively.
Conclusions: Poorer initial visual acuity and episode of ocular
hypertension or glaucoma are the significant predictors of poor
visual outcome of BSRC patients. This warrants prompt aggressive
treatment and careful surveillance of intraocular pressure.
Commercial Relationships: Sukhum Silpa-archa, None; Joan
J. Lee, None; Sutasinee Boonsopon, None; Pranav Patel, None;
Charles S. Foster, None
Enhanced Depth Imaging Optical Coherence Tomography
(EDI-OCT) in Uveitis: An Intra-Session and Inter-Observer
Reproducibility Study
Jane S. Kim, Laurence Jaworski, Padmini Kaushal, Susan Vitale,
Jared E. Knickelbein, Robert B. Nussenblatt, H Nida Sen. National
Eye Institute, National Institutes of Health, Bethesda, MD.
Purpose: Prior studies have addressed the reproducibility of manual
subfoveal choroidal thickness measurements in healthy eyes, but
none have examined uveitic eyes. This study seeks to determine
intra-session and inter-observer reproducibility of manual subfoveal
choroidal thickness measurements using EDI-OCT in patients with
uveitis and to correlate subfoveal choroidal thickness with anatomical
location and disease activity.
Methods: EDI-OCT images were collected for patients with anterior
(n = 6), intermediate (n = 9), posterior (n = 13), and panuveitis (n =
6). Two separate scans were obtained at a single visit for each patient,
except for three. Manual measurements of subfoveal choroidal
thickness were performed by two masked ophthalmologists using the
manufacturer’s software to place calipers beneath the fovea at the
outer border of the retinal pigment epithelium and the inner scleral
border. These measurements were compared using paired t-tests,
and their relationships were assessed using Pearson’s correlation
coefficients.
Results: Sixty eyes from 34 patients were included in the study. Most
eyes (62%, 37/60) were quiet with 13% (8/60) minimally active and
25% (15/60) active at the time the scans were obtained. Nineteen
patients (56%) were taking systemic corticosteroids and/or steroidsparing immunosuppressive agents. No association was observed
between subfoveal choroidal thickness and anatomical location
(Table 1, P>0.41). Despite an observed trend of greater subfoveal
choroidal thickness with increasing disease activity, the association
between disease activity and choroidal thickness was not statistically
significant (Table 2, P>0.08). Manual EDI-OCT measurements in
uveitic eyes, however, showed substantial agreement within a session
(all r values >0.83, P<0.001) and between observers (all r values
>0.76, P<0.001).
Conclusions: EDI-OCT is a non-invasive imaging method that can
be used to obtain detailed images of the choroid in patients with
uveitis. Although no statistically significant difference in subfoveal
choroidal thickness was found based on anatomical location or
disease activity, a trend of greater subfoveal choroidal thickness with
increasing disease activity was observed. Intra-session and interobserver manual EDI-OCT measurements were highly reproducible
in patients with uveitis.
Commercial Relationships: Jane S. Kim, None; Laurence
Jaworski, None; Padmini Kaushal, None; Susan Vitale, None;
Jared E. Knickelbein, None; Robert B. Nussenblatt, None; H Nida
Sen, None
Support: Grant support: NEI Intramural Research Program. This
research was also made possible through the National Institutes
of Health (NIH) Medical Research Scholars Program, a publicprivate partnership supported jointly by the NIH and generous
contributions to the Foundation for the NIH from Pfizer Inc, The
Doris Duke Charitable Foundation, The Newport Foundation, The
American Association for Dental Research, The Howard Hughes
Medical Institute, and the Colgate-Palmolive Company, as well as
other private donors. For a complete list, please visit the Foundation
website at: http://fnih.org/work/education-training-0/medicalresearch-scholars-program.
Effect of adalimumab on visual functioning (VFQ-25) in
VISUAL-1 trial patients with non-anterior non-infectious uveitis
John D. Sheppard1, 2, Avani Joshi3, Manish Mittal3, Keith Betts5,
Samir R. Tari3, Yanjun Bao3, Andrew D. Dick4. 1Ophthalmology,
Eastern Virginia Medical Sch, Norfolk, VA; 2Virginia Eye
Consultants, Norfolk, VA; 3AbbVie, North Chicago, IL; 4University
of Bristol, Bristol, United Kingdom; 5Analysis Group, Boston, MA.
Purpose: To compare the effects of adalimumab and placebo on the
National Eye Institute Visual Functioning Questionnaire 25 (VFQ25) in subjects requiring high dose corticosteroids for active noninfectious intermediate-, posterior-, or pan-uveitis.
Methods: The VISUAL-1 clinical trial (NCT01148225) was a phase
3, randomized placebo-controlled study. It investigated the efficacy
and safety of adalimumab (80 mg loading dose followed by 40 mg
every other week) as maintenance therapy in subjects with active
non-infectious intermediate-, posterior- or pan-uveitis. The VFQ-25
is a validated measure for assessing the impact of visual impairment
from the patient’s perspective. The VFQ-25 total score is calculated
as the mean of 11 vision-related domains and scores range from 0
(worst vision functioning) to 100 (best vision functioning). The VFQ25 was administered at every scheduled study visit of the 80 Week
trial. As a ranked secondary outcome, the change in VFQ-25 from
best state achieved prior to Week 6 to the final / early termination
visit was compared between adalimumab and placebo using ANOVA.
To investigate the temporal effects of adalimumab and placebo
on VFQ-25 in a robust manner, a longitudinal GEE model (which
incorporated all VFQ-25 measurements) was estimated.
Results: The VISUAL-1 clinical trial enrolled a total of 217 subjects
(110 adalimumab, 107 placebo). The mean VFQ-25 total scores for
adalimumab and placebo are similar through the tapering period
but subsequently diverge and maintain separation through week 80
(Figure 1). The average change in VFQ-25 total score from best state
achieved prior to Week 6 to the final / early termination visit was
-5.50 for placebo and -1.30 for adalimumab. This corresponds to a
statistically significant and clinically meaningful1 increase of 4.20
(95% confidence interval [CI]: 1.02 – 7.38; P = 0.010) associated
with adalimumab relative to placebo. The longitudinal model
estimated a statistically significant treatment effect of adalimumab of
3.07 (95% CI: 2.09 – 4.06; P<0.001).
Conclusions: Treatment with adalimumab is associated with
statistically significant improvements in visual functioning for
subjects with active non-infectious non-anterior uveitis.
Figure 1: Mean VFQ-25 over time
Commercial Relationships: John D. Sheppard, Abbvie (C), Alcon
(C), Aldeyra (C), Allergan (C), Bausch & Lomb (C), EyeGate (C),
EyeGate (I); Avani Joshi, AbbVie (E); Manish Mittal, AbbVie
(E); Keith Betts, Analysis Group (F); Samir R. Tari, AbbVie (E);
Yanjun Bao, AbbVie (E); Andrew D. Dick, Abbvie (C)
243 Viral, fungal, and parasitic infections
Monday, May 04, 2015 11:00 AM–12:45 PM
Physiology/Pharmacology, Retina
Role of atypical chemokine receptor-2 in murine corneal herpes
simplex virus type 1 infection
Tian Yu1, Gerard J Graham2, Lucia Kuffova1, John V. Forrester1,
3 1
. Immunity,Infection and Inflammation, School of Medicine and
Dentistry, University of Aberdeen, Aberdeen, United Kingdom;
2
Institute of Infection, Immunity and Inflammation, College of
Medical, Veterinary and Life Sciences, University of Glasgow,
Glasgow, United Kingdom; 3Ocular Immunology Program, Centre
for Ophthalmology and Visual Science, The University of Western
Australia, Perth, WA, Australia.
Purpose: The atypical chemokine receptor-2 (ACKR2) is known to
scavenge pro-inflammatory CC chemokines and is expressed mainly
on lymphatic endothelial cells, but also on some leukocyte subsets.
As well as a role in clearance of excess inflammatory chemokines and
a contribution to the resolution phase of inflammation, ACKR2 has
been proposed to facilitate the selective expression of homeostatic
chemokines which are essential for the migration of antigenpresenting cells (APC) from peripheral tissues to the secondary
lymphoid organs (SLO). Therefore, the current study was conducted
to investigate this hypothesis and the role of ACKR2 during corneal
herpes simplex virus type 1 (HSV-1) infection.
Methods: C57BL/6 wild type (wt) and ACKR2-/- mice were
infected with topically applied HSV-1 after abrasion of the corneal
epithelium. Herpetic stromal keratitis (HSK) was evaluated clinically
and the degree of corneal opacification was scored. Corneal whole
mounts were stained with CD31 and LYVE-1 for quantification of
neovascularization by confocal microscopy. Cell infiltration into
the cornea, trigeminal ganglion (TG) and eye-draining lymph node
(DLN) were analyzed by flow cytometry.
Results: Wt mice typically developed mild and transient HSK
indicated by increased corneal opacification at d2 to d7 post infection
(p.i.). In contrast, ACKR2-/- mice developed severe HSK of longer
duration (onset d2 p.i., duration 21d) with increased HSK severity
at d7, 14 p.i. compared to wt (p<0.01) and some mice developed
hypopyon. Increased HSK severity in ACKR2-/- mice was followed
by more extensive neovascularization at d14, 21 and 42 p.i. (p<0.05)
compared to wt mice both clinically and immunohistologically
(corneal whole mounts), increased leukocyte infiltration was also
seen at d7 and 14 p.i. in the cornea. Concomitantly, fewer T cells
and myeloid cells were found in the DLN of ACKR2-/- mice at d5 p.i.
(p<0.05). In contrast, increased number of CD11b+ cells were found
in the TG of ACKR2-/- mice at d7 p.i.
Conclusions: Absence of ACKR2 allows development of HSK
which is more severe than in wt mice and characterized by increased
neovascularization and opacification together with increased
leukocytes infiltration in the cornea. We suggest that this may be
due to failed resolution of the HSV-induced inflammatory response
occurring at the tissue level, due to leukocyte crowding and impaired
trafficking in dysfunctional lymphatics.
Commercial Relationships: Tian Yu, None; Gerard J Graham,
None; Lucia Kuffova, None; John V. Forrester, None
The Role of Chlamydia in HLA-B27 Acute Anterior Uveitis and
Seronegative Arthritis
Shahriar Amjadi1, 2, Peter Robertson3, Peter J. McCluskey4, Denis
Wakefield1. 1School of Medical Science, University of New South
Wales, Turramurra, Sydney, NSW, Australia; 2Department of
Ophthalmology, Lyell McEwin Hospital, Adelaide, SA, Australia;
3
South Eastern Area Laboratory Services, Prince of Wales Hospital,
Sydney, NSW, Australia; 4Save Sight Institute, University of Sydney,
Sydney, NSW, Australia.
Purpose: This study aimed to determine the prevalence of positive
serology to Chlamydia in patients with Acute Anterior Uveitis
(AAU) and Seronegative Arthritis (SNA) as cross-reactivity between
self-peptides and bacterial antigens such as Chlamydia may be a
pathogenic mechanism for HLA-B27 disease.
Methods: Serum was obtained from patients with a past history
of AAU and/or SNA (n=188). The patients were further stratified
as follows: All patients with AAU (n=176), all HLA-B27 positive
patients (n=116), HLA-B27 positive patients with AAU (n=102),
HLA-B27 positive patients with SNA (n=49), HLA-B27 positive
patients with both AAU and SNA (n=37), HLA-B27 positive patients
with AAU but no SNA (n=65), HLA-B27 positive patients with
SNA but no AAU (n=12) and HLA-B27-negative patients (all AAU)
(n=72). Healthy age and sex-matched HLA-B27 negative controls
were recruited (n=13) as well as controls from MEDAC GmbH
(Hamburg, Germany) (n=416). Serology was undertaken using a
commercially available recombinant ELISA kit that detects IgG, IgM
and IgA directed against a Chlamydia-specific lipopolysaccharide
(MEDAC GmbH). Statistical analysis was performed using a Fisher
exact test.
Results: The prevalence of IgG positivity to Chlamydia in patients
who were HLA-B27 positive with both AAU and SNA (48.65%) was
significantly higher compared to healthy controls (15.38%) (p<0.05).
In comparison to the healthy controls recruited by MEDAC, the
following groups had significantly higher proportions of positive
IgA and IgM titers compared to controls from MEDAC (13.0%,
3.0%, respectively): all HLA-B27 positive patients (35.1%, 11.7%),
HLAB27 positive patients with AAU (32.4%, 10.8%), HLA-B27
positive patients with SNA (32.7%, 10.2%), HLA-B27 positive
patients with both AAU and SNA (35.1%, 10.8%) (p<0.05).
Conclusions: Patients with HLA-B27 AAU and SNA disease have
evidence of exposure to Chlamydia. HLA-B27 patients with both
AAU and SNA have a higher rate of seropositivity compared to
controls.
Commercial Relationships: Shahriar Amjadi, None; Peter
Robertson, None; Peter J. McCluskey, None; Denis Wakefield,
None
Vitamin D Down-regulates the Pro-inflammatory Cytokine
Response to Aspergillus Fumigatus through PRRs in Corneal
Epithelium
Guiqiu Zhao, Jing Lin, Qian Wang, Lin Cong. Ophthalmology,
Affiliated Hosp of Qingdao University, Qingdao, China.
Purpose: To observe the expression of vitamin D receptor (VDR) in
human corneal epithelial cells. Meanwhile, we wanted to discover
the role of 1,25(OH)2D3 when challenged with Aspergillus fumigatus
(AF).
Methods: Immunohistochemistry was taken to examine the VDR
in corneal epithelium. PCR was performed to observe the mRNA
change of VDR when immortalized human corneal epithelial
cells (HCEC) were challenged with AF antigen. When treated
with 1,25(OH)2D3, the TLR2, TLR4, Dectin-1 and cathelicidin
antimicrobial peptide (CAMP) expression were detected by PCR and
western blot. The expression of pro-inflammatory cytokines were
detected at both mRNA and protein levels.
Results: VDR was expressed in human corneal epithelium. The
VDR mRNA expression increased when stimulated with AF antigen,
and the TLR2 monoclonal antibody could inhibit the expression
specifically (P<0.01). When pretreated HCEC with 1,25(OH)2D3
before AF stimulation, the TLR2, TLR4 and Dectin-1 expression
were decreased apparently (P<0.01) while the expression of CAMP
was risen (P<0.01). The expression of pro-inflammatory cytokines
were down-regulated by 1,25(OH)2D3.
Conclusions: VDR exists in human corneal epithelium and the
expression can be increased via TLR2/1-VDR pathway when
stimulated with AF antigen. 1,25(OH)2D3 can inhibit the expression
of TLR2, TLR4 and Dectin-1 while increasing CAMP production.
The effects can reduce the damage caused by pro-inflammatory
cytokines and enhance the antibacterial effects by CAMP.
Commercial Relationships: Guiqiu Zhao, None; Jing Lin, None;
Qian Wang, None; Lin Cong, None
Support: National Natural Science Foundation of China (81170825)
Dexamethasone Prevents the Loss of Corneal Nerves in Response
to HSV-1 Infection
Ana J. Chucair-Elliott1, Daniel J. Carr1, 2. 1Ophthalmology, Univ
of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Microbiology
and Immunology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK.
Purpose: Herpes simplex virus type 1 (HSV-1) is the leading
cause of infectious corneal blindness due to chronic episodes
of viral reactivation leading to herpetic stromal keratitis (HSK).
Associated with the immunopathology of HSK is the development of
neurotrophic keratitis, characterized by decreased or absent corneal
sensation, blink reflex and tear secretion as a consequence of damage
to the sensory fibers innervating the cornea. Our previous results
revealed regression of the corneal nerves during the acute phase of
HSV-1 infection. The aim of this study is to determine whether the
loss of nerves upon HSV-1 infection is caused directly by the virus or
indirectly by other means including the local immune response.
Methods: C57BL/6J mice were infected with 1,000 plaque forming
units of HSV-1. At 2 h post infection (pi), mice were topically
applied 0.1% dexamethasone ophthalmic solution (DEX) or artificial
tears as controls onto their corneas (QID). Corneas were harvested
at 2, 4, and 8 days pi and assessed for viral content by plaque
assay and infiltrating myeloid-derived cells and T cells by flow
cytometry. At 8 days pi corneal sensitivity was evaluated in mice
using a Cochet-Bonnet esthesiometer and corneas processed for
immunohistochemistry to analyze corneal nerves (β III tubulin+) via
confocal imaging.
Results: Treatment with DEX significantly preserved innervation in
the cornea and corneal sensitivity at day 8 pi compared to the controltreated group. DEX treatment was also found to reduce macrophage
and T cell populations but increase PMNs in the cornea in the
presence of an increase in infectious virus recovered at the studied
time points pi.
Conclusions: Our data suggest the nerve regression and loss of
corneal sensitivity observed at 8 days pi correlates with infiltrating
monocytes/macrophages in response to HSV-1 infection and is not a
direct effect of viral replication in the cornea.
Commercial Relationships: Ana J. Chucair-Elliott, None; Daniel
J. Carr, None
Support: National Institutes of Health (NIH) grant: R01 EY021238,
Oklahoma Center for Adult Stem Cell Research (OCASCR) grant
through the Oklahoma Tobacco Settlement Endowment Trust, and an
unrestricted grant from Research to Prevent Blindness.
Aspirin-triggered Resolvin D1 reduces the severity of HSVinduced corneal immunopathology
Naveen K. Rajasagi, Barry T. Rouse. Biomedical And Diagnostic
Sciences, University of Tennessee, Knoxville, TN.
Purpose: The purpose of this study is to evaluate the therapeutic
potential of Aspirin-triggered Resolvin D1 (AT-RvD1) for the
treatment of herpes simplex virus (HSV)-induced stromal keratitis
(SK), an immunopathological disease of the eye.
Methods: Balb/c mice were ocularly infected with HSV-1 strain RE.
AT-RvD1 (100 ng per eye; 5ml drop) was topically applied to the
cornea two times daily starting from day 6 until day 12 post infection.
The eyes were examined on different days postinfection (dpi) with a
slit-lamp biomicroscope (Kowa), and the clinical severity of stromal
keratitis and angiogenesis of individually scored mice were recorded.
Mice were sacrificed on day15pi and the infiltration of inflammatory
cells into the corneas and production of pro-inflammatory cytokines,
chemokines were compared with untreated animals using flow
cytometry and quantitative PCR.
Results: Topical administration of AT-RvD1 resulted in a significant
reduction in the severity and incidence of SK as well as the extent
of corneal neovascularization in the treated animals compared to
their untreated counterparts at day15dpi. We observed that treatment
with AT-RvD1 reduced the infiltration of neutrophils and pathogenic
CD4+ T cells into the cornea, along with fewer numbers of CD4+
T cells that could be induced ex vivo to produce IFN-γ, IL-2 and
IL-17. These cells are considered to be the primary orchestrators of
the lesions. Additionally, treatment with AT-RvD1 diminished the
production of pro-inflammatory cytokines and chemokines such as
IL-6 and CXCL1 in the corneas of infected animals.
Conclusions: Taken together, these results show that treatment with
AT-RvD1 significantly reduced the infiltration of leukocytes into
the infected corneas and reduced the severity of SK lesions and
neovascularization. Thus, our results indicate that AT-RvD1 treatment
may represent a useful approach in controlling lesion severity in
virally induced immunopathological diseases.
Commercial Relationships: Naveen K. Rajasagi, None; Barry T.
Rouse, None
Support: NIH Grant R21EY023027
Role of Vasoactive Intestinal Peptide (VIP) in modulating Herpes
Simplex Virus-1 (HSV-1) induced corneal lesion
Andrew D. Jerome1, Kate Israel3, Pushpa Rao1, Subhash Gaddipati1,
Susmit Suvas2, 1. 1Anatomy and Cell Biology, Wayne State University
School of Medicine, Detroit, MI; 2Opthalmology, Wayne State
University School of Medicine, Detroit, MI; 3Microbiology and
Immunology, Tulane University School of Medicine, New Orleans,
LA.
Purpose: Corneal infection with herpes simplex virus -1 (HSV-1)
could lead to the development of a chronic immunoinflammatory
condition called Herpetic Stromal Keratitis (HSK). The purpose of
this study was to examine the role of vasoactive intestinal peptide
(VIP) in reducing the development of severe HSK lesions in the
C57BL/6 mouse model.
Methods: C57BL/6 mice aged 8-12 weeks were used for corneal
HSV-1 infections. Experimental techniques used were flow
cytometry, Enzyme-linked Immunosorbent assay (ELISA) of tissue
lysates, semi-quantitative polymerase chain reaction (PCR) of
corneal tissue, immunofluorescence of frozen corneal sections, and
viral titration of corneal swabs. Systemic and localized treatment
of VIP was given as intra-peritoneal or subconjunctival injections,
respectively. Corneal opacity (scored from 0-5) and angiogenesis
(scored from 0-16) were measured with a hand-held slit lamp.
Results: Our data demonstrates the presence of VIP in both naïve and
HSV-1 infected corneas. In naïve corneas, VIP is largely localized
in the corneal endothelium, whereas in the infected corneas, VIP
staining was noted in the corneal stroma. With VIP receptors, VPAC1
is expressed at higher levels than VPAC2 on both CD45+ and
CD45-ve cell populations. Intra-peritoneal (i.p.) administration of
rVIP (5nm) given from day -1 through day 2 or day 4 post-infection
reduced the influx of innate immune cells in infected corneas
resulting in an increased viral load at early (day 3), but not late
time-point (day 5) post-infection. On the other hand, rVIP treatment
given from day 5 post-infection resulted in the development of
encephalitis in more than 50% of infected mice. Unexpectedly, both
systemic (i.p.) and subconjunctival injection of rVIP did not decrease
the severity of HSK lesions. In fact, VIP treatment resulted in an
increased incidence of eyes with severe HSK lesions when compared
with the PBS treated control group.
Conclusions: Our results concur with the immunosuppressive
property of VIP as its administration during active viral replication
increases the viral load in infected corneas. The ongoing research
is examining the immunosuppressive action of VIP in trigeminal
ganglia to determine the underlying mechanism for the development
of encephalitis and an increased incidence of severe HSK in VIP
treated groups.
Commercial Relationships: Andrew D. Jerome, None; Kate
Israel, None; Pushpa Rao, None; Subhash Gaddipati, None;
Susmit Suvas, None
Support: NH Grant EY020625 and NH Grant EY022417
Disruption of Lymphatic Vessel Genesis in the Cornea Suppresses
the Development of the Adaptive T Cell Immune Response in the
Draining Lymph Node Following HSV-1 Infection
Meghan Carr1, Hem R. Gurung2, Daniel J. Carr1, 2. 1Ophthalmology,
University of Oklahoma Health Science Center, Oklahoma City, OK;
2
Microbiology and Immunology, University of Oklahoma Health
Sciences Center, Oklahoma City, OK.
Purpose: To determine the role of corneal lymphatics in the localized
adaptive immune response to ocular HSV-1 infection.
Methods: VEGF-A floxed C57BL/6 mice were infected with 200
pfu/cornea of HSV-1, either parental (SC16) or Cre-expressing (SC16
ICP0-Cre). At times post infection (pi) mice were exsanguinated and
the cornea was removed and assessed for viral titer and leukocyte
subpopulations by plaque assay and flow cytometry, respectively.
In addition, the draining (mandibular) lymph nodes (MLN) were
investigated for immune, blood endothelial (BEC), and lymphatic
endothelial (LEC) cell content as well as blood lymphatic (LYVE1+) vessel structures by flow cytometry and confocal imaging
respectively.
Results: Previously, we reported VEGF-A floxed mice infected
with Cre-expressing HSV-1 displayed a significant loss in corneal
lymphatic vessel development by day 7 pi through day 30 pi. In the
current study, we report no differences in the viral load or number
of myeloid-derived or NK cell numbers residing in the cornea at day
7 pi. In contrast, there was a significant loss of CD8 T cells and a
trend toward fewer CD4+ T cells in the cornea of the SC16 ICP0Cre infected VEGF-A floxed mice. More pronounced differences
correlative with that found in the cornea were observed in the MLN
including a dramatic loss of HSV-specific CD8+ T cells. By day 14
pi, there was a reduction in T lymphocytes residing in the cornea of
the SC16 ICP0-Cre infected VEGF-A floxed mice consistent with a
loss of T cells but not B cells in the MLN at this time point. Changes
in the dynamics of T lymphocyte expansion in the MLN of the
SC16 ICP0-Cre infected VEGF-A floxed mice was associated with a
significant loss of BEC and LEC numbers as well as lymphatic vessel
structure in the MLN.
Conclusions: These results suggest corneal lymphatic vessels and/
or local VEGF A expression contributes to the development of the
adaptive T cell response to ocular HSV-1 infection.
Commercial Relationships: Meghan Carr, Genentech (F); Hem R.
Gurung, Genentech (F); Daniel J. Carr, Genentech (F)
Support: NIH R01EY021238
Dexamethasone suppresses HSV-1- induced corneal
neovascularization
Hem R. Gurung1, Daniel J. Carr1, 2. 1Microbiology and Immunology,
University of Oklahoma Health Sciences Center, Oklahoma City,
OK; 2Opthalmology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK.
Purpose: Herpes simplex virus type 1 (HSV-1) infection of murine
cornea induces lymphatic vessel genesis (lymphangiogenesis)
through the production of VEGFA by epithelial cells acting through
a VEGFR2- dependent pathway. Lymphangiogenesis progresses
and is maintained well past the resolution of infection (> 30 days
post infection, pi). The present study was undertaken to identify
the contribution of cells and pro-angiogenic factors that promote
lymphangiogenesis past virus clearance.
Methods: C57BL6/J mice were infected with 1,000 plaque forming
units (PFU) of HSV-1 per eye. Ten mg/kg dexamethasone (DEX)
or vehicle was administered by intraperitoneal injection at day 10
pi. Mice were euthanized at day 14 and day 30 pi and the corneas
were removed. Corneal lymphatic vessels (LYVE-1+) and blood
vessels (CD31+) were visualized by confocal microscopy following
immunohistochemical staining. Infiltrating leukocyte populations
were assessed by flow cytometry of corneal single-cell suspensions.
Pro-angiogenic protein levels were determined by bioplex multiplex
array or ELISA.
Results: A single bolus of DEX at day 10 pi resulted in a significant
reduction of blood vessel but not lymphatic vessel development in the
cornea at day 14 pi. When DEX-treated mice were euthanized at day
30 pi, there was a remarkable reduction of both blood and lymphatic
vessel development into the cornea compared to vehicle-treated
control mice. Flow cytometry analysis revealed fewer neutrophils
and CD4+ T cells in the corneas of the animals that received DEX
with no apparent impact on macrophages and CD8+ T cells compared
to vehicle-treated controls at day 14 and day 30 pi. There was a
significant decrease in IL-6 and hepatocyte growth factor (HGF)
levels at day 14 pi in the corneas of animals that received DEX
relative to vehicle-treated controls.
Conclusions: A single bolus of DEX at the time of HSV-1 clearance
completely reverses the incidence of angiogenesis in the cornea
of mice. This process may involve IL-6 and/or HGF as well as
neutrophils and/or CD4+ T cells.
Commercial Relationships: Hem R. Gurung, None; Daniel J.
Carr, None
Support: NIH R01 EY021238
HVEM in corneal epithelial cells modulates the innate immune
response against HSV type 1
Hui Guo, Xinyi Wu, Kunpeng Pang. Ophthalmology, Qilu Hospital of
Shandong University, Jinan, China.
Purpose: Herpes simplex virus (HSV) type 1 infection of the eye
is one of the leading infectious causes of vision impairment. Recent
studies provide evidence for HSV-1 latency in corneal cells, thus
HSV-1 may utilize unique mechanism to
escape from innate immunity and to be latent at the cornea. Infection
of cells with HSV-1 is initiated by
specific interactions of viral envelope glycoprotein D with host cell
surface receptor herpes virus entry mediator (HVEM). The objective
of the present study was to investigate the role of HVEM in the viral
entry and innate immune response to HSV-1 challenge in human
corneal epithelial cells.
Methods: HVEM expression in human corneal epithelial cells was
detected by immunofluorescence and flow cytometry. Viral entry
assays were performed to detect HSV-1 entry into human corneal
epithelial cells.
Then real time PCR and Enzyme Linked Immunosorbent Assay was
performed to detect the levels of cytokines and chemokines in corneal
epithelial cells treated with control or HVEM siRNA in response to
HSV-1 challenge.
Results: Human corneal epithelial cells were positive for HVEM
expression and showed high susceptibility to HSV-1 entry. Silencing
of HVEM did not alter viral entry dramatically. However, levels
of the cytokine IFN-gamma and chemokines MIP-1 alpha, MIP-1
beta were measured to be higher in HVEM siRNA-treated cells than
control siRNA-treated cells after HSV-1 challenge.
Conclusions: Our results suggest that HVEM in human corneal
epithelial cells may act to dampen these responses and may modulate
the innate immune response against HSV-1,apart from allowing the
virus to enter cells. This may provide a novel mechanism
for immune evasion and latency by HSV-1 in corneal epithelial cells.
Commercial Relationships: Hui Guo, None; Xinyi Wu, None;
Kunpeng Pang, None
Therapeutic Immunization with Herpes Simplex Virus Type
1 Glycoprotein D Derived “Asymptomatic” Human CD8+
T-cell Epitopes Protects Against Recurrent Ocular Herpetic in
Latently Infected HLA Transgenic Rabbits: Correlation with low
Frequency of Exhausted PD-1+TIM-3+CD8+ T Cells
Anthony Nesburn, Arif A. Khan, Ruchi Srivastava, Steven Wechsler,
Lbachir BenMohamed. Gavin Herbert Eye Institute, UC Irvine,
Irvine, CA.
Purpose: Most blinding ocular herpetic disease is due to reactivation
of herpes simplex virus type 1 (HSV-1) from latency rather than
to primary acute infection. No herpes simplex vaccine is currently
available for use in humans. Past therapeutic vaccine clinical trials
that used whole HSV glycoprotein D (gD) have failed to prevent
herpetic disease.
Methods: In this study, we used the HLA-A*02:01 transgenic (HLA
Tg) rabbit model of ocular herpes to assess the therapeutic efficacy of
HSV-1 gD epitopes that are mainly recognized by CD8+ T cells from
“naturally” protected HLA-A*02:01 positive, HSV-1 seropositive
healthy asymptomatic (ASYMP) individuals (who have never had
clinical herpes disease). Three ASYMP CD8+ T cell epitopes (gD53-61,
gD70-78 and gD278-286) were linked with a promiscuous CD4+ T-cell
epitope (gD49-82) to create 3 separate pairs of CD4-CD8 peptides,
which were then each covalently coupled to an Ne-palmitoyl-lysine
moiety, a toll like receptor 2 (TLR-2) ligand. This resulted in the
construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected
HLA-Tg rabbits were immunized with a mixture of these 3 ASYMP
lipopeptide vaccines, delivered as eye drops in sterile PBS.
Results: The therapeutic vaccine significantly reduced HSV-1
shedding detected in tears and reduced recurrent ocular herpetic
disease. The protection was associated with: (i) a boost in the number
and function of HLA-restricted HSV-1 gD-specific CD8+ T cells in
draining lymph nodes (DLN), cornea, conjunctiva, and trigeminal
ganglia (TG); and (ii) a decreased number of exhausted HSV-1 gDspecific PD-1+TIM-3+CD8+ T-cells.
Conclusions: The results underscore the potential of an
ASYMPTOMATIC CD8+ T cell epitope-based therapeutic vaccine
strategy against recurrent ocular herpetic disease.
Commercial Relationships: Anthony Nesburn, None; Arif A.
Khan, None; Ruchi Srivastava, None; Steven Wechsler, None;
Lbachir BenMohamed, None
Support: Public Health Service research grants EY019896,
EY14900 and EY024618 from the NIH, The Discovery Center for
Eye Research, Discovery Eye Foundation and a Research to Prevent
Blindness grant.
Plasmacytoid Dendritic Cells Mediate Adaptive Immunity in
Acute Herpes Simplex Virus Keratitis
Victor Sendra1, Arsia Jamali1, Deshea L. Harris1, Pedram Hamrah1,
2 1
. Schepens Eye Research Institute-Mass eye and ear, Boston, MA;
2
Department of Ophthalmology-Harvard Medical School, Cornea
& Refractive Surgery Service-Massachusetts Eye & Ear Infirmary,
Boston, MA.
Purpose: Plasmacytoid dendritic cells (pDCs) represent a highly
functional subset of bone marrow (BM)-derived cells and play a key
role in linking the innate and adaptive immune responses. The aim
of this study is to investigate the role of corneal pDCs in adaptive
immune responses after acute herpes simplex virus (HSV)-1 infection
Methods: Corneal HSV infection was induced by inoculation of
HSV-1 McKrae strain after scarification. Corneal pDCs were depleted
by subconjunctival (s.c.) injection of diphtheria toxin (DT) or by PBS
(sham controls) into BDCA-2-DTR mice 24h before virus infection
and repeated every 2 days. Clinical corneal opacity was scored on a
0-4 scale. Submandibular draining lymph nodes (dLNs) were excised
and quantified for IL-6 and TGF-β1 mRNA or assessed by flow
cytometry for CD45 (pan-leukocyte marker), NK 1.1 (NK cells),
CD11c (conventional dendritic cells [cDCs]), CD19 (B cells), CD3
(T cells), CD8, CD4, CD25 FoxP3 (Treg), IL-17 (Th17) and IFN-γ
(Th1)
Results: We observed a significant increase in corneal opacity
in pDC-depleted corneas at days 3 (3) and 7 (3.4) as compared
to sham controls (1.2 and 1.5 respectively, P<0.001) after HSV
inoculation. Similarly, a significantly increased influx of total T cells
(2.5-fold) and B cells (2.3-fold) on day 3, and 1.6-fold and 2.3-fold
respectively on day 7 (P<0.05) was seen in dLNs of pDC-depleted
corneas, as compared to sham controls. Further, dLNs from pDCdepleted corneas showed increased Th17 cells (1.6-fold and 2.1-fold)
compared to sham and non-infected controls respectively, while a
reduced frequency of Treg cells (0.74-fold) at day 3 as compared to
both controls. In contrast, CD8+ T cells, Th1 cells, cDCs, and NK
cells did not show significant changes in dLNs. Moreover, corneal
pDC-depletion resulted in up-regulation of TGF-β1 (P<0.001) and
IL-6 (P<0.05) mRNA at day 3 in dLNs compared to sham and noninfected controls. Similarly, corneal IFN-α depletion showed an
increased Th17 cells (2.2 fold) at day 7 with a reduced Treg cells at
day 5 (0.7-fold) in dLNs (P<0.01)
Conclusions: Our data showed that corneal pDC-depletion results
in increased frequency of total T cells and B cells, with shift in the
balance of Treg cells toward Th17 cells in local dLNs and downregulation of IL-6 and TGF-β1. Corneal pDCs may regulate the
adaptive immune responses in dLNs through IFN-α in acute corneal
HSV-1 infection
Commercial Relationships: Victor Sendra, None; Arsia Jamali,
None; Deshea L. Harris, None; Pedram Hamrah, None
Support: NIH-R01- EY022695 (PH), NIH K08-EY020575 (PH),
Research to Prevent Blindness Career Development Award (PH),
Falk Medical Research Foundation (PH), MEEI Foundation
Ex vivo quantification and memory phenotyping of HSV-1specific CD8+ T-cells in Herpes Simplex Keratitis
John Curnow1, 2, Lindsay Durant1, Sai Kolli3, Peter McDonnell3,
Saaeha Rauz2, 1, Geraint P. Williams1, 2. 1Centre for Translational
Inflammation Research, University of Birmingham, Birmingham,
United Kingdom; 2Academic Unit of Ophthalmology, University
of Birmingham, Birmingham, United Kingdom; 3Queen Elizabeth
Hospital Birmingham, Birmingham, United Kingdom.
Purpose: Demonstration of human Herpes Simplex Virus (HSV)
recognition by CD8+ T cells is challenging. They have been reported
using MHC class I-peptide tetramers, but largely following expansion
in vitro. We present a pilot study to quantify ex vivo the CD8+ T cell
response to reported immunodominant HSV-1 peptides in recurrent
herpes simplex keratitis (HSK) and healthy controls (HC).
Methods: 20 patients with HSK (median age 66 [range 35-90]) and
20 age-matched (58 [36-80]) HC were enrolled following ethical
approval. Clinical phenotyping was undertaken together with
peripheral blood/serum samples for HLA-A*0201 typing, HSV-1
sero-status and mononuclear cell isolation. CD8+ T cell memory
status was demonstrated with antibodies specific for CD45RA
and CCR7, and HSV-1 viral epitope recognition using MHC class
I pentamers (HLA-A*02:01; gD53–61, gD70 –78, gD278 –286,
gB183–191, gB441–449, gB561–569 and gB342–350). Samples
were analysed by flow cytometry.
Results: The median duration of symptoms was 21 years [173] with 8/20 HSK patients requiring long-term oral Acyclovir
prophylaxis (19 [12-36] months duration). The time since last
relapse was significantly shorter in the prophylaxis group (p<0.05).
There were no significant differences in the total CD8+ naïve and
memory compartment frequencies between the HSK and HC groups.
We were able to detect gD70 –78-specific memory CD8+ T cells
only in HLA-A*02:01+ and HSV-1 seropositive individuals, with
frequencies of 0.05% (0-1.74%) and 0.085% (0-6.39%) within the
CD8+ cells, for HSK and HC respectively (p=0.82). The HSV-1specific CD8+ expansions were predominantly of an effector memory
(CD45RA-CCR7-) or effector memory RA+ (CD45RA+CCR7+)
phenotype.
Conclusions: In this cross-sectional study of patients we have
detected ex vivo HLA-A*02:01-restricted CD8+ T cells specific
for gD70 –78 of HSV-1. These responses were only present in
HLA-A*02:01+ HSV-1 seropositive individuals, and were found
in both HC and those with HSK. These investigations suggest that
further characterisation in a larger cohort is warranted, to establish
any differences in epitope recognition during HSK.
Commercial Relationships: John Curnow, None; Lindsay Durant,
None; Sai Kolli, None; Peter McDonnell, None; Saaeha Rauz,
None; Geraint P. Williams, None
Support: Fight for Sight New Lecturers’ Award (1481/1482)
HLA-A02:01-Restricted Epitopes Identified from the Herpes
Simplex Virus Tegument Protein VP11/12 Preferentially Recall
Polyfunctional Effector Memory CD8+ T Cells from Seropositive
Asymptomatic Individuals and Protect “Humanized”
HLA-A*02:01 Transgenic Mice Against Ocular Herpes
Ruchi Srivastava, Arif A. Khan, Doran Spencer, Sumit Garg, Anthony
B. Nesburn, Steven Wechsler, Lbachir BenMohamed. Laboratory of
Cellular and Molecular Immunology, Gavin Herbert Eye Institute,
University Of California Irvine, Irvine, CA.
Purpose: The Herpes Simplex Virus type 1 virion tegument
phosphoprotein 11/12 (HSV-1 VP11/12) is a major antigen targeted
by CD8+ T cells from HSV-seropositive individuals. However,
whether and which VP11/12-epitope-specific CD8+ T cells play a role
in the “natural” protection seen in seropositive healthy asymptomatic
(ASYMP) individuals (who have never had clinical herpes disease)
remain to be determined.
Methods: In this study, we used multiple prediction computerassisted algorithms to identify 10 potential HLA-A*02:01-restricted
CD8+ T cell epitopes from the 718 amino acids sequence of VP11/12.
Results: Three out of ten epitopes exhibited high to moderate binding
affinity to HLA-A*02:01 molecules. In ten sequentially studied
HLA-A*02:01 positive and HSV-1-seropositive ASYMP individuals,
the most frequent, robust and polyfunctional effector CD8+ T-cell
responses, as assessed by a combination of tetramer frequency,
granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation,
IFN-g and multiplex cytokines assays, were predominantly directed
against three epitopes: VP11/1266-74, VP11/12220-228 and VP11/12702-710.
Interestingly, ASYMP individuals had significantly higher proportion
of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+
effector memory T cells (TEM) specific to the three epitopes,
compared to symptomatic (SYMP) individuals (with a history of
numerous episodes of recurrent ocular herpetic disease). Moreover,
immunization of HLA-A*02:01 transgenic mice with the three
ASYMP CD8+ T cell epitopes induced robust and polyfunctional
epitope-specific CD8+ TEM cells, in both cornea and trigeminal
ganglia (TG), that were associated with a strong protective immunity
against ocular herpes infection and disease.
Conclusions: Our findings outline phenotypic and functional features
of protective HSV-specific CD8+ T cells that should guide the
development of an effective T-cell-based herpes vaccine.
Commercial Relationships: Ruchi Srivastava, None; Arif
A. Khan, None; Doran Spencer, None; Sumit Garg, None;
Anthony B. Nesburn, None; Steven Wechsler, None; Lbachir
BenMohamed, None
Support: EY14900, EY019896, EY024618
complications than controls (12.9% and 10%, respectively), although
the association was not significant (p=0.67). Cases had an increased
prevalence of atopic disease (28.6% and 21.7%, respectively),
although significance was not established (p=0.27). Univariate
analysis identified atopic dermatitis (OR 4.2, 95% CI: 1.3 to 14.1)
and HIV (OR 3.2, 95% CI: 1.1 to 9.3) as significant risk factors for
the presence of HSV eye disease. Using multivariate analyses to
control for age and gender, atopic dermatitis was no longer significant
(OR 3.0, 95% CI: 0.85 to 10.4), while HIV remained significant (OR
3.5, 95% CI: 1.2 to 10.5). Among the HIV-positive group of HSV
eye disease patients 5 (n=6, 83.3%) were on HAART therapy and 6
(100%) presented with a form of keratitis or retinitis.
Conclusions: The multivariate analysis of potential risk factors
provides evidence for an increased risk of active HSV eye disease
for individuals with HIV when controlling for age and gender. To our
knowledge, this study represents the first time strength of association
has been calculated for HIV and HSV eye disease.
A Case-Control Study of Herpes Simplex Eye Disease: Bronx
Epidemiology of HIV Eye Studies (BEHIVES)
Ethan K. Sobol, Robert Fargione, Marianna Atiya, Jose Diaz,
Jonathan Powell, David C. Gritz. Albert Einstein College of
Medicine, Bronx, NY.
Purpose: To determine whether HIV/AIDS, diabetes mellitus, and
atopic disease are associated with active herpes simplex virus (HSV)
eye disease.
Methods: This retrospective case-control study utilized Montefiore’s
Clinical Looking Glass software to capture cases and controls from
the entire hospital system from June 1, 2010 through May 31, 2014.
Case inclusion criteria were the clinical diagnosis of HSV eye
disease and residency in the Bronx, NY. A hospital-based control
group was randomly chosen using a 4:1 ratio of controls to cases.
Medical records were reviewed to confirm inclusion criteria and
gather demographic and clinical data. HIV/AIDs, diabetes mellitus,
and atopic disease diagnosis (including asthma and atopic dermatitis)
were noted for the study participants. Odds ratio (OR) estimates and
95% confidence intervals (95% CI) were calculated for potential risk
factors.
Results: HSV eye disease was confirmed in 70 patients, who were
compared to 280 controls. Patients with ocular HSV had a greater
prevalence of HIV/AIDS compared to controls (8.6 and 2.9%,
respectively). Cases had a higher prevalence of diabetes with chronic
ml of the HHV-1. By using this protocol, our samples were negative
for HHV-1 DNA.
Conclusions: This technique may be of clinical importance in the
study of HHV-1 influence in corneal graft failure.
Commercial Relationships: Maria Valeria C. Silva, None; Erna
G. Kroon, None; Fernando C. Trindade, None; Mauricio L.
Nogueira, None; Eduardo A. Bambirra, None
Commercial Relationships: Ethan K. Sobol, None; Robert
Fargione, None; Marianna Atiya, None; Jose Diaz, None;
Jonathan Powell, None; David C. Gritz, None
Support: I received a Fight For Sight summer student fellowship for
this research.
Detection of Human herpes virus 1 (HHV-1) in graft failure
corneas: a study of 24 cases using polymerase chain reaction
(PCR)
Maria Valeria C. Silva1, Erna G. Kroon2, Fernando C. Trindade2,
Mauricio L. Nogueira2, Eduardo A. Bambirra2. 1Ophthalmology,
Federal University of Minas Gerias, Belo Horizonte, Brazil; 2Federal
University of Minas Gerias, Belo Horizonte, Brazil.
Purpose: The purpuse of this study was to evaluate the presence of
Human herpes virus 1 (HHV-1) in paraffin-embedded corneas of
graft failure using a polymerase chain reaction protocol.
Methods: Twenty four corneas obtained by penetratin keratoplasty
(PK) were fixed in 70% alcohool and paraffin embedded. From each
patient clinical data was collected such as gender, age and surgical
indications.In cases of previous corneal graft failure, we collected
information about previous PK indication, such as: how long the
graft remained transparent, whether the patient had glaucomaand the
time interval between the corneal graft failure and the new surgery.
The samples were submitted to DNA extraction and PCR DNA
amplification.Primers for HHV-1 thymidine kinase and interferon
beta gene were used in PCR. Clinical data from 18 patients were
analyzed. In six cases the clinical data was not available.
Results: The male/female ratio was 50%. The age average of
analyzed patients was 53.9 years with a standard deviation of 17.4
years. The corneal transplant indication occurred in 13 (72%) cases
because of graft failure, the other 5 cases (27.5%) due to one of the
following pathologies each: bullous kerathopathy, fungus keratitis,
traumatic corneal opacity, bacterial ulcer and corneal opacity
presumed to be secondary to herps keratitis happened six years
before. The time period in which the graft remained clear in failure
cases varied from immediate, primary failure to 34 months after
surgery, with an average of 10.0 months. Seven patients (54%) had
glaucoma diagnosis among patients that had a corneal graft because
of graft failure. The time period between the graft failure and the
new surgery varied from one to 132 months, with an average of 30.8
months with a standard deviation of 34.6 months. The developed
protocol showed to be able to detect up to 0.001 plaque forming unit/
Relationship between viral genotype and frequency of relapses in
patients with herpetic keratitis
Andrea Alcocer1, Hector J. Perez-Cano2, Alejandro Babayan1.
1
Cornea, Hospital de la Luz, Mexico, Mexico; 2Centro de
Investigación Biomédica, Fundacion Hospital “Nuestra Señora de la
Luz” IAP, Mexico City, Mexico.
Purpose: To determine the herpes virus genotype using polymerase
chain reaction in patients with herpetic keratitis. Compare the
results obtained in patients with herpetic keratitis to assess response
to treatment in different genotypes found. Establish whether there
is a relationship between the genotype found by chain reaction
polymerase and relapsing herpetic keratitis.
Methods: Descriptive, longitudinal, analytical, prospective, and
observational study. We obtained a scrapping corneal sample from
patients diagnosed with herpetic keratitis who attended the outpatient
department and the Department of Cornea at Hospital de la Luz, to
determine Herpes Simplex Virus 1, Herpes Simplex Virus 2, Varicella
Zoster<span style=”line-height:20.7999992370605px”> Virus<\>>
and Cytomegalovirus using the Polimerase Chain Reaction.
Results: We studied 11 patients, 5 women, 6 men, mean age 50
years, who met the inclusion criteria, with recent initial diagnosis
of herpetic keratitis. Prior informed consent, an epithelial sample
was taken with a rayon swab, and placed in transport medium with
antibiotics. After DNA extraction and quantification, we performed
detection of Herpes Simplex Virus 1, Herpes Simplex Virus 2,
Cytomegalovirus and Varicella Zoster Virus by Polymerase Chain
Reaction amplification of specific viral secquences, followed by
agarose gel electrophoresis, revealing the results with UV light
transilluminator.
We found 2 positive patients to Herpes Simplex Virus type 1,
5 positive patients to Varicella Zoster Virus, 4 patients positive
for Herpes Simplex Virus type 2 and none was positive to
Cytomegalovirus. All responded favorably to treatment indicated and
none had recurrences.
Conclusions: The genotype most frequently found in our study
was Varicella Zoster Virus. So far there have been no recurrences in
our study and all have responded favorably to treatment indicated.
Patients will continue to be included until February 2015 and we
will follow patients to assess recurrence and thus determine which
genotypes tend to present them.
Commercial Relationships: Andrea Alcocer, None; Hector J.
Perez-Cano, None; Alejandro Babayan, None
Proteomic analysis of the effects of Amniotic Membrane on
Human Lymbal Myofibroblasts
Yonathan Garfias1, 2, Alfredo Dominguez1, Juan Pablo ReyesGrajeda3, Victor M. Bautista1. 1Research Unit, Institute of
Ophthalmology, Mexico City, Mexico; 2Department of Biochemistry,
Faculty of Medicine, Universidad Nacional Autónoma de México,
Mexico City, Mexico; 3Instituto Nacional de Medicina Genómica,
Mexico City, Mexico.
Purpose: Corneal damage observed in herpes simplex keratitis
(HSK) is mainly caused by exacerbated immune response to viral
components. One of the main lines of defense are the pattern
recognition receptors (PRRs) such as TLR3, MDA5 and RIG-1which
recognize viral dsRNA and after activation, they are able to promote
the production of pro-inflammatory cytokines via NF-κB nuclear
translocation. We have previously reported that amniotic membrane
(AM) decreases the production of proinflammatory cytokines and
PRRs on poly I:C stimulated human limbal myofibroblast (HLM)
through the inhibition of NF-κB nuclear translocation. These results
explain in part the anti-inflammatory microenvironment that induces
the AM transplantation in HSK. The objective of this study was to
anlayze the protein changes favored by amniotic membrane on HLM.
Methods: HLM were obtained from cadaveric sclero-corneal
rims. Frozen AM was enzimatically de-epithelized (dAM). All the
assays were carried out using 106 HLM incubated or not with dAM.
After dAM exposition, the HLM were recovered to obtain total
protein extract and 2D electrophoresis assays were then performed.
Subsequently, differential protein sequencing was done by means of
MALDI-TOF and Mascot software.
Results: Thirteen proteins were differentially found in HLM with
dAM in comparison to HLM seeded on plastic wells. dAM favored
de novo expression of three proteins, identified as serum albumin,
with three different isoelectric points. In contrast, 6 out of the thirteen
differentially expressed proteins were downregulated by dAM: actin
4.7 fold decrease, heat shock cognate 5.4 fold decrease, α-enolase
4.8 fold decrease, glucose-regulated protein 3.8 fold decrease,
collagen α-1 4.5 fold decrease, calreticulin 2.9 fold decrease, and
the expression of 3 proteins were completely inhibited: 2 variants of
cytoplasmic actin and glyceraldehyde-3-phosphate dehydrogenase.
Conclusions: Taken together these results indicate that the AM
exerted overlapping functions on HLM given by the protein pattern
identified by proteomics. More experiments are needed to determine
the nature and functional activity of such molecules.
Financial Support: This project was supported by CONACYT:
SALUD 160286; CIENCIA BASICA 167438; DGAPA-PAPIIT
IA203514; CVU: 406706
Commercial Relationships: Yonathan Garfias, None; Alfredo
Dominguez, None; Juan Pablo Reyes-Grajeda, None; Victor M.
Bautista, None
Support: This project was supported by CONACYT: SALUD
160286; CIENCIA BASICA 167438; DGAPA-PAPIIT IA203514;
CVU: 406706
Incidence of Herpes zoster ophthalmicus (HZO) at Massachusetts
Eye and Ear Infirmary (MEEI) from 2007-2013
Emma Davies, Deborah Langston, James Chodosh. Ophthalmology,
Massachusetts Eye and Ear Infirmary, Boston, MA.
Purpose: This study investigated the incidence of HZO in patients
presenting to MEEI from 2007 until 2013 to define current trends in
disease incidence across age groups.
Methods: A total of 1,283 potential cases were identified by
searching the MEEI electronic medical record for patient charts with
ICD-9 codes for Herpes zoster, shingles, and varicella from 2007
to 2013. The cases were reviewed to confirm diagnosis of acute
HZO, requiring documentation of a skin rash or pain in the V1 or
V2 distribution, resulting in 913 patients. The data were analyzed
to determine the number of cases of HZO per year as well as
stratified by age group. The mean age of HZO patients each year was
determined, with a comparision of the 2007 mean age to the 2013
mean age completed by a t test. The number of HZO patients with an
immunodeficiency state (including HIV, leukemia, organ transplant,
and steroid use) were also analyzed.
Results: The incidence of HZO at MEEI increased from 71 cases in
2007 to 195 cases in 2013. The mean age of patients with acute HZO
reduced significantly from 61.2 years in 2007 to 55.8 years in 2013
(P=0.0168). When subdivided by age, a non-statistically significant
trend of decreasing number of patients aged 75-84 years and aged
85-94 years was found. The number of patients aged 35-44 years
and aged 55-64 years trended up over time but was not statistically
significant. Finally, the number of patients with acute HZO and an
immunodeficiency state did not change over the study.
Conclusions: The research demonstrated an increased incidence of
HZO with a reduction in mean age that may be explained by several
factors. An increased awareness of Herpes zoster could increase the
number of patients presenting to MEEI, but this factor is unlikely to
be the sole cause for increased incidence as awareness stems from
high disease burden. A greater prevalence of immunodeficiency
conditions is a plausible factor, but the current data demonstrates no
increase in the number of HZO patients with immunodeficiency. The
contribution of vaccination against zoster in reducing mean age of
HZO patients is apt to be minimal as only 14.4% of eligible patients
received the vaccination by 2010. The remaining factor of pediatric
varicella vaccination reducing population exposure and limiting
immunologic boost remains a possible explanation for both the
increased imcidence in HZO as well as the reduction in mean age.
Commercial Relationships: Emma Davies, None; Deborah
Langston, None; James Chodosh, None
Support: N&J Johnstone Fund, G. Stevens Fund
Cytomegalovirus Anterior Uveitis in Immunocompetent Patients
Natasha V. Nayak, Emile Sharifi, C M. Samson, Sanjay Kedhar. New
York Eye and Ear Infirmary of Mount Sinai, New York, NY.
Purpose: To describe the clinical features and management
of cytomegalovirus (CMV) associated anterior uveitis in
immunocompetent patients within the United States.
Methods: Retrospective chart review of immunocompetent patients
with anterior uveitis who had tested positive for CMV by polymerase
chain reaction (PCR) of aqueous humour. Records were reviewed
for demographics, ocular findings at presentation, laboratory results,
presence of elevated intraocular pressure, and treatment.
Results: Medical records of nine immunocompetent patients with
PCR positive CMV anterior uveitis were reviewed (median age
55 years, 66% male). Ethnicity of patients were as follows: 55%
East Asian, 22% Caucasian, 11% Hispanic, 11% South Asian. All
patients had unilateral anterior uveitis (55% right eyes) associated
with hypertensive crisis. Median age of onset of anterior uveitis was
36 years. Median best-corrected visual acuity on presentation was
0.12 (logMAR). Prior to PCR results, working diagnoses included
Posner-Schlossman syndrome, Fuchs heterochromic iridocyclitis,
herpes simplex virus iritis, CMV irits, and HLA-B27 associated
iritis. Median interval between initial episode of anterior uveitis to
PCR-based diagnosis was 3 years (range of 1-20 years). All nine
patients were started on oral valgancyclovir based on PCR results.
Withdrawal of oral valgancyclovir resulted in recurrence in 5 of
5 patients, when attempted. Three patients were managed with
topical valgancyclovir after discontinuation of oral medication, and
two patients remain on oral valgancyclovir. Eight of nine patients
required long term glaucoma management, including two patients
who required glaucoma surgery. Median duration of follow-up was
48 months.
Conclusions: This study highlights the presence of CMV associated
anterior uveitis in immunocompetent patients in the United States,
across various ethnicities. Management includes long-term oral or
topical valgancyclovir and control of intraocular pressure.
Commercial Relationships: Natasha V. Nayak, None; Emile
Sharifi, None; C M. Samson, None; Sanjay Kedhar, None
UVEITIS IN PSORIASIC PATIENTS: A DESCRIPTIVE
STUDY
Mónica Delgado Riveira, Nicolás Alejandre Alba. fundacion jimenez
diaz, Madrid, Spain.
Purpose: Psoriasis is a chronic T cell-mediated autoimmune disease,
but its pathogenesis remains unknown. Since viral infections
appear to be increased in patients with psoriasis, viral antigens have
been proposed as potential triggers. The aim of our study was to
describe features of eye inflammation in patients with psoriasis and
to compare them with previously described patterns of uveitis. We
sought for findings that could support the influence of viruses in the
development of uveitis in patients with psoriasis.
Methods: We retrospectively evaluated 14 patients diagnosed with
psoriasis (10 men and 4 women with an average age of 51 years),
suffering an episode of uveitis. Of them, 50% had pustular psoriasis
and 33% had psoriatic arthritis, while 2 patients had both.
Results: Slit lamp revealed retro-keratic precipitates in 50% of
the patients. All of them followed a similar pattern, consisting of
pancorneal, white, thin and star-shaped lesions. Four out of the 7
patients with retro-keratic precipitates had an increased intraocular
pressure. Nine out of the 14 patients had unilateral eye involvent.
The most frequent diagnosis was Recurrent Acute Anterior Uveitis
(RAAU) (43%).There was one patient diagnosed with Chronic
Bilateral Ischemic Vasculitis (CBIV) and another with Chronic
Intermediate Anterior Uveitis (CIAU). A sample of aqueous humor
(AH) was available in 4 of the 14 patients. Interestingly, 2 of them
yielded a positive result for type I HSV, as studied by genome
amplification with polymerase chain reaction. The patient with CIAU
had AH high CD4+ and CD8+ T cell counts, with an inverted CD4+/
CD8+ ratio (0.69). In addition, vitreous humor from the patient with
a CBIV showed also high CD4+ and CD8+ T counts, with a ratio of
1.37.
Conclusions: In our cohort, patients with psoriasis showed some
characteristics resembling those of viral-induced uveitis. These
included a predominantly unilateral involvement, presence of starshaped retro-keratic precipitates, ocular hypertension and high local
counts of CD4+ and CD8+ T cells. All these features have been found
in patients with uveitis caused by cytomegalovirus, herpes simplex
and rubella viral infections.
In spite of the small sample size of our study, our findings point to an
antiviral-like cell response during episodes of uveitis in patients with
psoriasis, and abound in the global contribution of viral antigens to
the pathogenesis of psoriasis.
Commercial Relationships: Mónica Delgado Riveira, None;
Nicolás Alejandre Alba, None
Support: Do not use n/a
The role of RIP3 in innate immune responses and death of
bystander cells during MCMV retinitis
Ming Zhang, Juan Mo, Brendan Marshall, Sally S. Atherton, Sylvia
B. Smith. Georgia Regents University, Augusta, GA.
Purpose: Recent studies suggest that RIP kinases, which are key
decision makers in cell death and innate immunity against invading
pathogens, are activated during MCMV ocular MCMV infection. The
purpose of this study is to determine if RIP3 plays a significant role in
innate immune responses and death of bystander cells by comparing
MCMV retinal infection, innate immune response and cell death in
Rip3-/- mice and control mice.
Methods: RIP3-/- and RIP3+/+ mice were immunosuppressed (IS)
with methylprednisolone acetate (2mg per mouse, i.m. every 4
days) and inoculated with 5 Χ103 PFU of MCMV K181 strain,
via the supraciliary route. Virus injected and control eyes were
removed at days 4, 7 and 10 post-infection and analyzed by plaque
assay, electron microscopy (EM), H & E staining, TUNEL assay,
western blot (for caspase 3, caspase 1, caspase 12, NFκB), as well
as immunohistochemical staining for MCMV early antigen, cleaved
caspase 3 and AIF.
Results: Compared to RIP3+/+ mice, significantly more MCMV was
recovered and more MCMV infected RPE cells were observed in
injected eyes of RIP3-/- mice at day 4 and day 7 p.i. In contrast, fewer
TUNEL-stained photoreceptors were observed in injected RIP3/eyes than in injected RIP3+/+ eyes at these times. EM results also
confirm that significantly more apoptotic photoreceptor cells were
observed in the outer nuclear layer of RIP3+/+ wild type mice than
in the outer nuclear layers of RIP3−/− mice. Furthermore, the results
of immunohistochemistry showed that the majority of TUNELstained photoreceptors died via AIF-mediated, caspase 3-independent
apoptosis, although significantly lower levels of cleaved caspase 3
were also detected in injected eyes of RIP3-/- mice than in injected
eyes of RIP3+/+ mice by western blot. Expression of cleaved caspase 1
was increased in RIP3+/+ eyes after MCMV infection as early as day 4
p.i. In contrast, increased cleaved caspase 1 expression was observed
in RIP3-/- eyes only at day 10 p.i. In addition, significantly higher
levels of activated NFκB were observed in RIP3+/+ eyes compared to
RIP3-/- eyes at days 4, 7 and 10 p.i.
Conclusions: Our results suggest that RIP3 enhances innate immune
responses against ocular MCMV infection via activation of the
inflammasome and NF-κB, which, also leads to inflammation and
death of bystander cells by multiple pathways including apoptosis
and necroptosis.
Commercial Relationships: Ming Zhang, None; Juan Mo, None;
Brendan Marshall, None; Sally S. Atherton, None; Sylvia B.
Smith, None
Support: Fight for Sight/International Retina Research Foundation
Both canonical and non-canonical inflammasome systems may
operate during development of murine cytomegalovirus (MCMV)
retinitis in mice with retrovirus-induced immunodeficiency
(MAIDS)
Richard D. Dix1, 2, Christine I. Alston1, 2, Hsin Chien1. 1Viral
Immunology Center, Department of Biology, Georgia State
University, Atlanta, GA; 2Ophthalmology, Emory University School
of Medicine, Atlanta, GA.
Purpose: NLR proteins are innate immune sensors that can form
large complexes called canonical inflammasomes that activate
caspase-1 and induce production of active IL-1beta and IL-18 that
can lead to pyroptosis, an inflammatory cell death program. A second
inflammatory caspase, caspase-11, may also mediate pyroptosis
through a non-canonical inflammasome system. We therefore
performed studies to test the hypothesis that key molecules required
for both canonical and non-canonical inflammasome systems are
stimulated during development of experimental MCMV retinitis in
mice with MAIDS.
Methods: Groups of C57BL/6 mice with MAIDS of 4-weeks
(retinitis resistant) or 10 weeks (retinitis susceptible) duration were
injected subretinally with MCMV or mock injected (control). At days
3, 6, and 10 postinfection, whole eyes were collected and subjected
to real time RT-PCR and/or western blot assays for quantification
of AIM2, NLRP1b, NLRP3, NLRC4, caspase-1, IL-1beta, and
caspase-11 mRNAs and/or proteins, respectively.
Results: When compared with mock-infected eyes, MCMV-infected
eyes from MAIDS-10 mice exhibited a significant increase in AIM2,
NLRP1b, NLRP3, and NLRC4 mRNAs and caspase-1, IL-1beta
and caspase-11 proteins at day 6 postinfection prior to retinitis
development. In contrast, MCMV-infected eyes from MAIDS-4 mice
showed minimal or no detectable changes in production of these
molecules at day 6 postinfection.
Conclusions: Key molecules associated with canonical
inflammasomes as well as non-canonical inflammasomes that
are involved in the development of pyroptosis were significantly
stimulated within MCMV-infected eyes of MAIDS-10 mice that
develop retinitis, but were only minimally stimulated or not detected
within MCMV-infected eyes of MAIDS-4 mice that do not develop
retinitis. These findings suggest that both canonical and noncanonical inflammasome systems may operate during development of
MCMV retinitis in mice with MAIDS and lead to pyroptosis, another
cell death mechanism by which retinal tissue destruction may occur
during MAIDS-related MCMV retinitis.
Commercial Relationships: Richard D. Dix, None; Christine I.
Alston, None; Hsin Chien, None
Support: NIH Grant EY010568, NIH/NEI Core Grant P30/
EY006360, NIH/NEI Training Grant 5T32/EY007092-28, GSU
Department of Biology Bridge Grant, and Fight for Sight
Mutations within the pathogenic region of HSV-1 gK signal
sequences alter cell surface expression and neurovirulence
Homayon Ghiasi. Ophthalmology Reseach, Cedars-Sinai Medical
Center, Los Angeles, CA.
Purpose: Glycoprotein K (gK) is a highly conserved and essential
HSV protein. We previously demonstrated that immunization of mice
with gK exacerbates eye disease following ocular HSV-1 infection
independent of mice or virus strain. We also have reported that a
recombinant HSV-1 expressing two extra copies of gK exacerbated
eye disease in both mice and rabbit, further suggesting that gK is
pathogenic. In addition, we had shown that the pathogenic region of
the gK is located within the 8mer signal sequence of gK. In this study
we investigated the role of the gK 8mer in corneal scarring (CS) and
neurovirulence by constructing recombinant viruses with and without
mutations within the 8mer region of the gK.
Methods: We constructed two recombinant viruses expressing two
additional copies of the mutated (MgK) or native (NgK) form of the
gK gene with a myc epitope tag, to facilitate detection, at their 3’
end. The replication of MgK, NgK, and revertant (RgK) viruses were
determined in vitro and in vivo. In addition, the levels of gB and gK
in the corneas, trigeminal ganglia (TG), and brains of infected mice
on days 3, 5, and 28 post infection (PI) were determined. Finally,
LD50, CS and extent of explant reactivation in infected mice were
determined.
Results: The replication of MgK virus was similar to that of NgK
virus both in vitro and in vivo as well as in the TG of latently-infected
mice. The levels of gB and gK transcripts in the corneas, TG, and
brains of infected mice on days 3 and 5 PI were markedly virus- and
time-dependent as well as tissue-specific. Point mutations in the 8mer
region of gK in MgK virus blocked cell surface expression of gKmyc in rabbit skin (RS) cells and reduced LD50 and CS in ocularly
infected mice as compared with the NgK or RgK virus. MgK and
NgK viruses and not the RgK virus had reduced extent of explant
reactivation at the lower dose of ocular infection but not at the higher
dose of infection. However, time of reactivation was not affected by
overexpression of different forms of gK.
Conclusions: Taken together, these results strongly suggest that
the 8mer peptide (ITAYGLVL) within the signal sequence of gK
promotes cell surface expression of gK in infected cells and ocular
pathogenesis in infected mice. Thus, these studies point to a key role
for the 8mer within the signal sequence of gK in HSV-1-induced
pathogenicity.
Commercial Relationships: Homayon Ghiasi, None
Support: This work was supported by Public Health Service grant
RO1 EY13615 from the National Eye Institute.
Epidemic Keratoconjunctivitis-causing Adenoviruses Induce
MUC16 Ectodomain Release in Corneal Epithelial Cells
Balaraj B. Menon1, Xiaohong Zhou2, Sandra Spurr-Michaud1, James
Chodosh2, Ilene K. Gipson1. 1Harvard Med Sch-Ophthal, Schepens
Eye Rsch Inst, MEEI, Boston, MA; 2Harvard Medical School MEEI, Boston, MA.
Purpose: Membrane-associated mucins (MAMs) that are expressed
on the apical side of all mucosal epithelia of the body interfere with
pathogen adhesion and invasion. Nevertheless, certain pathogens
overcome the MAM barrier prior to initiating infection and associated
inflammation. The purpose of this study is to test the hypotheses that
1) ectodomain release of MAMs, primarily MUC1 and MUC16, is
a pre-requisite step for establishment of adenoviral infections at the
ocular surface and 2) MUC1 and MUC16 regulate the inflammatory
response associated with adenoviral keratoconjunctivitis.
Methods: Telomerase-immortalized human corneal-limbal epithelial
(HCLE) cells, grown to confluence and allowed to differentiate
for optimal mucin expression, were exposed to HAdV-D37 (EKCcausing) and HAdV-D19p (non EKC-causing, control), each at
an MOI of 3, for 2 h. Following exposure, equal volumes of cell
culture supernatants were harvested, concentrated, and analyzed for
released ectodomains of MUC1 and MUC16 by immunoblotting. To
determine if MUC1 and MUC16 regulate the inflammatory response
associated with HAdV-D37 infection, HCLE cells and those knocked
down for MUC1 (HCLE-shMUC1) or MUC16 (HCLE-shMUC16),
and corresponding scramble-transfected cells (HCLE-scrMUC1 and
HCLE-scrMUC16) were exposed to empty HAdV-D37 capsids for
1 h, following which mRNA was extracted from the different cells,
and IL-8 message levels quantified by qRT-PCR as an outcome of
inflammatory response.
Results: 1) The EKC-causing HAdV-D37, but not the non EKCcausing HAdV-D37, induced MUC16 ectodomain release. No
ectodomain release of MUC1 was observed. 2) HCLE-shMUC1
and HCLE-shMUC16 cells exhibited a 2-2.5 fold increase in IL-8
message levels upon exposure to empty HAdV-D37 capsids in
comparison to HCLE-NT, HCLE-scrMUC1, and HCLE-scrMUC16
cells.
Conclusions: Results suggest that adenovirus-induced ectodomain
release of MUC16 may be a pre-requisite step for establishment
of adenoviral infections at the ocular surface and that MUC1 and
MUC16 may be involved in suppressing the inflammatory response
associated with such infections.
Schematic summarizing the hypothesis that keratoconjunctivitiscausing adenoviruses (HAdV-D) bind to MUC16 (Step 1) and induce
release of the ectodomain (Step 2) to infect corneal epithelial cells
(Step 3).
Keratoconjunctivitis-causing adenoviruses (HAdV-D37) induce
MUC16 ectodomain release.
Commercial Relationships: Balaraj B. Menon, None; Xiaohong
Zhou, None; Sandra Spurr-Michaud, None; James Chodosh,
None; Ilene K. Gipson, None
Support: Harvard Cornea Center of Excellence Research
Investigator Award
Dynamin-2 negatively regulates adenovirus trafficking in corneal
cells
Jaya Rajaiya, JiSun Lee, Jeong Yoon Lee, James Chodosh.
Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard
Medical School, Boston, MA.
Purpose: We have earlier shown that human adenovirus type D37
(HAdV-D37) used caveolae mediated entry into human corneal
fibroblasts (HCF) causing keratitis. Our earlier reports also suggested
a central role for activated Src in viral entry and infection. Dynamins
are associated with multiple cellular processes including endocytosis
of caveolae, fission and release of transport vesicles, and regulating
the dynamics of microtubules. Dynamin has also been shown to be
important in the entry of other viruses. Here we studied the role of
dynamin-2 in the intracellular trafficking of HAdV-D37 in HCF.
Methods: HCF were pretreated with DMSO, bafilomycin A1, or
cytochalasin B for 30min and then infected with HAdV-D37 for 1h.
Cells were also transfected with siRNA against dynamin-2 or scRNA
prior to infection. Cells were Trizol treated for RNA isolation or lysed
for subsequent real time-PCR or protein studies, respectively. For
microtubule visualization, cells grown on slide chambers were treated
with DMSO or nocodazole for 1hr, and then infected with Cy3labeled HAdV-D37. Cells were then fixed in 4% paraformaldehyde,
washed in PBS 2% BSA, permeabilized in 0.1% Triton X-100,
blocked in 2% BSA-PBS, incubated in anti-tubulin antibodies for
1hr at room temperature, washed again, and mounted for confocal
microscopy.
Results: Neither the endosomal acidification inhibitor, bafilomycin
A1 or the actin polymerization inhibitor, cytochalasin B had an
effect on adenoviral infection of HCF. However, the microtubule
inhibitor, nocodazole repressed viral gene expression in a dose
dependent manner, consistent with reduced trafficking of virus to the
nucleus. Dynamin-2 siRNA increased Src activation, microtubule
accumulation, and viral gene expression. Upon dynamin-2
overexpression, Src activation and virus trafficking in corneal cells
were both reduced.
Conclusions: Dynamin-2 knock down results in stability and
accumulation of microtubules, which in HCF, enhances HAdV-D37
trafficking. These results are consistent with a negative role for
dynamin-2 in HAdV-D37 infection and suggests that microtubule
stability is a potential target for anti-adenoviral therapy in the cornea.
Commercial Relationships: Jaya Rajaiya, None; JiSun Lee, None;
Jeong Yoon Lee, None; James Chodosh, None
Support: NIH grants EY013124, EY021558, and P30EY014104,
Research to Prevent Blindness, The Massachusetts Lions Eye
Research Fund, and the Falk Foundation.
ADenoVirus Initiative Study in Epidemiology (ADVISE) Interim Analysis
Eric Tuil1, Louis Hoffart2, Pierre Gabisson3, Stéphanie Baillif4,
Tristan Bourcier5, Marc Labetoulle6, Bruno Mortemousque7, Cedric
Schweitzer8, Pierre-Jean Pisella9. 1Emergency Ophthalmology
Department, XV-XX Hospital, Paris, France; 2La Timone Hospital,
Marseille, France; 3Private practice, Marseille, France; 4Saint-Roch
Hospital, Nice, France; 5Civil Hospital, Strasbourg, France; 6KreminBicêtre Hospital, Kremlin-Bicêtre, France; 7Pontchaillou Hospital,
Rennes, France; 8Pellegrin Hospital, Bordeaux, France; 9Bretonneau
Hospital, Tours, France.
Purpose: Adenoviral conjunctivitis is extremely contagious and
may be severe. It is challenging to make an accurate early diagnosis
based on clinical signs and symptoms only. AdenoPlus® is a CE
marked rapid differential diagnostic IVDMD. ADVISE is a noninterventional, observational epidemiology study. Its main objectives
are to assess clinical characteristics and incidence of adenoviral
conjunctivitis in patients who present with signs and symptoms of
acute conjunctivitis.
Methods: This interim analysis included data collected from 332
patients from 16 sites in France. Prior to inclusion, patients gave their
oral consent. Inclusion criteria were: presence of acute signs and
symptoms of conjunctivitis inferior or equal to 7 days duration, and
a minimum age of 1 year-old. Patients already treated with topical
antiviral therapies, steroids or immunomodulators were excluded.
Results: The percentage of positive tests was 36.1% (120/332). Two
invalid test results were reported (0.6%), 49.1% of the investigators
had their clinical assessment confirmed by the test whilst it was not
confirmed in 50.9% of the cases. Patients were mostly active adults
(mean age 42.2 yo) without any eye medical history, 68.4% of the
patients already received local antibiotics before the study visit.
In the population of patients who were tested positive with
AdenoPlus®, the visits occurred statistically significantly earlier
(2.9 vs.7.2 days, p=0.008), “red eye” was assessed as more severe
and overall, higher percentages of signs and symptoms were
reported (e.g. purulent eye discharge, chemosis, petechia, keratitis,
lymphadenopathy, photophobia), although none of them could be
identified as pathognomonic of the disease. Fifty percent of positive
patients reported a previous contact with someone with “red eye”.
Conclusions: Approximately 4 out of 10 patients presenting with
signs and symptoms of acute conjunctivitis, suspected to be from
viral origin, had a positive result with AdenoPlus®. These study
results also pointed out the severity of adenoviral conjunctivitis and
its contagiousness, highlighting the need of implementing preventive
measures to contain the virus spread.
Commercial Relationships: Eric Tuil, Nicox (C); Louis Hoffart,
None; Pierre Gabisson, None; Stéphanie Baillif, None; Tristan
Bourcier, None; Marc Labetoulle, None; Bruno Mortemousque,
None; Cedric Schweitzer, None; Pierre-Jean Pisella, Nicox (C)
Clinical Trial: NTC02054234
The Evaluation of Topical SPL, a Novel Dendrimer Antiviral,
against Adenovirus in NZW Rabbit Ocular Models
Eric G. Romanowski1, Kathleen A. Yates1, Robert M. Shanks1, Oliver
K. Bernhard2, Jeremy R. Paull2, Regis P. Kowalski1. 1The Charles T.
Campbell Laboratory, UPMC Eye Center, University of Pittsburgh,
Pittsburgh, PA; 2Starpharma Pty Ltd, Melbourne, VIC, Australia.
Purpose: There is no FDA approved antiviral therapy for
adenovirus (Ad) ocular infections. Dendrimers are novel nanoscale
macromolecules that have the ability to be designed to interact
polyvalently with a target and act as virucidal agents. SPL is a
polyanionic dendrimer. SPL’s unique size, shape, and highly charged
surface allow attachment to targets on viruses which then prevent
viral attachment and/or adsorption to cells. The goals of the current
study were to evaluate the ocular tolerability and anti-adenoviral
efficacy of topical SPL in separate NZW rabbit ocular models.
Methods: Tolerability: 12 NZW rabbits were divided into 4 groups
(n=3/group): 1) 5% SPL; 2) 3% SPL; 3) Vehicle (VEH); and 4) 0.5%
Cidofovir (CDV). Rabbits were treated topically OU 4X/day for 5d
except for CDV which was instilled 2X/day for 5d. Eyes were graded
using the Draize scale on Days 1, 3, 5, 8, 10 and 12. Efficacy: 20
NZW rabbits were inoculated in both eyes after corneal scarification
with 1.5 x 106 PFU/eye of Ad5. On day 1, rabbits were divided into 4
groups (n=5/group): 1) 5% SPL; 2) 3% SPL; 3) VEH; 4) 0.5% CDV.
Rabbits were treated topically OU 8X/day for 4d, then 4X/day for 6d,
except for CDV (2X/day for 7d). All eyes were cultured for virus on
Days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Viral titers were determined.
Results: Tolerability: There were no differences in Draize scores
among 5% SPL, 3% SPL and VEH on any day (P>0.05, K-W).
CDV produced higher Draize scores on Day 12 than SPL and VEH
(P≤0.05, K-W). Efficacy: 5% SPL (Days 3, 5, 7, 9), 3% SPL (Days 3,
5, 7, 9) and CDV (Days 7, 9) reduced daily viral titers compared with
VEH (P≤0.05, K-W). 5% SPL (7d), 3% (4.5d) and CDV (5d) reduced
the Duration of Viral Shedding compared to VEH (9d) (P≤0.05,
K-W). 3% SPL was more effective than 5% SPL in several outcome
measures (Daily titers Day 5 and Duration of Viral Shedding)
(P≤0.05, K-W).
Conclusions: 5% SPL and 3% SPL demonstrated significant antiviral
efficacy compared with VEH in the Ad5/NZW rabbit ocular model.
Both 5% and 3% SPL demonstrated significantly better efficacy than
CDV, during the Early Phase of Infection (Days 1-5). SPL induced
“Minimal” to “Practically Non-Irritating” Draize scores in the NZW
rabbit ocular tolerability model. Further development of SPL as a
topical antiviral for adenoviral ocular infections is indicated.
Commercial Relationships: Eric G. Romanowski, Starpharma
Pty Ltd (F); Kathleen A. Yates, Starpharma Pty Ltd (F); Robert M.
Shanks, Starpharma Pty Ltd (F); Oliver K. Bernhard, Starpharma
Pty Ltd (E); Jeremy R. Paull, Starpharma Pty Ltd (E); Regis P.
Kowalski, Starpharma Pty Ltd (F)
Support: Starpharma Pty Ltd; NIH CORE Grant for Vision Research
EY08098; NIH Grant AI085570; The Eye and Ear Foundation of
Pittsburgh; and Research to Prevent Blindness
New method of typing and quantification for human adenoviruses
in tear fluid of patients with adenovirus conjunctivitis
Nobuyo Yawata1, 2, Yu-Chi Liu1, 3, Jay Siak3, 1, Makoto Yawata4, 5,
Jodhbir S. Mehta1, 3. 1Singapore Eye Research Institute, Singapore,
Singapore, Singapore; 2Duke-NUS Graduate Medical School,
Singapore, Singapore; 3Singapore National Eye Centre, Singapore,
Singapore; 4National University of Singapore, Singapore, Singapore;
5
Singapore Institute for Clinical Sciences, Singapore, Singapore.
Purpose: Currently, conjunctival swab samples are used in diagnostic
tests for epidemic keratoconjunctivitis (EKC) caused by group D
human adenoviruses (HAdVs) such as HAdV-8, 19a, 37, 53 and 54.
However, this method is not suitable to quantify virus load. We have
established novel methodology to type and quantify HAdVs using
small amounts of tear fluid obtained from patients with adenovirus
conjunctivitis.
Methods: Tear fluid was collected using Schirmer strips from 25 eyes
of 13 patients with acute follicular conjunctivitis in the Singapore
National Eye Centre. The viral gDNA was extracted and virus copy
numbers were determined using qPCR of the hexon gene. Virus type
was determined using sequence-based typing of the 554bp of hexon
gene and fiber gene. Clinical symptoms were studied in correlation
with virus types and virus load.
Results: In patients diagnosed with acute viral conjunctivitis, we
detected five different HAdV types and have found that HAdV-8
is a dominant type in Singapore. Of interest, HAdV-8 was detected
from all cases with corneal subepithelial infiltration. HAdV gDNA
was detected from as little as 1ul of tear fluid. Virus copy numbers
in the tear fluid were 2.5x10^4-1x10^12/ml at the first clinical visit.
Virus gDNA was not detected after two weeks. Of interest, we found
equivalent copy numbers of viral gDNA in both eyes although the
eye with first onset showed more severe inflammation, and this
observation implies that factors specific in the second onset eye may
suppress development of severe inflammation that is characteristic of
EKC.
Conclusions: The new method for HAdV quantification using tear
fluid makes maximum use of ocular material and will be useful in
understanding the mechanisms of inflammation in EKC. Further, the
method is suitable in clinical studies such as in evaluating the effects
of new anti-viral therapies.
Commercial Relationships: Nobuyo Yawata, None; Yu-Chi Liu,
None; Jay Siak, None; Makoto Yawata, None; Jodhbir S. Mehta,
None
Support: NMRC/TA/0010/2012, Agency for Science, Technology
and Research (A*STAR) core funding, SERI Pilot Grant
R816/11/2011
Intraocular inflammation and hemorrhage associated with
Dengue virus infections in Paris France
Pablo L. Goldschmidt1, Marie Hélène Errera3, Christine Chaumeil1,
Christine Fardeau2. 1Laboratoire d’Ophtalmobiologie, Centre Hospit
Nat d’Ophtal des Quinze-Vingts, Paris, France; 2Ophtalmologie,
CHU Pitié-Salpétrière, Paris, France; 3Service 4, Centre Hospit Nat
d’Ophtal des Quinze-Vingts, Paris, France.
Purpose: Viruses provoking Dengue fever have become endemic
in Europe and their growing incidence is the result of developing
urbanization, tourism, and trade. The goal of this work is to show
the need to relate intraocular inflammation (retinitis, chorioretinitis,
retinal vasculitis, optic nerve involvement, etc.) to emerging
infections (Rickettsia,West Nile virus,Rif-valley virus,Dengue virus,
Chikungunya virus, etc).
Methods: Patients with intraocular inflammatory diseases attending
the National Eye Center (CHNO) and the University Hospital Pitie
Salpetriere in Paris France were evaluated according to symptoms,
signs and epidemiological data. Ocular examination included visual
acuity, slit lamp examination, fundus examination through dilated
pupil and color photography, enhanced depth optical coherence
tomography, indocyanine green angiography, autofluorescence and
fluorescein angiography. This was followed by sampling for specific
antibodies titrations (including testing for Herpesviridae, Treponema,
Toxoplasma, Mycobacteria, Bartonella and Lyme disease) and for
Herpesviridae-DNA and Toxoplasma-DNA detection by real-time
polymerase chain reaction
Results: Three females aged 17; 30 and 21 presented fundus
alterations (January-September 2014).The first reported blurred
vision, and showed scotoma and retinal hemorrhage.The second
visual impairment,reduction in blood perfusion assessed by
indocyanine green fluorescence.The third showed perfusion increase,
hemorrhages and macular edema associated with images suggesting
outer retinal and retinal pigment epithelial involvement. For these 3
cases antibody titers (Ig and IgM) and titer evolution let to establish
Dengue-virus associated intraocular inflammation. Prognosis varied
from full resolution to partial vision loss despite intervention with
symptomatic treatments.
Conclusions: Air travel and globalization have overturned the
traditional patterns of geographic distribution of viral infections.
Among others, Denguevirus associated infections require to be
searched in people presenting intraocular inflammatory processes
with or without hemorrhage, especially with history of travelling to
endemic or epidemic regions.
Commercial Relationships: Pablo L. Goldschmidt, None; Marie
Hélène Errera, None; Christine Chaumeil, None; Christine
Fardeau, None
Viral Profile in Pterygium using Polymerase Chain Reaction
Hector J. Perez-Cano1, Aidee Mendoza2, Monica Reyes-Santos1,
Javier Acosta-Gonzalez1. 1Genetics and Microbiology, Hosp
Foundation “Nuestra Senora de la Luz””, Mexico City, Mexico;
2
Orbita, Fundacion Hospital “Nuestra Señora de la Luz” IAP, Mexico
City, Mexico.
Purpose: Pterygium is a fibrovascular lesion originating from the
conjunctiva that often extends on the corneal surface, this is a treath
for the vision. The risk factors involves UV light exposure and
viral infections. Many studies have been conducted to investigate
the involvement of a variety of oncogenic viruses, including HPV,
CMV, HSV or EBV, in the development of pterygium. In this study
we determine the presence of oncogenic viruses in pterygium and
phenotypically normal conjunctiva and the possible relation between
viral presence and this clinical entity
Methods: Twenty-five pterygia samples were obtained by surgery
and fifty scraping healthy conjunctival were analyzed like control.
HSV1, VZV, CMV, EBV and HPV detection were accomplished by
polymerase chain reaction amplification of viral sequences followed
by electrophoresis in agarose gel.
Results: EBV was detected in 7 (28%), HPV in 2 (8%), HSV (type
1) 1 (4%), VZV 0 (0%), and CMV 0 (0%) in pterygia samples. The
healthy conjunctival specimen were negatives for HSV1, VZV, CMV
and EBV, whereas, HPV was detected in two controls (4%)
Conclusions: Pterygium is associated with not only viral infections
but also to exposure to UV rays and genetic predisposition. The
detection of potentially oncogenic viruses, such as EBV, HPV and
HSV-1, supports the concept that the pterygium can be considered
a multifactorial neoplastic condition. Detection of EBV, HPV and
HSV1 between patients and control subjects implies a possible viral
participation to develop pterygium.
Commercial Relationships: Hector J. Perez-Cano, None; Aidee
Mendoza, None; Monica Reyes-Santos, None; Javier AcostaGonzalez, None
Human Papillomavirus Detection in Soft Contact Lens Cases by
DNA Deep Sequencing
Dallin Andersen, Thuy Doan, Lakshmi Akileswaran, Narae Ko,
Angira Shrestha, Russell Van Gelder. Ophthalmology, University of
Washington, Seattle, WA.
Purpose: Contact lenses are a significant risk factor for microbial
keratitis. Contact lens storage hygiene may be a significant factor in
this risk. The microbial environment on contact lenses and cases has
not been re-investigated using deep DNA sequencing. We applied
Biome Representational in Silico Karyotyping (BRiSK) and 16s
rDNA deep DNA sequencing techniques to samples collected from
the conjunctiva, soft contact lenses, and contact lens cases of nine
subjects.
Methods: Conjunctiva, buccal mucosa and skin were swabbed using
forensic DNA recovery swabs. Contact lenses and the interior of
cases were soaked in phosphate buffered saline and sampled. DNA
was purified and analyzed by BRiSK and 16s rDNA. 33 bp DNA
sequence tags from BRiSK and 16s rDNA gene sequences were
compared to a comprehensive database for species identification.
Results: 16s rDNA was applied to 85 samples from 9 subjects
(including left and right contact lens cases, left and right contact
lenses, upper and lower conjunctiva from both eyes, buccal, and
skin samples). BRiSK was applied to 41 samples from 5 subjects
(contact lens cases, contacts, and conjunctival samples). High
quality DNA was found in 26 samples from BRiSK. Both 16s
and BRiSK demonstrated organisms found in common and in
high proportions in conjunctiva, contact lenses, and cases, which
include Propionibacterim, coagulase negative Staphylococcus,
and Pseudomonas genera. Remarkably, BRiSK revealed multiple
sequence tags corresponding to seven serotypes of human
papillomavirus (HPV) in the contact lens cases from three of five
subjects, although HPV was not recovered from lens or conjunctival
samples.
Conclusions: Deep DNA sequencing of samples from conjunctiva,
contact lenses, and contact lens cases demonstrate a rich microbial
environment in contact lens cases and a general correlation of genera
between sample locations. HPV sequences were recovered from
numerous contact lens cases suggesting that cases are a potential
source of viral infection.
Commercial Relationships: Dallin Andersen, None; Thuy Doan,
None; Lakshmi Akileswaran, None; Narae Ko, None; Angira
Shrestha, None; Russell Van Gelder, None
Support: NIH Grant R01EY022038, P30EY001730, Unrestricted
Department Grant from Research to Prevent Blindness.
Seasonal trends and demographic variation of viral conjunctivitis
across the US
Karen Christopher1, Bryan A. Stear2, Christopher A. Andrews1,
Joshua D. Stein1. 1Ophthalmology, University of Michigan, Ann
Arbor, MI; 2University of Colorado, Boulder, CO.
Purpose: Viral conjunctivitis is a highly contagious condition that
affects all age groups. Little data exists regarding the timing and
location of outbreaks of this condition. The purpose of this study is to
determine whether there are seasonal and socio-demographic trends
in viral conjunctivitis diagnoses in the United States (US) and how
they vary over time.
Methods: Using eleven years (2002-2012) of longitudinal data from
a nationwide US healthcare claims database, all healthcare encounters
that included an ICD-9-CM diagnosis code suggestive of viral
conjunctivitis (0773, 37200, 37202) were identified for enrollees age
≤21. Spotfire data visualization software was used to plot seasonal
variation of conjunctivitis diagnoses by variables including year of
diagnosis, patient age, sex, household income, race, and geographic
region of the US to identify trends for further analysis.
Results: During the 11 years, there were 671,086 eligible
encounters with a patient ≤21 years old receiving a diagnosis code
of probable viral conjunctivitis. The monthly rate of diagnosis of
viral conjunctivitis per 100,000 enrollees increased from 150.0±27.8
in 2002 to 206.8±63.3 in 2012; p=0.0005. We identified a cyclical
pattern of spikes in viral conjunctivits diagnoses which varied
regionally across the country. The South and West regions of the US
typically have a peak in diagnosis rate in March and April, whereas
the Midwest and Northeast have an additional distinct peak during
December of most years. Over the course of the study, the diagnosis
rates in the South and West regions were most tightly correlated
(r=0.92) whereas the rates in the Northeast and West were the least
tightly correlated (r=0.71). Overall, other demographic factors
including sex, household income, and race did not have a substantial
influence on the seasonal variation.
Conclusions: From year to year, we observe cyclical patterns of
spikes in viral conjunctivitis diagnoses. In a given year, there is a
rise in viral conjunctivitis cases in southern and western states during
March and April, followed by spikes in cases in midwestern and
northeastern states. Researchers can use this data to build predictive
models of the timing and location of future outbreaks and coordinate
efforts with public health officials to educate the public of ways to
protect themselves from the spread of this condition.
Commercial Relationships: Karen Christopher, None; Bryan
A. Stear, None; Christopher A. Andrews, None; Joshua D. Stein,
None
Combination Therapy for Candida Endophthalmitis: An in vitro
study
Daniel Choi1, Harry W. Flynn1, Darlene Miller1, Benjamin D.
Wilson2. 1Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;
2
Ophthalmology, University of Miami School of Medicine, Miami,
FL.
Purpose: Antifungal treatment options for Candida endophthalmitis
are evolving. Combination therapy is increasingly being used to
manage cases. The value of monotherapy versus combination therapy
with azoles and amphotericin B remains unclear. We conducted a
pilot experiment to evaluate the in vitro efficacy of amphotericin
B, voriconazole, and posconazole alone and in combination
(amphotericin B plus voriconazole) against Candida isolates from
patients with endophthalmitis.
Methods: A comparative, experimental microbiological study of
antifungal agents against Candida was performed. Isolates included
Candida parapsilosis (N=2) and Candida albicans (N=9) collected
from 7 vitreous samples and 4 anterior chamber samples recovered
in the last 10 years. E tests were used to document the antifungal
MICs of the Candida isolates and 2 controls Candida parapsilosis
ATCC 22019 and Candida krusei ATCC 6758. Inoculum preparations
to achieve a turbidity equivalent of 0.5 McFarland standard were
made for each sample and control. The inoculum was spread onto
agar plates and allowed to soak for 15 minutes. E test strips (2
amphotericin B, 2 voriconazole, and 1 posconazole) were placed
on each agar plate. After one hour, one of the initial amphotericin
B and voriconazole E tests were switched for voriconazole and
amphotericin B respectively. E test MICs were interpreted after 48
hours described in the E test interpretation protocol. Quality control
strains achieved appropriate MIC levels.
Results: All isolates were tested for MIC90 levels at comparable time
points. MIC90s were: amphotericin B-0.38 ug/ml ( range 0.047 ug/
ml to 0.5 ug/ml), voriconazole 0.008 ug/ml ( 0.003-0.032 ug/ml),
posconazole-0.047 ug/ml (0.023-0.125). The MIC90 for amphotericin
B plus voriconazole was the same as for voriconazole alone- 0.008
ug/ml (range 0.003-0.032) (ie no additive effect).
Conclusions: This pilot in vitro study demonstrated that voriconazole
had the lowest in vitro MICs for these select Candida isolates from
patients with endophthalmitis. Additionally, the combination of
amphotericin B and voriconazole has no additive effect or increased
efficacy against Candida species using the current E test technology.
Commercial Relationships: Daniel Choi, None; Harry W. Flynn,
None; Darlene Miller, None; Benjamin D. Wilson, None
Chitinase 3-like-1 Promotes Candida albicans Killing and
Preserves Corneal Structure and Function by Controlling Host
Antifungal Responses
Nan Gao, Fushin X. Yu. Ophthalmology, Wayne State Univ/Kresge
Eye Inst, Detroit, MI.
Purpose: To elucidate the role of chitinase 3-like 1 (Chi3l1), a
conserved prototypic chitinase-like protein, in controlling corneal
innate immune response to Candida albicans (CA) infection in
C47BL/6 mice.
Methods: Scarified B6 mouse corneas were pretreated with
flagellin and then inoculated with 105 C. albicans. At 6 hpi,
epithelial cells were scraped off the cornea and chi3l1 expression
was detected by PCR and immunohistochemistry. The function of
Chi3l1 was determined by applying recombinant protein or siRNA
subconjunctivally prior to C. albicans inoculation. Disease progress
was monitored by digital photograph, ELISA determination of
cytokine expression, and MPO measurement for PMN infiltration.
Cryostat sections of mouse corneas were immunostained with
antibodies against CXCL10 and CXCR3. For the NF-κB inhibition
experiment, human corneal epithelial cells were pretreated with
NF-κB inhibitor (kamebakaurin) for 30 min before stimulating with
200ng/ml CHI3L1. IκB-α, p-IκB-α and CXCL10 were determined
by Western blotting or dot blotting.
Results: Flagellin-pretreatment markedly induced Chi3l1 expression,
coinciding with greatly enhanced innate immunity against CA
keratitis in B6 mouse corneas. While CA keratitis was more severe
in the corneas treated with Chi3l1 siRNA, the cornea treated with
recombinant Chi3l1 had greatly reduced fungal burden, markedly
decreased PMN infiltration, and much reduced expression of CXCL2.
Chi3l1 treatment resulted in the induction of CXCL10 and IL-17c in
corneal epithelial cells. Exposure of human corneal epithelial cells to
Chi3l1 induced CXCL10 expression in a NF-κB dependent manner.
Conclusions: These data demonstrate that Chi3l1 is induced during
infection, where it promotes fungal clearance and regulates the
appropriateness and intensity of antifungal innate immune responses
in the cornea.
Commercial Relationships: Nan Gao, None; Fushin X. Yu, None
Support: NIH grants R01 EY017960, EY010869
Ubiquitin-Like Protein ISG15 in Host Defense against Candida
albicans Infection in a Mouse Model of Fungal Keratitis
Fushin X. Yu, Chen Dong, Nan Gao. Dept of Ophthalmology, Wayne
State Univ/Kresge Eye Inst, Detroit, MI.
Purpose: ISG15, a di-ubiquitin-like protein, is critical for controlling
certain viral and bacterial infections. We sought to determine if
ISG15 plays a role in corneal innate immunity against fungal
infection.
Methods: Scarified corneas of adult B6 mice were pretreated with
the TLR5 ligand flagellin and then inoculated with C. albicans. The
expression of ISG15 and other genes involved in ISGylation was
determined by realtime PCR and ISG15 expression and distribution
in infected corneas was assessed by immunohistochemistry.
ISGylation was examined by Western blotting. SiRNA knockdown
was used to elucidate the function of ISG15 in controlling fungal
keratitis, through characterization on digital photography and clinical
scoring, fungal counting, ELISA cytokine determination, and PMN
infiltration measurement.
Results: C. albicans infection induced the expression of ISG15,
ISGylation associated genes (UBE1L, UBCH8 and HERC5),
and ISG15 conjugation in mouse cornea epithelial cells. flagellin
pretreatment enhanced innate immunity against C. albicans and
augmented ISG15 expression and ISGylation. ISG15 was most highly
expressed in epithelial cells while infiltrated neutrophils were also
ISG15 positive. ISG15 downregulation increased keratitis severity
and dampened flagellin-induced protection in B6 mouse corneas.
Conclusions: These findings, for the first time, demonstrate that
ISG15-mediated ISGylation plays a critical role in controlling fungal
keratitis. Additional studies are needed to clarify the role of this
molecule in corneal innate immunity and infectious keratitis.
Commercial Relationships: Fushin X. Yu, None; Chen Dong,
None; Nan Gao, None
Support: NIH EY017960, EY010869
Protective Role and Mechanism of NLRP3/Caspase-1
Inflammasome Induced IL-33 Production by Corneal Epithelium
in Fungal Keratitis
Xia Hua1, 2, Xiaoyong Yuan1, 2, Wei Chi1, Jin Li1, Zongduan Zhang1,
Stephen C. Pflugfelder1, De-Quan Li1. 1Ophthalmology, Baylor
College of Medicine, Houston, TX; 2Cataract, Tianjin Eye Hospital,
Tianjin Medical University, Tianjin, China.
Purpose: To explore the protective role and mechanism of innate
immunity by corneal epithelium through NLRP3/caspase-1
inflammasome-induced IL-33 production in response to fungal
infection using Candida albicans (C. albicans) infected fungal
keratitis (FK) mouse model and in vitro human corneal epithelial
cells (HCECs) exposed to heated killed C. albicans (HKCA).
Methods: Fungal keratitis was established in C57BL/6 mice by 106
CFU of C. albicans strain SC5314 isolated from patients. Primary
HCECs, established from donor limbal explants, were challenged
by HKCA (104-106 cells/ml) with or without prior incubation of
inhibitors to NLRP3 (glybenclamide), caspase-1 (z-YVAD fmk) or
dectin-1 (laminarin and syk inhibitor). The production and/or activity
of NLRP3 inflammasome associated molecules and IL-33 were
evaluated by reverse transcription and quantitative real time PCR
(RT-qPCR), immunofluorescent staining, caspase activity assays,
ELISA and Western blot analysis. FK mice were also treated by
recombinant mature form of mouse IL-33 (rmIL-33) to evaluate its
efficacy.
Results: The mRNA and protein levles of IL-33 was found to be
significantly induced, which accompanied by increased production
and activity of NLRP3, ASC, caspase-1, in FK mice compared
with the mock mice as controls. The dose-dependent production
and activity of NLRP3 inflammasome and IL-33 were also
observed in HCECs exposed to HKCA compared to normal control.
Glybenclamide inhibited NLRP3 activation, and also blocked the
activation of caspase-1. Both inhibitors to NLRP3 or caspase-1
suppressed maturation of IL-33. Interestingly, IL-33 maturation and
NLRP3 activation were also suppressed in HCECs exposed to HKCA
with pretreated dectin-1 inhibitors (laminarin and syk inhibitor) and
NF-B inhibitors (BAY11-7082 and quinazoline). Furthermore, the
keratitis was attenuated in severity and recovered rapidly by rmIL-33
subconjunctival injection, while it was exacerbated with treatment of
inhibitors to NLRP3 or caspase-1in FK mice.
Conclusions: Our findings reveal a novel mechanism and signaling
pathway of innate immunity by corneal epithelium, which produces
IL-33 through dectin-1 mediated activation of NLRP3/caspase-1
inflammasome to defense against C. albicans invasion in fungal
keratitis. The studies provide new insight in the innate immunity that
protects cornea from fungal infection.
Commercial Relationships: Xia Hua, None; Xiaoyong Yuan,
None; Wei Chi, None; Jin Li, None; Zongduan Zhang, None;
Stephen C. Pflugfelder, None; De-Quan Li, None
Support: NIH NEI Grants EY023598 (DQL) and EY11915
(SCP), Allergan Inc., Research to Prevent Blindness, Oshman
Foundation, William Stamps Farish Fund; National Natural Science
Foundations of China (81170829, 81400375), Tianjin Research
Program of Application Foundation and Advanced Technology
(12JCYBJC15400), Supporting Project for Key Specialty from
Bureau of Health, Tianjin.
Effect of pretreatment with antifungals on clinical outcomes in
the Mycotic Ulcer Treatment Trial I
Catherine Sun1, Namperumalsamy Venkatesh Prajna2, Lalitha
Prajna2, Muthiah Srinivasan2, Michael E. Zegans3, Stephen D.
McLeod4, Nisha Acharya1, 4, Thomas M. Lietman1, 4, Jennifer RoseNussbaumer1, 4. 1Proctor Foundation, University of California, San
Francisco, San Francisco, CA; 2Aravind Eye Care System, Madurai,
India; 3Dartmouth Medical School, Hanover, NH; 4Ophthalmology,
University of California San Francisco, San Francisco, CA.
Purpose: To determine if pretreatment with antifungal medications is
predictive of worse clinical outcome in a fungal keratitis clinical trial.
Methods: The Mycotic Ulcer Treatment Trial I was a randomized,
double-masked, NEI-funded trial to determine the optimal treatment
for filamentous fungal keratitis. 323 smear-positive fungal ulcer
cases with enrollment visual acuity of 20/40 (0.3 logMAR) to 20/400
(1.3 logMAR) were randomized to receive 5% topical natamycin or
1% topical voriconazole at the Aravind Eye Care System in India.
Prior topical and systemic antifungal use, dose and duration were
collected at enrollment. Corneal scrapings were obtained for smears
and cultures at enrollment and day 7 of treatment. Enrolled patients
had positive smears for filamentous fungus and negative Gram stain
for bacteria. Pre-specified outcomes included 3-month visual acuity
(primary), 3-month infiltrate or scar size, corneal perforation and/or
transplant, and re-epithelialization time.
Results: Of the 323 patients enrolled, 44% presented on an
antifungal with 24% using only topical natamycin, 5% using only
topical or systemic azole, and 15% using both topical natamycin and
an azole. At enrollment, patients pretreated had on average 9 days of
symptoms compared to 6 days for those not pretreated (P<0.001).
Pretreated patients had larger mean baseline infiltrate size (P<0.001)
and larger mean baseline epithelial defect size (P=0.02) compared to
those not pretreated. Pretreated ulcers were significantly less likely
to be fungal-culture positive at day 1 (P=0.04) and day 7 (P=0.046)
compared to ulcers not pretreated. Multivariate regression analysis
demonstrated that pretreatment with an antifungal was associated
with significantly worse 3-month visual acuity (0.36 logMAR, 95%
CI 0.11 to 0.62, P=0.006), larger 3-month scar size (1.47mm, 95%
CI 0.82 to 2.13, P<0.001) and an increased odds of perforation and/
or corneal transplant (OR 12.87, 95% CI 2.68 to 61.70, P=0.001).
Pretreatment was not significantly associated with longer time to reepithelialization.
Conclusions: In our analysis, we found that patients who were
pretreated had longer duration of symptoms, had worse clinical
characteristics at presentation, and were more likely to be fungalculture negative. Study participants who were pretreated had
significantly worse clinical outcomes, likely as a result of initial ulcer
severity.
Commercial Relationships: Catherine Sun, None;
Namperumalsamy Venkatesh Prajna, None; Lalitha Prajna,
None; Muthiah Srinivasan, None; Michael E. Zegans, None;
Stephen D. McLeod, None; Nisha Acharya, Santan (C), Xoma (C);
Thomas M. Lietman, None; Jennifer Rose-Nussbaumer, None
Support: NEI U10EY018573
Acyclovir downregulate TNF-alpha in human limbal fibroblasts
infected in vitro with Fusarium solani isolated from patient with
keratitis
Herlinda Mejia-Lopez, Daniela Castro Farias, Victor M. Bautista.
Research Unit, Inst of Ophthal ““Conde de Valenciana””, Mexico
City, Mexico.
Purpose: Keratomycosis can cause damage with risk the loss of the
eye. The treatment requires early and aggressive approaches based on
the evolution of the lesion and the type of etiologic agent.1 In a first
approach, we found the Acyclovir of inhibiting growth of Fusarium
solani and Aspergillus fumigatus2 and significant decrease was
ejected in MICs by amphotericin B and Natamicyn in presence of
Acyclovir.3
The aim of this work was to assess the expression of IL-6 and TNF-α
from human limbal fibroblasts (HLF), in the presence of Fusarium
solani, treated with Acyclovir, in an in vitro model.
Methods: Human limbal fibroblast from cadaveric donors were
obtained and were infected with conidia of a Fusarium solani strain
isolated from a patient with keratomycosis. Some were treated with
Acyclovir. The expression of IL-6, TNF-α and β-actin by means of
RT PCR and 2-ΔCt was evaluated.
Results: IL-6 is expressed at similar concentrations like HLF without
Acyclovir, also, infection of HLF with conidia e hyphae of F. solani,
induced TNF-α expression to 4.99, and 4.6 times, respectively
compared to control; while the presence of Acyclovir significantly
downregulated the TNF-α expression.
Conclusions: To our knowledge, this is the first study to demonstrate
the Acyclovir modulates the TNF-α expression from HLF infected
with Fusarium solani.
References. 1. Cornea 2009;28:856–859); 2.Invest Ophthalmol Vis
Sci ARVO Abstract 51:E 2432; 3. Invest Ophthalmol Vis Sci. ARVO.
Abstract 55:2833.
Commercial Relationships: Herlinda Mejia-Lopez, None; Daniela
Castro Farias, None; Victor M. Bautista, None
Support: Conde de Valenciana Fundation.
Human Corneal microRNA Expression Profile in Fungal
Keratitis
Bharanidharan Devarajan1, Hemadevi Boomiraj2, VIDYARANI
MOHANKUMAR2, Lalitha Prajna2. 1Bioinformatics, Aravind
Medical Research Foundation, Madurai, India; 2Ocular Microbiology,
Aravind Medical Research Foundation, Madurai, India.
Purpose: Keratitis can progress rapidly with corneal ulceration
through pathological wound healing within 24-48 hours. The
predominant cause of corneal ulceration in India is fungi, may be
responsible for 60-70% of infectious keratitis cases. Many of the
fungal keratitis cases are refractory to medical treatment and will
require corneal transplantation. MicroRNAs (miRNAs) are small,
stable non-coding RNA molecules with regulatory function and
marked tissue specificity that post-transcriptionally regulate gene
expression, however their role in fungal keratitis remain unknown.
Here, our purpose is to identify the miRNAs in human cornea from
fungal keratitis patients and understand their key role in regulation of
pathogenesis.
Methods: Corneal samples from normal cadaver (n=3) and fungal
keratitis (n=5) patients were pooled separately. Deep sequencing was
done using illumina platform to identify miRNA profile. NOISeq
R package was applied to identify the differentially expressed
significant miRNAs. Select miRNAs were validated by Real-time RTPCR (Q-PCR). The regulatory functions of miRNAs were predicted
by combining miRNA target genes and pathway analysis. mRNA
expression levels of select target genes were further analysed by
Q-PCR.
Results: Using deep sequencing, seventy five miRNAs were
identified as differentially expressed with fold change >2 and the
probability score >0.9 in fungal keratitis corneas. MiRNAs with
same isomiRs, showing high fold difference (>5), were confirmed
by Q-PCR. In addition, we predicted their role in regulating target
genes in several pathways. Among those, miR-21-5p, miR-223-3p,
miR-146b-5p, miR-155-5p, miR-511-5p were found to be involved
in inflammatory and immune responses, regulating Toll like receptor
signalling pathways, which is of particular interest. MiR-451a with
an increased expression in keratitis may have a role in wound healing
by targeting Macrophage Migration Inhibitory Factor (MIF). Further,
we highlighted that Neurotrophin signalling pathway may play a role
in wound healing process. One novel miRNA was detected only in
keratitis cornea.
Conclusions: Several miRNAs with high expression in fungal
keratitis corneas point towards their potential role in regulation of
pathogenesis. Further insights in understanding miRNAs role in
wound healing and inflammation may help design new therapeutic
strategies.
Commercial Relationships: Bharanidharan Devarajan, None;
Hemadevi Boomiraj, None; VIDYARANI MOHANKUMAR,
None; Lalitha Prajna, None
γδ T cells regulate the expression of cytokines but not the
manifestation of fungal keratitis
Liya Wang, Siyu He, Hongmin Zhang, Susu Liu, Yanting Xie,
Guoming Chen, Hui Liu. Henan Eye Institute, Henan Eye Hospital,
Zhengzhou, China.
Purpose: As an important immunoregulatory cell type, the role of γδ
T cells in fungal
keratitis(FK) is unclear. We observed the distribution of γδ T cells in
infected corneas in vivo by twophoton microscopy.
Methods: The γδ T cells were depleted by neutralizing antibodies.
The cytokine expression profile was obtained by protein arrays to
determine the cytokines regulated by γδ T cells. ICAM-1, MIP-2
and IL-17A were evaluated by ELISA assays to confirm the role of
γδ T cells in FK. We counted the number of neutrophils, evaluated
the volume of fungal hypha and analyzed the manifestation of the
disease.
Results: The γδ T cells increased significantly at 36 h and 72 h
post fungal infection (P< 0.05) and migrated from the limbus to the
infection site. The neutralizing antibodies completely depleted the
γδ T cells in 24 h. The depletion of γδ T cells led to upregulation of
25 cytokines and downregulation of 3 cytokines. ICAM-1, MIP-2
and IL-17A changed significantly because of the depletion of γδ
T cells (P< 0.05). However, the number of neutrophils, volume of
fungal hypha and manifestation of the disease was not affected by the
depletion of γδ T cells.
Conclusions: Our results demonstrated that γδ T cells have a
role in FK via regulation of some cytokines but did not affect the
manifestation of this disease, suggesting that γδ T cells are not the
key regulator cells in this disease
Commercial Relationships: Liya Wang, None; Siyu He, None;
Hongmin Zhang, None; Susu Liu, None; Yanting Xie, None;
Guoming Chen, None; Hui Liu, None
PAMPs, PRRs and AMPs in Human Corneal Epithelial Innate
Response to Fusarium solani
Satya Sree N. Kolar, Hasna Baidouri, Alison M. McDermott. The
Ocular Surface Institute, UH College of Optometry, Houston, TX.
Purpose: Antimicrobial peptides (AMPs), including defensins and
cathelicidins are known to have antifungal activity against Fusarium
solani (FS). We investigated the effect of fungal and PAMP challenge
on AMP expression and the involvement of pattern recognition
receptor (PRR), TLR2 in human telomerase corneal epithelial cells
(hTCEpi).
Methods: hTCEpi cells were treated with or without heat
inactivated FS, 10mg/ml of fungal derived PAMPs Zymosan (Z) or
Zymosan depleted (ZD) alone or in combination for 6, 12 and 24h.
Supernatants and cell lysates were used to determine protein or
mRNA expression of AMPs hBD2 and LL37 via immunoblotting
and RT-PCR. Micro-broth dilution assays were used to determine
antifungal activity of PAMP treated cell culture supernatants.
To address the involvement to TLR2 in fungal stimulated AMP
expression, 10nM of specific siRNA was used to knock-down TLR2.
Knock-down efficiency, AMP mRNA and protein expression was
determined using PCR and immunoblotting.
Results: Exposure of hTCEpi to heat inactivated FS for 6h caused
significant upregulation of hBD2 and LL37 mRNA expression by 4.55.6 fold relative to control (p<0.0001; n=3). LL37 and hBD2 protein
expression was significantly increased by 6 and 16.5 fold at 24h in
FS treated cells (n=3). Z, ZD and their combination significantly
increased hBD2 and LL37 mRNA expression by 10-45 fold (p<0.001,
n=3) at 6h. AMP mRNA expression was not significantly different
from control at 12 or 24h with any of the treatments (p>0.1). Culture
supernatant from Z and ZD treated cells had greater anti-fungal
activity than that from control cells (n=2). TLR2 siRNA knock-down
efficiency was over 65%. Control scrambled siRNA followed by
FS challenge resulted in a significant increase in hBD2 and LL37
expression (p<0.002). However, knock-down of TLR2 followed by
FS challenge demonstrated no difference in expression compared to
scrambled siRNA control (p>0.05).
Conclusions: hTCEpi respond to fungal challenge with increased
LL37 and hBD2 production which may be responsible for the
anti-fungal activity of culture media from PAMP treated cells. Z
(TLR2 and dectin-1 agonist) and ZD (dectin-1 agonist only) gave
comparable results and knock-down of TLR2 by siRNA did not
significantly modulate FS induced AMP upregulation suggesting
that this PRR is not involved in mediating the AMP response to FS
exposure and that Dectin-1 is more likely involved.
Commercial Relationships: Satya Sree N. Kolar, None; Hasna
Baidouri, None; Alison M. McDermott, None
Support: NIH grants EY13175 (AMM), UHCO Vision Grant to
Advance Research (AMM), EY07551 (UHCO CORE grant)
Cytopathic Mechanisms of Acanthamoeba
Ji-Eun Lee1, Haksun Yu2. 1Ophthalmology, Pusan National University,
Yangsan; 2Parasitology, Pusan National University, Busan, Korea (the
Republic of).
Purpose: To report the mechanism of cytolysis of corneal epithelial
cells by Acanthamoeba using streroid.
Methods: After quantifying the corneal epithelial cell cytolysis
capacity of steroid-treated Acanthamoeba by a spectrophotometric
assay, collagenase activity of Acanthamoeba was measured. In
addition, flow cytometric analyses with annexin V were used
to identify the nature of the corneal epithelial cell response to
Acanthamoeba. Corneal epithelial cells were preincubated with
various concentrations of caspase inhibitors and apoptotic response
was assayed by LDH assay.
Results: Steroid-treated Acanthamoeba induced a significant
cytopathic effect on corneal epithelial cells compared with untreated
organisms. The enhanced cytolysis of epithelial cells by steroidtreated Acanthamoeba was due to increase elaboration of collagenase.
Apoptotic changes in corneal epithelial cells were detected by flow
cytometry and specific inhibitor of caspase-3 significantly reduced
the LDH activity induced by Acanthamoeba.
Conclusions: These findings indicated that exposure of
Acanthamoeba to steroid increased the pathogenicity of the
organisms, and Acathamoeba induced apoptosis in corneal epithelial
cells associated with caspase-3 pathway.
Commercial Relationships: Ji-Eun Lee, None; Haksun Yu, None
Characterization of A. castellanii Ligand that is Recognized by
TLR4 on Corneal Epithelial Cells
Hassan Alizadeh, Trivendra Tripathi, Mahshid Abdi. Cell Biology
and Immunology, UNTHSC Fort Worth TX, Fort Worth, TX.
Purpose: Purpose: Previous results indicate that Acanthamoeba
trophozoites activate TLR2 and TLR4 on human and Chinese hamster
corneal epithelial cells and produce chemokines such as IL-8 or
MIP-2 respectively. However, the components of the Acanthamoeba
trophozoites that induce cytokine and chemokine production remain
unknown. We sought to identify the trophozoite molecules that
interact with TLR4 on the human corneal epithelial cells (HCE) and
trigger the production proinflammatory chemokines.
Methods: Methods: HCE cells and TLR4 expressing HEK293
cells were incubated with or without Acanthamoeba castellanii,
lipopolysaccharide for 12 and 24 h. Inhibition of TLR4 involved
preincubating HEK293 and HCE cells for 1 h with neutralizing TLR4
antibody or with the control antibody followed by incubation with
A. castellanii or LPS for 24 h. Acanthamoeba-membrane protein
(AcMP) was isolated by homogenization of trophozoites using sterile
glass beads in PBS and the resultant homogenates were centrifuged
at 1000×g for 20 min at 40C. The supernatants were collected,
solubilized, and membrane fractions separated by centrifugation
using Mem-PERTM plus kit. AcMP chromatographed by fast protein
liquid chromatography (FPLC) on Superdex 200 10/300 GL column.
Fractions were pooled into four peaks and protein determined by
polyacrylamide gels electrophoresis and western blotting. The
ability of the membrane protein to stimulate the induction of IL-8 in
HEK293 and HCE cells was examined by RT-PCR and ELISA.
Results: Results: mRNA of several TLRs is expressed constitutively
in HCE cells; however, only TLR2 and TLR4 are involved in
Acanthamoeba recognition. A. castellanii induced significant
upregulation of IL-8 production in HCE and TLR4 expressing
HEK293 cells (P< 0.05). Anti-TLR4 antibody attenuated the
production of IL-8 induced by A. castellanii treatment. Four fraction
were identified by chromatography and were subjected to SDS-PAGE
analysis. Only fraction #2 contain one band at approximately 20
KDa. Western blotting of AcMP fractions isolated by FPLC showed
TLR4-antigen in fractions #2. Treatments of HEK293 and HCE cells
with AcMP fractions 1-4 induce significant upregulation of IL-8
production by fraction# 2.
Conclusions: Conclusion: A.castellani recognize TLR4 on HCE
and HEK293 cells by Acanthamoeba-cell membrane protein with a
molecular mass of 20 KDa and induced up-regulation of IL-8.
Commercial Relationships: Hassan Alizadeh, None; Trivendra
Tripathi, None; Mahshid Abdi, None
Support: NEI grant Ro109756
An analysis of the incidence, management, clinical outcomes and
risk factors of Acanthamoeba keratitis infections in a tertiary
hospital in the UK over the last 5 years
Zoe K. Johnson1, Manjusha Narayanan2, We Fong Siah1, Hamed
Anwar1, Francisco C. Figueiredo1. 1Newcastle Eye Centre, Newcastle
Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom;
2
Department of Microbiology, Newcastle Hospitals NHS Trust,
Newcastle upon Tyne, United Kingdom.
Purpose: Acanthamoeba keratitis (AK) is typically characterized by
a rapidly progressive course with increasing pain and deterioration of
vision as the disease progresses. The aim of this study is to report the
incidence of AK at the Royal Victoria Infirmary (RVI), Newcastleupon-Tyne, between 2011 to 2014 and analyze the factors associated
with both good and poor visual outcomes.
Methods: A retrospective case note review was performed of all
microbiology confirmed consecutive patients who underwent corneal
scrapes specifically for acanthamoeba keratitis at the RVI, between
November, 2011 to November, 2014. Cases that were lost to followup or moving out of the area were excluded from the study.
Results: Fourteen cases of AK were identified, i.e. mean age 32;
range 21-76; M=8 F=6). The mean time to diagnosis from symptom
onset to first presentation to an ophthalmologist was 15 days (range
1-21 days). The mean time from first ophthalmic examination to
securing a diagnosis of AK was 25 days (range 0-124 days), with an
average of 3 physician visits (range 1-9) needed before a diagnosis
of AK was suspected clinically. Fifty seven percent of patients
were initially treated as herpetic keratitis prior to diagnosis. Two
patients received topical steroids prior to diagnosis, which led to
considerable worsening of their condition. In all cases, corneal
scraping and culture confirmed the diagnosis. Cysts were seen on in
vivo confocal microscopy in 4 cases. The mean duration of topical
treatment was 7.5 months. A reactivation of AK occurred in 1 case.
There were 4 cases where an early diagnosis of AK was established,
all of whom had best corrected visual acuity of 20/30 or better at last
follow-up. Corneal scarring necessitating penetrating keratoplasty
(PKP) occurred in 4 patients, while cataract and glaucoma due to AK
occurred in 2 patients.
Conclusions: A moderate increase in the incidence of AK was noted
(2014 - 5 cases, 2012 - 2 cases). Seventy nine percent of all subjects
used soft monthly contact lenses. In all cases, corneal scraping and
acanthamoeba culture primarily made a diagnosis of AK. Better
visual outcomes were associated with earlier diagnosis, while
cases with poorer visual outcomes were diagnosed later. Steroid
administration prior to diagnosis of AK was associated with a poorer
prognosis.
Commercial Relationships: Zoe K. Johnson, None; Manjusha
Narayanan, None; We Fong Siah, None; Hamed Anwar, None;
Francisco C. Figueiredo, None
The efficacy of Polihexanide (PHMB) eye drops against
Acanthamoeba polyphaga investigated by an ATPbioluminescence assay and a rat model of keratitis
Antonino Asero1, Andrea Sudano Roccaro1, Loic Favennec2, Julie
Gueudry2, Laetitia Le Goff2, Anna Rita Blanco1. 1SIFI SPA, Lavinaio,
Italy; 2Universitè de Rouen, Rouen, France.
Purpose: To assess the efficacy of different PHMB concentrations,
for potential use in humans, against Acanthamoeba polyphaga using
in vitro and in vivo test systems.
Methods: In vitro: An ATP-bioluminescence assay was adapted
to screen the activity of PHMB against Acanthamoeba polyphaga
cysts. Previously viability studies and reported minimal cysticidal
concentrations of PHMB, ranging from 0.0001-0.005%, allowed us
to select 1:10 and 1:100 dilutions of 4 different PHMB concentrations
(0.02, 0.04, 0.06 and 0.08%) to be tested against Acanthamoeba
polyphaga cysts for exposure times of 0.5, 1, 3 and 7 hours.
In vivo: Acanthamoeba polyphaga was inoculated into rat corneal
stroma and animals treated 4 times a day with PHMB at 0.02, 0.04,
0.06 and 0.08% or with PHMB 0.02% and propamidine 0.1%
(combination therapy). Rat corneas were examined and scored
clinically every week until day 28. At the end of the experiment
superficial corneal epithelium was removed for cultured and PCR and
the eye then assessed histologically for Acanthamoeba polyphaga
invasion.
Results: In vitro: Killing curves of all the 10-fold dilutions of PHMB
concentrations showed the same profile with a 90% reduction of
Acanthamoeba polyphaga cyst viability at 3 h. The 100-fold dilutions
of 0.04, 0.06 and 0.08% PHMB, showed a 60% reduction of cyst
viability. The 100-fold dilution of 0.02% PHMB was the least
efficacious with 40% reduction of cyst viability at 3 hours.
In vivo: Monotherapy with 0.02% PHMB drops decreased the clinical
severity grading, but not Acanthamoeba polyphaga viability as
graded by culture, PCR or histology. Monotherapy with 0.04, 0.06 or
0.08% PHMB, as well as combination therapy, significantly reduced
the clinical severity and/or Acanthamoeba polyphaga viability by
culture, PCR and histology.
Conclusions: These findings suggest that PHMB 0.02% is less
effective than either of the other PHMB concentrations tested, or of
combination therapy. However the discriminating power of these
test systems could be further improved by the inclusion of more
quantitative read outs. We think these are promising test systems
for investigating the efficacy of antiamoebic drugs and that their
refinement will increase their sensitivity and value in preclinical
testing.
Commercial Relationships: Antonino Asero, SIFI SpA (E);
Andrea Sudano Roccaro, SIFI SpA (E); Loic Favennec, None;
Julie Gueudry, None; Laetitia Le Goff, None; Anna Rita Blanco,
SIFI SpA (E)
Support: The ODAK project is funded by the EU under the Seventh
Framework Programme (Grant N. 305661)
Expression of Angiogenic Regulators by Human Retinal Cells
Infected with Toxoplasma gondii: Understanding the Clinical
Course of Ocular Toxoplasmosis
Shervi Lie, Liam M. Ashander, Binoy Appukuttan, Justine R. Smith.
Eye & Vision Health, Flinders University, Adelaide, SA, Australia.
Purpose: Retinal infection with the parasite, T. gondii, induces
substantial inflammation and results in sizeable scarring. However,
unlike other inflammatory posterior segment diseases that
involve scarring, ocular toxoplasmosis is rarely complicated by
neovascularization. To understand this phenomenon, we investigated
the expression of angiogenic regulators, VEGF-A and -B, PEDF and
TSP-1, and transcription factor, HIF-1α, by retinal cells that modulate
the development of neovessels.
Methods: The natural T. gondii strain, GT-1, was maintained in
tachyzoite form by culture in fibroblasts. Monolayers of human
retinal pigment epithelial (ARPE-19) cells or human Mueller
(MIO-M1) cells were infected with freshly egressed tachyzoites
(MOI 5:1) in DMEM with up to 5% FBS or cultured with medium
alone. Cell invasion and intracellular replication were confirmed by
microscopy, and parasite viability was determined by plaque assay.
Total RNA or protein was isolated at 4 and 24 hours post-infection
(n=6/condition) and analyzed by qRT-PCR (RPLP0 and PPIA as
references) or Western blot (β-actin as reference), respectively.
Statistical significance was defined by p<0.05 on t-test.
Results: VEGF-A mRNA was significantly induced 1.9- to 2.4-fold
in infected ARPE-19 cells compared to controls at 4 and 24 hours
post-infection; isoforms 121, 165 and 189 increased (≥2.6-fold)
at 4 and/or 24 hours. PEDF and TSP-1 mRNA rose significantly
(≥2.2 fold) at one or both time points. Consistently, HIF-1α protein
increased by 24 hours in infected cells. Simultaneously, VEGF-B
mRNA did not change. In MIO-M1 cells, infection with T. gondii
significantly elevated PEDF mRNA at 4 hours (1.8-fold), but induced
no rise in VEGF-A and its isoforms, VEGF-B or TSP-1 at the tested
time points. Parasite viability was >15% for all infections, consistent
with published measurements for natural isolates.
Conclusions: Although infection with T. gondii increased expression
of pro-angiogenic VEGF-A isoforms in human retinal pigment
epithelial cells, these changes were balanced by high expression of
anti-angiogenic PEDF and TSP-1, and lack of rise in VEGF-B, as
well as increased expression of PEDF by human Mueller cells. Our
findings may explain the rare occurrence of neovascularization in
ocular toxoplasmosis, in comparison to other inflammatory posterior
segment diseases associated with scarring.
Commercial Relationships: Shervi Lie, None; Liam M. Ashander,
None; Binoy Appukuttan, None; Justine R. Smith, None
Support: NHMRC (GNT1066235) and ARC (FT130101648)
Cell death of gamma interferon (IFNg)-stimulated human
macrophages upon Toxoplasma gondii infection limits parasite
replication
Eleni Konstantinou1, 2, Jeroen P.J. Saeij2, Lucy H. Young1.
1
Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard
Medical School, Boston, MA; 2Biology, Massachusetts Institute of
Technology, Cambridge, MA.
Purpose: Toxoplasmosis is the most common cause of infectious
retinochoroiditis. It is caused by the parasite Toxoplasma gondii,
which affects both immune compromised and immune competent
patients. The cytokine interferon gamma (IFNg) plays an important
role in the inhibition of Toxoplasma growth. In some cell types
(such as HeLa cells) IFNg induces the enzyme indoleamine
2,3-dioxygenase (IDO) leading to tryptophan depletion and restriction
of Toxoplasma growth while other cell types restrict Toxoplasma
growth through an unknown mechanism. Macrophages and other
innate immunity cells that infiltrate the retina in ocular toxoplasmosis
play an important role in fighting the infection but can also contribute
to the inflammation in the eye. It is unclear how human macrophages
inhibit Toxoplasma growth. The goal of this study is to determine
how IFNg-stimulated human cells inhibit Toxoplasma replication.
Methods: An in vitro study using the MM6 and THP-1human
macrophage cell lines was performed. Cells were stimulated
with IFNg for 24 h and subsequently infected with Toxoplasma
expressing luciferase and GFP. Parasite growth over time was
determined by microscopy and by measuring luciferase activity.
Macrophage cell death was determined by measuring the release of
lactate dehydrogenase into the culture medium. To assess whether
tryptophan depletion was implicated in cell death, cells were
supplemented with tryptophan or treated with an inhibitor of IDO.
Results: IFNg-stimulated human macrophages rapidly died upon
Toxoplasma infection and thereby restricted parasite growth. We
obtained similar results in two different human macrophage cell lines
and in human fibroblasts. Cell death was independent of tryptophan
depletion. Cell death of human fibroblasts was also independent of
necroptosis and apoptosis. We are currently determining the role of
a variety of candidate genes in the induction of cell death by making
knockout macrophage cell lines using CRISPR/Cas9.
Conclusions: Increased death of IFNg-stimulated cells upon
Toxoplasma infection is an effective mechanism of Toxoplasma
growth restriction. Unraveling this mechanism of cell death could
eventually lead to better therapeutics to treat ocular toxoplasmosis.
Commercial Relationships: Eleni Konstantinou, None; Jeroen P.J.
Saeij, None; Lucy H. Young, None
The intraocular cytokinome is linked to clinical characteristics in
human toxoplasmosis
Claudia Thieme1, Stephan Schlickeiser2, Claudia Dames2, Sylvia
Metzner1, Hans-Dieter Volk2, Uwe Pleyer1. 1Department of
Ophthalmology, University Hospital Berlin, Berlin, Germany;
2
Immunology, University Hospital Berlin, Berlin, Germany.
Purpose: To investigate the cytokine profiles of aqueous humor
samples of patients with primary (p) or recurrent (r) ocular
toxoplasmosis (OT) and potential correlations with clinical
characteristics.
Methods: We investigated aqueous humor samples of 62 individuals/
patients (27 male, 35 female, mean age: 36 yrs). All patients
included in this study were confirmed as OT by intraocular positive
toxoplasma gondii antibodies (Goldman/Witmer coefficient>3).
The Bioplex-Immunoassay was used to analyze for 27 chemokines/
cytokines. Our study cohort consisted of 22 pts. with primary (p)
OT infection and 29 pts. with recurrent (r)OT and 11 age matched
individuals as controls. The Wilcoxon-Mann-Whitney-Test was
applied to compare cytokine levels between groups, whereas the
spearman correlation was used for a subgroup analysis of recurrent
OT.
Results: Significant differences of the intraocular cytokine profile
could be related to clinical features. Firstly, we demonstrate that
several cytokines are highly elevated in pOT as well as in rOT as
compared to controls. IFNγ as an important primary indicator for
intraocular immune response was higher in pOT (p=0.0002) and
rOT (p=0.0017). We also observed that IL-17-levels were increased
in pOT (p=0.05), but not in rOT (p=0.2996). IL-12-levels are not
significantly elevated in pOT (p=0.75) and even slightly decreased
in rOT (p=0.38). Moreover, we could correlate the number of
recurrences with a reduction of IL-9 (p=0.01), TNFα (p=0.0326), IL13 (p=0.0326) and IFNγ (p=0.0315).
Conclusions: The role of the intraocular cytokinome is still under
debate. Our results confirm the significant contribution of IFNγ in
both pOT and rOT. In contrast, intraocular IL-12, as major inducer
of IFNγ, remained low. This may imply that other pathways of OT
activation are important. IL-17 as a potential target for specific
therapy, was significantly upregulated in active pOT but not rOT.
As an interesting feature we first demonstrate an impaired IL-9
production which highly correlated with rOT. Currently, the role of
TH9 cells in OT is unknown and warrants further investigation.
Strategies designed to identify certain cytokine clusters could
potentially select patients at risk for recurrent retinitis.
Commercial Relationships: Claudia Thieme, None; Stephan
Schlickeiser, None; Claudia Dames, None; Sylvia Metzner, None;
Hans-Dieter Volk, None; Uwe Pleyer, None
Trimethoprim/Sulfamethoxazole and Azithromycin combination
in the treatment of presumed ocular toxoplasmosis
Zakia Berkani3, Yacine kitouni2, Abdelhak Lakehal1, Daoud
Roula2. 1Service de médecine préventive et d’épidémiologie
CHU Constantine Dr Benbadis, Faculté de médecine, Université
Constantine 3, Constantine, Algeria; 2Service de médecine interne
CHU Constantine Dr Benbadis, Faculté de médecine, Université
Constantine 3, Constantine, Algeria; 3service d’ophtalmologie,
CHU de Constantine Dr Benbadis, Faculté de médecine, Université
Constantine 3, Constantine, Algeria.
Purpose: To evaluate the efficacy and safety of trimethoprim/
sulfamethoxazole and azithromycin combination for the treatment of
presumed ocular toxoplasmosis.
Methods: Sixteen patients, treated for presumed ocular
toxoplasmosis (OT), from september 2012 to january 2014, in
a tertiary referral hospital’s Ophtalmology department, were
retrospectively reviewed.
The treatment consisted on an association :Trimethoprim
-Sulfamethoxazole 2 times daily (160mg / 800mg for 6 weeks) and
azithromycin (500mg the first day followed by 250 mg for 5 weeks)
Corticosteroids: 48 hours after the beginning of treatment according
to the associated inflammatory signs and location of retinochoroidal
lesion.
Main outcome measures: Changes in retinochoroidal lesion size after
treatment, the time interval until resolution of inflammation, best
corrected visual acuity (BCVA) before and after intervention, adverse
drug reactions during follow-up, and rate of recurrence.
Results: The mean follow-up time of the patients was 20.43 ± 7.2
(range; 6–32) months. Final VA improved in all patients (100%) with
a mean BCVA of 0.7 ± 0.5 (P= 0.01). Inflammatory findings began to
subside within 14 ± 9.0 days. The reduction of lesion’s size was seen
in 75% cases; the mean lesion size was 1.6 ± 0.8 (p =0.0001).
One patient (0.16%) had recurrent attack. One patient (0.16%) had
side effects from therapy.
Conclusions: Trimethoprim/sulfamethoxazole and azithromycin
combination seems to be effective and safe alternative therapy for the
treatment of ocular toxoplasmosis.
Commercial Relationships: Zakia Berkani, None; Yacine kitouni,
None; Abdelhak Lakehal, None; Daoud Roula, None
270 Inflammation / Infection and imaging
Monday, May 04, 2015 3:45 PM–5:30 PM
Multimodal evaluation of Vogt-Koyanagi-Harada disease: A
12-month prospective longitudinal study from the acute onset
Viviane M. Sakata, Carlos E. HIrata, Maria K. Oyamada, Smairah
F. Abdallah, Ever E. Rodriguez, Celso Morita, Joyce H. Yamamoto.
Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil.
Purpose: To describe Vogt-Koyanagi-Harada disease (VKHD)
course during a 12-month follow-up from the acute onset concerning
clinical, morphological and functional aspects.
Methods: Nine VKHD patients, diagnosed according to Revised
Diagnostic Criteria, were included from the acute onset following
a predefined protocol: 3-day pulsetherapy with methylprednisolone
followed by slow tapering of oral prednisone over at least 1 y; folow
up protocol included clinical exam, imaging exams (autofluorescence,
indocyanine green and fluorescein angiography-FA- and enhanced
depth-optical coherence tomography-OCT) performed with Spectralis
HRA+OCT and ERG (RETI-port system; Roland Consult, Germany).
The following disease activity signs defined “flare”: anterior chamber
cells (ACC), choroidal neovascular membrane, perivascular or
optic disc leakage on FA, dark dots (DD), fuzzy vessels, increase
in choroidal thickness (CT≥30%) or worsening of ERG parameters
(≥30%) in two consecutive visits, after 2 mo of quiescence, without
change in scheduled treatment. After 6 mo from disease onset,
three patterns of disease evolution were characterized: A: no flare,
B: subclinical flare or C: clinical flare (ACC and/or FA signs).
Two trained readers analyzed autofluorescence patterns. The study
protocol followed the statements of the Declaration of Helsinki and
was approved by local Institutional Review Board.
Results: Nine patients (7F/2M) median age of 33 y/o and median
time to treatment of 12 d (3-46) were included. At day 30, all eyes
had visual acuity ≥ 1,0 (logMar) and 12/18 eyes resolved retinal
detachment. At 12th mo, DD were attenuated, though still observed
in all eyes. Concerning disease pattern of evolution: 1 eye had no
flare (A), 5 eyes had only subclinical (B) and 12 eyes had clinical
flare (C). In 17/18 eyes (94%) at least one sign of inflammation was
observed at 8 mo with a median prednisone dose of 0.3mg/kg/d.
16/17 eyes had ≥2 inflammatory signs. CT and autofluorescence
pattern at one mo were related to chronic evolution with clinical
flares (p=0.03; kappa=0.7, CI95%0.3-1.0).
Conclusions: 8/9 VKHD patients had subclinical or clinical flares
during the 12-mo follow up even when treated with early high-dose
corticosteroids. Oral prednisone dose around 0.3mg/kg/d was a
critical point when flares were observed. Autofluorescence pattern
and CT analysis at 1 mo can help to identify more severe cases.
Commercial Relationships: Viviane M. Sakata, None; Carlos E.
HIrata, None; Maria K. Oyamada, None; Smairah F. Abdallah,
None; Ever E. Rodriguez, None; Celso Morita, None; Joyce H.
Yamamoto, None
Support: FAPESP 2011/50936-7;2011/19194-4;2014/01222-0
The retinal pigment epithelium as a gateway for healing
macrophages into the retina
Inbal Benhar1, Kitty Reemst1, Vyacheslav Kalchenko2, Michal
Schwartz1. 1Neurobiology, Weizmann Institute of Science, Rehovot,
Israel; 2Veterinary Resources, Weizmann Institute of Science,
Rehovot, Israel.
Purpose: A self-perpetuating process of neuronal degeneration takes
place in the retina following mechanical or biochemical insults to
the eye or optic nerve. Monocyte-derived macrophages have been
identified as important players in arresting this process. Since the eye
is an immune-privileged site, endowed with specialized barriers to
preserve its integrity, we hypothesized that the entry route of immune
cells to the eye dictates their fate. Specifically, we envisioned that
“healing” monocyte-derived macrophages enter the eye through the
retinal pigment epithelium (RPE).
Methods: C57BL/6J mice were subjected to either acute biochemical
or mechanical injury (retinal glutamate intoxication or optic nerve
crush, respectively). Monocyte augmentation was accomplished by
either intravenous or intravitreal adoptive transfer, or by CNS-based
neuroprotective vaccination. The RPE response to injury and the
interaction of monocytes with the RPE and retina were monitored by
immunohistochemistry, quantitative Real Time PCR, flow cytometry
and live imaging.
Results: Monocytes, injected either intravenously or intravitreally
following glutamate intoxication to the retina, conferred retinal
ganglion cell protection. The injected monocytes localized to the
site of damage, as well as to the subretinal space, adjacent to the
RPE. Mechanical injury in the form of optic nerve crush resulted in
immune cell infiltration into the retina, which was further augmented
under experimental manipulations that lead to better neuronal
survival. The RPE responded to injury with the elevated expression
of adhesion and trafficking molecules. Live imaging showed the
sequential accumulation of monocytes in the RPE and retina after
ONC.
Conclusions: Our results emphasize the relationship between the
activation of the RPE for leukocyte trafficking and the recruitment
of monocyte-derived macrophages, which display a beneficial
effect within the retina following injury. They further highlight the
possibility that the RPE serves as a selective and educative gateway
for “healing” immune cell recruitment to the retina, similar to the role
displayed by the brain’s choroid plexus in the context of CNS injury.
Commercial Relationships: Inbal Benhar, 61/915,069 (P); Kitty
Reemst, None; Vyacheslav Kalchenko, None; Michal Schwartz,
61/915,069 (P)
Support: EU FP7 Grant 279017
Pathogenic Bacteria Disrupt the Immunomodulatory Function of
Conjunctival Goblet Cells
Laura Contreras-Ruiz1, Qiang Shan2, Mihaela G. Gadjeva2, Sharmila
Masli1. 1Department of Ophthalmology, Boston University School
of Medicine, Boston, MA; 2Department of Medicine, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA.
Purpose: Conjunctival goblet cells (GC) confer ocular surface
protection via mucin secretion. Previously we reported their
immunomodulatory role based on their ability to release soluble
factors like active TGFβ and support tolerogenic phenotype of
dendritic cells. In this study we determined the effect of cytotoxic
vs. commensal bacteria on the immunomodulatory function of
conjunctival GCs.
Methods: Primary cultures of GCs were generated from WT
(C57BL/6) conjunctival explants. Cultured GCs were challenged
with heat-inactivated cytotoxic (Pseudomonas aeruginosa, PA14) or
commensal (Streptococcus sp.) bacterial strains for 24h. Proliferation
and apoptosis of GCs was assessed by BrdU/7-AAD staining and
flow cytometry. Culture supernatants were evaluated for levels of
MUC5AC by ELISA and active TGFβ using MFB11 reporter assay.
Expression of TGFβ2 and thrombospondin-1 (TSP1) in GCs was
determined by RT-PCR. Expression of activation markers MHC class
II, CD80, CD86 and CD40 was assessed by RT-PCR on bone marrow
derived dendritic cells (BMDCs) co-cultured with GCs in a transwell
system for 24h.
Results: Viability of GCs challenged with either the cytotoxic or the
commensal strain at MOI=60 was comparable to that in untreated
control cells. At this concentration, the commensal strain induced
38% increase in GC proliferation and no change in apoptosis
as compared to untreated control cells. However, the challenge
with the cytotoxic strain induced a 17% increase in GC apoptosis
without changing proliferation as compared to untreated control
cells. Supernatants from GCs challenged with the cytotoxic strain
contained significantly diminished levels of MUC5AC and active
TGFβ compared to untreated controls, while the commensal strain
did not induce such change. The decline in active TGFβ correlated
with significantly reduced expression of TSP1 in GCs challenged
with the cytotoxic but not the commensal strain. Consistently,
a significant increase in the expression of MHC class II and costimulatory molecules was detected in BMDCs co-cultured with GCs
challenged with the cytotoxic but not the commensal strain.
Conclusions: Cytotoxic strains of bacteria, but not commensals,
disrupt ocular surface homeostasis by altering mucin secretion as
well as the immunomodulatory function of conjunctival goblet cells.
These results underscore the significant contribution of conjunctival
goblet cells in preventing ocular surface inflammation.
Commercial Relationships: Laura Contreras-Ruiz, None; Qiang
Shan, None; Mihaela G. Gadjeva, None; Sharmila Masli, None
Support: NEI grant EY015472
Characterization of YFP+ cells in the retina of CD11c-eYFP
transgenic mice: the effects of breeding out the Crb1rd8 mutation
Samantha Dando1, Xiangting Chen1, Marc Ruitenberg2, Paul G.
McMenamin1. 1Department of Anatomy and Developmental Biology,
School of Biomedical Science, Monash University, Clayton, VIC,
Australia; 2School of Biomedical Sciences, University of Queensland,
St Lucia, QLD, Australia.
Purpose: The normal CNS parenchyma is traditionally thought to
be devoid of professional antigen presenting cells (APCs); however,
a population of putative dendritic cells (DCs) expressing yellow
fluorescent protein (YFP) are present in the neural retina of C57Bl/6N
CD11c-eYFP mice. We have previously shown that these mice carry
the Crb1rd8 mutation, which results in retinal dystrophic lesions. We
hypothesized that the accumulation of YFP+ putative DCs within the
retina may be the result of the pathology associated with the Crb1rd8
mutation.
Methods: C57Bl/6N CD11c-eYFP mice were outcrossed with
C57Bl/6J mice to create Crb1rd8/wt offspring; heterozygous offspring
were then intercrossed to generate CD11c-eYFP Crb1wt/wt mice. At 6
weeks of age, CD11c-eYFP Crb1wt/wt mice (n=50) underwent clinical
fundus examination in brightfield and fluorescent modes. At 8 weeks
of age, eyes were collected from perfusion-fixed mice (n=5) and
retinal wholemounts were processed for confocal microscopy. Single
cell suspensions were prepared from pooled retinas (n=10 mice),
stained with an antibody panel for the identification of APC surface
markers and subsequently characterized by flow cytometry.
Results: In vivo examination of the eyes of CD11c-eYFP Crb1wt/
wt
mice revealed normal appearance of the fundus in brightfield
mode, with no retinal lesions observed. However, fluorescent fundus
imaging demonstrated the continued presence of YFP+ cells in the
retina. Quantitative analysis of retinal wholemounts by confocal
microscopy revealed a density of 94±3.3 (mean ± SEM) YFP+ cells/
mm2. These YFP+ cells, which were predominantly distributed within
the plexiform and nerve fiber-ganglion cell layers of the retina, coexpressed the microglial markers CD11b, F4/80 and Iba-1, whereas
few YFP+ cells expressed the APC markers CD11c, CD80, CD86,
CD103, CD8, DEC205, 33D1 and MHC class II.
Conclusions: The detection of YFP+ cells in the retina of CD11ceYFP Crb1wt/wt mice indicates that the presence of these cells is not
wholly a consequence of the Crb1rd8 mutation. Retinal YFP+ cells
displayed an immunophenotype that is consistent with microglia, and
not DCs, suggesting that the YFP+ cells in the retina of CD11c-eYFP
Crb1wt/wt mice represent a subset of microglia.
Commercial Relationships: Samantha Dando, None; Xiangting
Chen, None; Marc Ruitenberg, None; Paul G. McMenamin, None
Support: National Health and Medical Research Council of Australia
grant: APP1069979
Targeting P-TEFb recruitment and super-enhancers function
suppress the development of Th1-mediated EAU disease
Malihe Eskandarpour2, Arnulf Hertweck1, Catherine Evans1,
Jonathan Lau1, Audrey Kelley3, Peter S. Adamson2, David Cousins3,
Virginia L. Calder2, Graham Lord3, richard jenner1. 1UCL
Cancer Institute, London, United Kingdom; 2UCL, Institute Of
Ophthalmology, London, United Kingdom; 3King’s College London,
London, United Kingdom.
Purpose: We aimed to determine how T-bet acts through the
enhancer elements to drive Th1 differentiation and also to provide
further insight into the mechanism of super-enhancer function using
human and mice models.
Methods: To identify the lineage-specific genes and groups of
genes repressed by JQ1 and/or Flavopiridol in Th1 cells, wild-type
C57BL/6 mouse naive CD4+ T cells were cultured under Th1 and
Th2 polarizing conditions and run for gene expression profiling
using the Mouse GE 8x60K microarrays (Agilent).The libraries
were generated for RNA-seq and RNA-Chip and quantified and then
sequenced on an Illumina HiSeq (50bp paired-end) and Illumina
GAIIx ro HiSeq 2000 sequencer, respectively. To induce EAU,
B10RIII mice aged 5-8 weeks were immunized with 300 mg IRBP161–
emulsified with Complete Freund’s adjuvant, supplemented with
180
1.5 mg/ml Mycobacterium tuberculosis complete H37 Ra (1:1
v/v). Each mouse also received 0.4mg Bordetella pertussis toxin
intraperitoneally. Flavopiridol and JQ1 were then administered by
daily intraperitoneal injection and disease progression scored by
retinal funduscopy and histology.
Results: We revealed that T-bet acts through super-enhancers
to recruit P-TEFb and activate lineage-specific transcriptional
elongation. We showed that recruitment of P-TEFb to Th1 genes
depends on T-bet activity and both T-bet and P-TEFb function at
super-enhancers to activate transcription of enhancer RNAs. We
also demonstrated that P-TEFb activity was required for Ifng eRNA
production and treating Th1 cells with JQ1 and Flavopiridol resulted
in a marked reduction in eRNA levels. This requirement for P-TEFb
function renders lineage-specific genes hyper-sensitive to inhibitors
of transcriptional elongation both in vitro and in vivo in a mouse
model of uveitis. EAU mice treated with Flavopiridol and JQ1
showed suppression of disease which associated with inhibition of
super-enhancer function. Flow cytometry of CD4+ T cells sorted
from the retina and lymph node revealed that expression of the superenhancer-associated gene products Ifng, Tnf, Fasl, Il18r1 and Ctla4
were downregulated by Flavopiridol and JQ1.
Conclusions: Using in vitro and in vivo models, we concluded that
P-TEFb binds extensively to the super-enhancers of lineage-specific
genes and revealed a molecular mechanism for the selective effect of
those drugs on genes involved in mediating inflammation.
Commercial Relationships: Malihe Eskandarpour, None; Arnulf
Hertweck, None; Catherine Evans, None; Jonathan Lau, None;
Audrey Kelley, None; Peter S. Adamson, None; David Cousins,
None; Virginia L. Calder, None; Graham Lord, None; richard
jenner, None
Dendritic cells are recruited to the outer retina by cone death in a
mouse model for Type 2 Leber congenital amaurosis
Peter H. Tang, Mark J. Pierson, Neal D. Heuss, Dale S. Gregerson.
Ophthalmology & Visual Neurosciences, University of Minnesota,
Minneapolis, MN.
Purpose: Using the CD11c-DTR/GFP mice, we have previously
shown that GFP+ dendritic cells (DC) were recruited to retina
following optic nerve crush injury (ONC). Cells were heavily
concentrated in the retinal ganglion cell/nerve fiber layer (RGC/
NFL), consistent with the site of injury, and in the outer plexiform
layer. Other studies showed they were the antigen-presenting
cells (APC) within the retina. To explore if GFP+ DC home to the
site of injury, mice exhibiting photoreceptor degeneration were
tested. Mutation of the gene encoding RPE65 protein leads to
disruption of innate retinoid metabolism and development of Type
2 Leber congenital amaurosis (LCA2), a form of retinal dystrophy
characterized by early-onset of cone death and progressive visual
impairment. Details of the mechanism for cone death in this disease
remain unknown. We investigated the timing, localization and role of
DC associated with cone death in a mouse model for LCA2.
Methods: To develop the animal model, Rpe65-/- mice were bred to
transgenic mice whose DC expressed a chimeric protein containing
GFP and diphtheria toxin receptor driven by a CD11c promoter
(CD11c-DTR/GFP mice). Progeny were screened by PCR and
immunoblotting to confirm transgenic Rpe65-/- mice along with
Rpe65+/+ and Rpe65+/- mice as controls. Cone survival was evaluated
by counts from retinal flatmounts stained for cone opsins, and
morphology was further evaluated by cryosections of transgenic
Rpe65-/- and Rpe65+/+ mice.
Results: Homozygous knockout of the Rpe65 gene from progeny
that was bred to the transgenic CD11c-DTR/GFP background
was confirmed through PCR and immunoblotting. In these mice,
cone counts showed massive cone death compared to Rpe65+/+
transgenic mice. Morphology showed increased GFP+ cells within
the outer retina in Rpe65-/- mice compared to controls. The DC were
concentrated within the areas of cone death, unlike their homing to
the RGC/NFL following an ONC.
Conclusions: These results show that the GFP+ DC were recruited to
the site of injury or stress. In contrast to the results with an ONC to
the RGC, the GFP+ DC in the RPE65-/- mice were recruited to outer
retina by the death of cone cells due to disruption of intrinsic retinoid
metabolism as seen in LCA2. The presence and activity of these cells
may have implications for novel therapeutics involving immune cells
in the treatment for congenital retinal dystrophies.
Commercial Relationships: Peter H. Tang, None; Mark J.
Pierson, None; Neal D. Heuss, None; Dale S. Gregerson, None
Support: Wallin Neuroscience Discovery Fund (DSG); NIH/NEI
grant R01EY021003 (DSG)
Atypical autophagic responses in sensory neurons induced by
HSV-1 infection and interferon signaling
David A. Leib, Sarah Katzenell. Microbiology and Immunology,
Geisel School of Medicine at Dartmouth, Lebanon, NH.
Purpose: Herpes simplex virus-1 (HSV-1) establishes lifelong
latent infections in the sensory neurons of trigeminal ganglia (TG)
wherein it retains the capacity to reactivate. Interferon β (IFNβ)
drives antiviral responses, which are crucial to control of HSV-1 lytic
replication and reactivation within the host neuron. Additionally,
autophagy may play a role in these neuronal antiviral responses. We
therefore sought to investigate the relationship between antiviral
signaling and autophagy during HSV-1 infection of TG neurons in
vitro and in vivo.
Methods: We cultured TG neurons from adult mice expressing
GFP fused to the autophagy protein LC3. GFP-LC3 allows rapid
and simple identification and quantification of fluorescent punctate
autophagosomes. We used this system to measure neuronal
autophagic responses to viral infection and IFNβ treatment.
Additionally, we infected mice via the cornea and examined
autophagic responses in TG during acute linfection.
Results: In the absence of viral infection we observed a basal level
of autophagy in the TG neurons that resulted in the formation of
standard-sized autophagic puncta (0.5-1.5mm). Following infection,
however we observed an accumulation of novel large puncta (≥4mm)
which high resolution microscopy has shown to be an aggregation of
autophagosomes. These structures co-localize with other autophagy
markers (p62 and lysosomes), and were disrupted by bafilomycin,
an inhibitor of autophagy maturation. Interestingly we showed that
these large autophagsomes were also induced by IFNβ, and were
dependent on STAT1 signaling. Moreover, these large puncta also
appeared in TG following in vivo infection of mice via the cornea,
and were preferentially present in neurons that were not expressing
viral antigens, suggesting that neurons containing autophagosomes
are resistant to productive viral gene expression.
Conclusions: Together these data suggest that these large
autophagosomes may be a novel and critical component of the
neuronal IFN-driven antiviral response that may regulate acute
infection and the maintenance of latency. The antiviral modulation
of autophagy may therefore be an attractive target for the control of
acute and recurrent HSV infections.
Formation of atypical autophagic puncta during acute infection of
trigeminal ganglia with HSV-1. Puncta were preferentially present in
neurons that were not expressing viral antigens.
Commercial Relationships: David A. Leib, None; Sarah
Katzenell, None
Support: NIH Grant EY09083
324 Ocular inflammation; clinical treatment and experimental
models
Tuesday, May 05, 2015 8:30 AM–10:15 AM
Dynamic changes of M1 and M2 macrophages in a murine model
of endotoxin-induced uveitis
Omar Delgado, Maura Crowley, Wei Zheng, Casey Lewis, Kathryn
McAllister, Bruce D. Jaffee, Sha-Mei Liao. Ophthalmology, Novartis
Institutes for BioMedical Research, Cambridge, MA.
Purpose: To investigate the roles of M1 and M2 macrophages in
TLR4-mediated acute ocular inflammation using a murine model
of endotoxin-induced uveitis (EIU). M1 macrophages promote
inflammation, while M2 macrophages have anti-inflammatory and
wound-healing properties. Both M1 and M2 macrophages have been
implicated in the pathogenesis of age-related macular degeneration.
Methods: EIU was induced by an intraperitoneal injection of
lipopolysaccharide (LPS), and tissues were collected at various time
points. RNA samples were prepared for microarray (transcriptional
profiling) and Taqman (gene expression) analysis and protein
extracts were prepared for Western blot and multiplex cytokine
assays (Aushon). To quantitate leukocyte infiltration, retinas were
immunostained for Gr-1+ neutrophils and F4/80+ macrophages, while
posterior eye cups (RPE-choroid-sclera) were stained for Iba1+
microglia cells.
Results: Within 24 hours after an intraperitoneal injection of LPS,
genes encoding M1 macrophage markers such as iNOS, CD40,
IL-6, and IL-1b were up-regulated in eye tissues both the retina and
in the RPE-choroid-sclera. In contrast, M2 macrophage markers
Arg1, CD163, and CD209 were reduced during the first 24 hours
as compared to PBS-treated controls. The M2 macrophage markers
were elevated after 24 hours, and peaked between 3-5 days after
LPS administration. Taqman analysis of several M1 and M2
genes correlated with the microarray results. In addition, the proinflammatory cytokine and chemokine proteins IL-6, Il-1b, IL-8,
and MCP-1 were elevated at the same time as M1 gene induction
(3-16 hours), followed by up-regulation of cell adhesion molecules,
complement activation proteins, and Gr1+-neutrophil infiltrates in the
retina (16 hours-2 days). The number of retinal F4/80+ macrophages
and subretinal Iba1+ microglia cells increased in response to LPS and
peaked around 3-5 days post LPS injection, correlating with the peak
of M2 macrophage gene expression.
Conclusions: Intraperitoneal injection of LPS induces an acute
inflammatory response in the eye by activating resident cells
and M1 macrophages within the first 24 hours followed by M2
macrophages on day 3 to 5. These M1 macrophages are associated
with inflammatory mediators and infiltrating neutrophils into the eye.
This work identifies in vivo markers for M1 and M2 macrophages in
the eye and demonstrates their role in the acute ocular inflammatory
processes.
Commercial Relationships: Omar Delgado, Novartis Institutes
for BioMedical Research (F); Maura Crowley, Novartis Institutes
for BioMedical Research (F); Wei Zheng, Novartis Institutes for
BioMedical Research (F); Casey Lewis, Novartis Institutes for
BioMedical Research (F); Kathryn McAllister, Novartis Institutes
for BioMedical Research (F); Bruce D. Jaffee, Novartis Institutes
for BioMedical Research (F); Sha-Mei Liao, Novartis Institutes for
BioMedical Research (F)
Alterations in tight junction expression and endothelial cell
density during a mouse model of sterile corneal inflammation.
Holly R. Chinnery. Optometry and Vision Sciences, University of
Melbourne, Parkville, VIC, Australia.
Purpose: We have previously reported that application of toll-like
receptor (TLR) ligands onto the injured corneal surface of C57BL/6
mice induces corneal edema at 24 hours post-treatment which
subsides by one week. We tested the hypotheses that endothelial
expression of the tight junction protein, zonula occludens-1 (ZO-1),
would be altered during experimental sterile corneal inflammation
and that the endothelial cell density (ECD) would remain unaffected.
Methods: C57BL/6J mice aged 6-10 weeks were anaesthetized and
received central 1mm corneal abrasions with an Alger Brush followed
by topical application of saline or CpG-ODN (TLR9 agonist). At 24
hours and one week post-treatment, eyes were enucleated and fixed
in paraformaldehyde and processed for immunofluorescence staining
of ZO-1. Confocal imaging of corneal flat mounts were analysed for
expression of endothelial ZO-1 (mean pixel intensity) and the density
of corneal endothelial cells was evaluated by masked observers.
Differences between groups were determined using ANOVA.
Results: Compared to untreated corneas, the expression of ZO-1 by
the corneal endothelium was unchanged in saline-treated corneas
at both 24 hours and 1 week. Treatment with CpG-ODN led to
a significant reduction in the expression of ZO-1 by the corneal
endothelium at 24 hours, which returned to normal levels by 1 week.
Endothelial cell density was similar to untreated corneas at 24h but
was significantly reduced in CpG-ODN treated corneas at 1 week
(2009 ± 46 [mean ± S.E.M] cells/mm2) compared to untreated eyes
(2426 ± 102 cells/mm2). There was no difference in the ECD between
CpG-ODN treated eyes and saline treated eyes at one week.
Conclusions: Experimental sterile corneal inflammation was
associated with a transient reduction in ZO-1 expression by the
corneal endothelium. The loss of corneal endothelial cells at later
stages of inflammation suggest that sterile insults to the cornea may
be associated with a preceding down-regulation of tight junction
proteins.
Commercial Relationships: Holly R. Chinnery, None
Support: NH&MRC grant 1042612
The Aldehyde Trap NS2 Reduces Ocular Inflammation in an
Endotoxin-Induced Model in Rats
Kenneth J. Mandell, Scott L. Young, Todd C. Brady. Aldeyra
Therapeutics, Lexington, MA.
Purpose: NS2 is an investigational drug in development for acute
noninfectious anterior uveitis and other ocular indications. The
mechanism of action of NS2 involves neutralizing toxic freealdehydes, which are mediators of inflammatory disease, both in the
eye and elsewhere. The purpose of this study was to evaluate efficacy
of a topical NS2 eye drop formulation in a rat model of ocular
inflammation.
Methods: Ocular inflammation was induced in Lewis rats by foot
pad injection of lipopolysaccharide (LPS). Thirty (30) female Lewis
rats were split in 3 groups to receive topical NS2 0.5%, balanced
salt solution (BSS) or dexamethasone 0.1% (DEX) at 1, 3, 7, 10 and
17 hours post-induction. Ocular exams were performed at 24 hours
post-induction and scored using a combined Combined Draize and
McDonald-Shadduck scoring system. Animals were euthanized to
collect ocular tissues, and levels of inflammatory markers ICAM-1
(Intercellular Adhesion Molecule 1), IL-6 (Interleukin 6) and MCP-1
(Monocyte Chemoattractant Protein 1) were measured from retinachoroid specimens by enzyme-linked immunosorbent assay (ELISA).
Results: The mean total ocular examination (OE) scores at 24
hours post-LPS induction were 21.4, 10.1 and 15.7 for the topical
BSS, DEX and NS2 groups, respectively. These reductions in mean
total OE scores were statistically significant for both NS2 and
DEX relative to the BSS control. Similarly, scores for individual
components of the OE revealed similar trends with statistically
significant reductions observed for conjunctival hyperemia, iris
hyperemia and anterior chamber flare for both NS2 and DEX.
Statistically significant reductions in ICAM-1 levels were observed
for both NS2 and DEX relative to BSS. However, a statistically
significant reduction in IL-6 was observed only for NS2. MCP-1
levels could not be detected in any group.
Conclusions: Together these findings provide symptomatic and
biochemical evidence of the anti-inflammatory effects of the aldehyde
trap NS2 in a model of inflammatory eye disease. The production of
toxic aldehydes is mechanistically linked to uveitis and a variety of
other inflammatory ocular diseases. The ability of NS2 to trap and
sequester free-aldehydes may prove to be a novel mechanism for the
treatment of ocular inflammatory diseases.
Commercial Relationships: Kenneth J. Mandell, Aldeyra
Therapeutics (C); Scott L. Young, Aldeyra Therapeutics (E); Todd
C. Brady, Aldeyra Therapeutics (E)
Experimental autoimmune uveoretinitis in Akita/slc mice
spontaneously developing diabetes
Kouzo Harimoto1, Masataka Ito2, Yoko Karasawa1, Yutaka Sakurai1,
Masaru Takeuchi1. 1Ophthalmology, National Defense Medical
College, Tokorozawa City, Japan; 2Developmental Anatomy
and Regenerative Biology, National Defense Medical College,
Tokorozawa, Japan.
Purpose: Recently, several studies have indicated that inflammatory
process is involved in the pathogenesis of diabetic retinopathy
provoking retinal tissue damage, and that progression of proliferative
diabetic retinopathy was mediated by various systemic and local
factors. In this study, we induced experimental autoimmune
uveoretinitis in Akita/slc mice spontaneously developing diabetes,
and evaluated an effect of diabetes on ocular inflammation.
Methods: Six to eight week-old female Akita/slc mice with
C57BL/6 background and the wild type mice were immunized with
human interphotoreceptor retinoid-binding protein (amino acid
sequence 1-20) peptide and complete Freund’s adjuvant to induce
EAU. Funduscopic examinations were performed on days 11, 14,
and 17 after immunization, and SD-OCT images of the retina was
obtained on day 20. In addition, after mice were euthanized on day
21, eyes were enucleated and the histopathological examination
was performed. The severity of EAU was assessed by clinical and
histopathological scores and by grades of SD-OCT images.
Results: Clinical scores of EAU on days 11, 14, and 17 were 1.25 ±
0.44, 1.50 ± 0.51, and 1.80 ± 0.41 in wild type mice, and 1.85 ± 0.37,
2.50 ± 0.51, 2.70 ± 0.4 in Akita/slc type mice, which was significantly
higher in Akita/slc type mice compared with wild type mice on each
observation day. Scores of EAU in SD-OCT was also significantly
higher in Akita/slc type mice (2.67±0.52) than in wild type mice
(1.83±0.41), which was corresponding to the histopathological scores
of Akita/slc type mice (2.16±0.38) and wild type mice (1.54±0.52).
Conclusions: It was indicated that EAU in mice developing diabetes
is more severe than that in non-diabetic mice.
Commercial Relationships: Kouzo Harimoto, None; Masataka
Ito, None; Yoko Karasawa, None; Yutaka Sakurai, None; Masaru
Takeuchi, None
Human ESC-derived Mesenchymal Stem Cells Attenuate
Experimental Autoimmune Uveitis
Yu Qin1, Ann M. Chan1, Nicholas Kouris2, Maria-Dorothea Nastke2,
Erin Kimbrel2, Negin Ashki1, Wei Wang1, Robert Lanza2, Ralph D.
Levinson1, Lynn K. Gordon1. 1Ophthalmology, UCLA, Los Angeles,
CA; 2Advanced Cell Technology, Marlborough, MA.
Purpose: Mesenchymal stem cells (MSCs) have significant tissue
regeneration potential as well as immunomodulatory properties,
exerted by direct contact and in a paracrine fashion; therefore MSC
therapy is explored as a promising treatment for autoimmune disease.
Uveitis is a category of inflammatory diseases that affects humans
and is a significant cause for vision loss. Here, we investigate the
effects of human embryonic stem cell-derived MSCs (hESC-MSCs)
on experimental autoimmune uveitis (EAU), a murine model of
uveitis.
Methods: EAU was induced in mouse by peptides of the
interphotoreceptor retinoid binding protein (IRBP). B10RIII mice
were immunized with 50μg IRBP 161-180 and C57BL6 mice with
500μg IRBP 1-20 in the presence of 1.5μg immune adjuvant pertussis
toxin. hESC-MSCs were obtained from Advanced Cell Technology
(ACT, Boston, MA). Intraperitoneal injections of 5 million hESC-
MSCs were performed on day 0 or day 7. Clinical exams were
performed at the peak of EAU and mice were euthanized for
histology analysis.
Results: Treatment of hESC-MSCs on day 0 significantly decreased
EAU histology scores in B10RIII (p=0.04) and C57BL6 (p=0.0001)
mice, and clinical exam scores in C57BL6 (p=0.0002) mice
compared to untreated control EAU mice. Treatment of hESC-MSCs
on day 7 had a tendency of reducing EAU inflammation in mice;
however, the effect was not statistically significant.
Conclusions: Early systemic treatment of hESC-MSCs ameliorated
both severe (B10RIII) and mild (C57BL6) EAU in murine models.
Additional work is needed to fully understand the mechanism of
attenuation and whether MSCs can be used during periods of active
uveitis to control disease.
Commercial Relationships: Yu Qin, None; Ann M. Chan, None;
Nicholas Kouris, None; Maria-Dorothea Nastke, None; Erin
Kimbrel, None; Negin Ashki, None; Wei Wang, None; Robert
Lanza, None; Ralph D. Levinson, None; Lynn K. Gordon, None
SOCS1-Mimetic Eye-drops inhibit ocular inflammation
and protect against ocular pathology during Experimental
Autoimmune Uveitis (EAU)
Chang He, Chengrong Yu, Lin Sun, Rashid M. Mahdi, Justine Yeung,
CHRALES E EGWUAGU. National Eye Institute, National Institutes
of Health, Bethesda, MD.
Purpose: Suppressor of cytokine signaling 1 (SOCS1) inhibits
inflammation by targeting JAK kinases and activated cytokine
receptors for degradation in the proteasome, thereby terminating
STAT signals activated by pro-inflammatory cytokines. Enhancing
SOCS1 activity has therefore been considered as a potential
therapeutic approach to mitigate pathology in inflammatory
and autoimmune diseases. However, a major impediment to
the therapeutic use of SOCS1 is its relatively short half-life.
Consequently there is significant interest in developing strategies
to efficiently deliver exogenous SOCS1 into cells. In this study,
we have synthesized a 16 amino acids lipophilic SOCS1 peptide
(SOC1-KIR) that penetrates cell membrane, interacts with JAK autophosphorylation loop and inhibits its kinase activity. We investigated
whether topical administration of SOCS1-KIR can be used as therapy
for uveitis.
Methods: We induced EAU in C57/BL6 mice by immunization with
interphotoreceptor retinoid-binding protein in complete Freund’s
adjuvant. Mice received 10mg SOCS1-KIR (diluted in PBS to 5ml/
eye) as eye drops, every day beginning Day 0 until Day 12. Disease
severity was assessed by fundoscopy, optical coherence tomography,
histological examination, and electroretinogram. The development of
inflammation or production of inflammatory molecules was assessed
by flow cytometry and mRNA analysis of cells in the draining lymph
nodes and retina.
Results: Treatment with SOCS1-KIR eye drops efficiently
suppressed EAU and protected mice from ocular pathology by
inhibiting lymphocyte proliferation and limiting infiltration of
inflammatory cells into the eye. In line with our in vivo results,
analyses of lymphocytes isolated from the lymph nodes and spleen
of the mice show that treatment with the SOCS1-KIR inhibited the
expression of chemokine receptors and integrins, which mediate
lymphocyte trafficking. Importantly, SOCS1-KIR had no effect on
systemic immune response.
Conclusions: Local administration of SOCS1-KIR peptide inhibited
the development of uveitis in the mouse EAU model by suppressing
the expansion of pathogenic cells that mediate uveitis and their
recruitment into the retina. SOCS1-KIR is nontoxic, suggesting that
topical administration of SOCS1-Mimetics can be exploited as a noninvasive treatment for human uveitis.
Commercial Relationships: Chang He, None; Chengrong Yu,
None; Lin Sun, None; Rashid M. Mahdi, None; Justine Yeung,
None; CHRALES E EGWUAGU, None
Mechanisms involved in the anti-inflammatory effect of
melatonin in experimental uveitis
Pablo Sande, Damian Dorfman, Magali Silberman, Diego
Fernandez, Monica Chianelli, Daniel Saenz, Ruth E. Rosenstein.
Human Biochemistry/Sch of Med, University of Buenos Aires,
Buenos Aires, Argentina.
Purpose: Uveitis is a prevalent intraocular inflammatory disease.
We have previously shown that melatonin not only prevents but
also counteracts LPS-induced uveitis in the Syrian hamster. The aim
of this work was to identify the mechanisms involved in the antiinflammatory effect of melatonin administered after the onset of
ocular inflammation.
Methods: Syrian hamster eyes were intravitreally injected with
vehicle or LPS. Melatonin was intraperitoneally supplied every 24
h, starting 12 h or 24 h post-LPS injection. Prostaglandin (PG) E2
and PGF2α levels were assessed (radioimmunoassay) in the aqueous
humor. Moreover, retinal nitric oxide synthase (NOS) activity
(using 3H-arginine), lipid peroxidation (thiobarbituric acid reactive
substance levels), and TNFα levels (enzyme-linked immunosorbent
assay) were examined. Light microscopy and immunohistochemistry
(Müller cell glial fibrillary acidic protein (GFAP)) were used to
evaluate the retinal structure.
Results: Concomitantly with an improvement of the clinical score
and retinal function (electroretinogram), both treatments with
melatonin significantly decreased PG levels in aqueous humor from
eyes injected with LPS. Moreover, both treatments with melatonin
protected the retinal structure, reduced Müller cell GFAP levels,
and the increase in retinal nitric oxide synthase (NOS) activity, lipid
peroxidation, and TNFα levels induced by LPS.
Conclusions: These results indicate the involvement of aqueous
humor prostaglandins, and retinal TNFα levels, NOS activity, and
oxidative stress in the attenuation of ocular inflammation induced
by LPS, and further support the use of melatonin as a therapeutic
resource for uveitis treatment.
Commercial Relationships: Pablo Sande, None; Damian
Dorfman, None; Magali Silberman, None; Diego Fernandez,
None; Monica Chianelli, None; Daniel Saenz, None; Ruth E.
Rosenstein, None
Support: CONICET PIP 0446, FUNDACION FLORENCIO
FIORINI, ANPCyT PICT 0610, UBA
Characterization of endotoxin-induced uveitis in two different
mouse strains.
Fátima Sofía Magaña1, Joaquín A. Quiroz1, Gibrán Estua-Acosta1,
Mariana García1, Yonathan Garfias1, 2. 1Research Unit, Institute of
Opthalmology, Mexico, City, Mexico; 2Biochemistry, Faculty of
Medicine, UNAM, Mexico, City, Mexico.
Purpose: Uveitis is a systemic disease in which the uvea is inflamed.
It has been reported that uveitis animal models can be generated
in the laboratory. In this context, the aim of the present work was
to determine the differences on clinical signs of uveitis as well
as the eye leukocyte infiltration in two different mouse strains
intraperitoneally exposed to LPS from E. coli.
Methods: Following the ARVO statement of animal care on
investigation for visual sciences, male BALB/c and C57BL/6
mice from 6-8 weeks old were intreperitoneally injected with LPS
from E. coli. Animals were clinically evaluated using stereoscopic
microscopy. After 24 or 48 h of treatment, the mice were euthanized
and the eyes were removed in order to determine histologic leukocyte
infiltration. Student t test was performed to determine differences
between groups taking a p<0.05 as statistically significative.
Results: Intraperitoneally LPS induced clinical signs of uveitis in
both mouse strains, which consisted in aqueous flare in the anterior
chamber, miosis, iriitis and retinitis. In both strains, retinal blood
vessels were igurgitated. These clinical signs appeared as soon as
24 h post LPS-injection and remain until 48 h. However, the clinical
severity was higher in BALB/c mice in comparison to C57BL/6
mice. As expected, histological leucocyte infiltration was significantly
higher (p<0.05) in BALB/c mice compared to C57BL/6 mice at 24
and 48 h post LPS-injection. Interesingly, there was a diferentially
infiltration pattern, presenting a higher number of leucocytes
infiltration in the posterior segment than in the anterior segment of
the eye.
Conclusions: Taken together these results, suggest that endotoxin
induced uveitis animal model depends not only on the administation
via but also on the genetic background of the experimental mouse
strain.
Financial Support: This project was supported by CONACYT:
SALUD 160286; CIENCIA BASICA 167438; DGAPA-PAPIIT
IA203514; CVU: 406452.
Commercial Relationships: Fátima Sofía Magaña, None; Joaquín
A. Quiroz, None; Gibrán Estua-Acosta, None; Mariana García,
None; Yonathan Garfias, None
Support: This project was supported by CONACYT: SALUD
160286; CIENCIA BASICA 167438; DGAPA-PAPIIT IA203514;
CVU: 406452.
Ischemia-reperfusion injury as a model of activation and
resolution of retinal neuroinflammation and vascular
permeability
Steven F. Abcouwer, Sumathi Shanmugam, Cheng-mao Lin, Heather
Lindner, Dolly A. Padovani-Claudio, Prathiba Jayaguru, Arivalagan
Muthusamy, David A. Antonetti. Ophthalmology & Visual Science,
Univ of Michigan Kellog Eye Ctr, Ann Arbor, MI.
Purpose: The retina is often considered an immune privileged
tissue with vascular endothelial tight junctions (TJ) that restrict
leukocyte trafficking. Despite the broad use of ischemia reperfusion
(IR) injury to investigate neuronal damage, little is known about
the neuroinflammatory and vascular responses in this model
of sterile retinal damage. We hypothesized that IR induces a
neuroinflammatory and vascular permeability response with
subsequent resolution.
Methods: Ischemia was induced in C57BL/6 mice for 90
min followed by natural reperfusion. The effect of IR on
neurodegeneration, vascular permeability and neuroinflammation
over 4 weeks was examined using measures of retinal cell death by
DNA fragmentation; integrity of the blood-retinal barrier (BRB) by
FITC-BSA leakage; the disassembly of endothelial TJ complexes by
immunofluorescence (IF); retinal edema and retinal layer thinning in
situ by optical coherence tomography (OCT); and luminal leukostasis
and tissue infiltration of leukocyte subsets by IF and flow cytometry
examining leukocyte, myeloid and granulocyte markers
Results: The IR model exhibited rapid neuronal cell death, BRB
permeability and neuroinflammation that was sustained for 2 weeks
and resolved by 4 weeks. OCT demonstrated a transient edema
followed by layer thinning up to 2 weeks. A dramatic increase in
leukostasis occurred at day 1, consisting of both lymphocytes and
myeloid leukocytes, composed primarily of Ly6C(hi) inflammatory
monocytes and Ly6G(+) granulocytes. A transition to leukocyte
infiltration occurred between days 1 and 4, continued for 2 weeks and
was fully resolved at 4 weeks. As myeloid inflammation progressed
toward resolution, granulocytes were replaced by Ly6C(neg)
reparative patrolling monocytes.
Conclusions: The mouse retinal IR-injury represents a model
of prolonged but self-limiting neuroinflammation and vascular
permeability that can be used to study mechanisms of resolution of
cellular inflammation and restoration of the BRB.
Commercial Relationships: Steven F. Abcouwer, None; Sumathi
Shanmugam, None; Cheng-mao Lin, None; Heather Lindner,
None; Dolly A. Padovani-Claudio, None; Prathiba Jayaguru,
None; Arivalagan Muthusamy, None; David A. Antonetti, None
Support: Supported by a grant from Novo Nordisk (SFA and DAA),
NIH R01 EY012021 (DAA), NIH R01 EY007739 (SFA) and NIH
P30 EY007003 Core Center for Vision Research.
Modulation of monocyte activation by retinal pigment epithelium
(RPE)-derived exosomes
Jared E. Knickelbein1, Susan Hannes1, Baoying Liu1, Anush
Arakelyan2, Jean Charles Grivel2, Arvydas Maminishkis3, H Nida
Sen1, Sheldon S. Miller3, Leonid Margolis2, Robert B. Nussenblatt1.
1
Laboratory of Immunology, National Eye Institute, Rockville,
MD; 2Section on Intercellular Interactions, National Institute of
Child Health and Human Development, Bethesda, MD; 3Section
on Epithelial and Retinal Physiology and Disease, National Eye
Purpose: Exosomes are important mediators of intercellular
communication and have been implicated in modulation of the
immune system. We sought to investigate if exosomes secreted from
retinal pigment epithelial (RPE) cells could alter the activation status
of immune cells in vitro.
Methods: ARPE-19 cells were stimulated or not with the
inflammatory cytokines interferon gamma (IFN-g), tumor necrosis
factor alpha (TNF-a), and interleukin 1 beta (IL-1b) for 48 hours.
Supernatants were harvested for isolation of exosomes with the
ExoQuick TC isolation kit, and ARPE-19 cells were assayed for
expression of surface and intracellular CD81 and CD107b (known
exosomal proteins) by flow cytometry. Isolated exosomes were
quantified with a NanoSight NS300 nanoparticle analyzer and
cultured for 24 hours with either THP-1 or enriched human donor
monocytes, which were assayed for expression of ICAM-1 (THP-1)
or the phenotypic markers CD14 and CD16 (human monocytes) by
flow cytometry.
Results: Stimulation of ARPE-19 cells with IFN-g, TNF-a, and
IL-1b reduced the expression of surface and intracellular CD81,
while levels of intracellular CD107b (LAMP-2) were unaltered,
compared to non-stimulated controls. The number of exosomes
secreted from ARPE-19 did not differ between stimulated and
non-stimulated cultures. THP-1 monocytes upregulated ICAM-1
expression upon exposure to exosomes isolated from non-stimulated
and cytokine-stimulated ARPE-19 cells compared to THP-1 cells not
exposed to exosomes. However, exosomes from cytokine-stimulated
ARPE-19 cells caused significantly higher ICAM-1 expression
per THP-1 cell compared to those from non-stimulated ARPE-19.
Exposure to exosomes from non-stimulated ARPE-19 cells induced
undifferentiated human monocytes into a more regulatory phenotype
with a significantly higher percentage of CD14++CD16+ cells
compared to human monocytes exposed to exosomes from ARPE-19
cells stimulated with cytokines.
Conclusions: RPE constitutively secrete exosomes. The quality but
not the quantity of exosomes secreted from ARPE-19 cells is altered
with cytokine stimulation. Exosomes from cytokine-stimulated
ARPE-19 cells activated THP-1 cells to express high levels of ICAM1, while exosomes from non-stimulated ARPE-19 cells induced
human monocytes toward a regulatory phenotype. These findings
suggest that the inflammatory milieu of the RPE can influence
monocyte activation in a paracrine fashion through exosomes.
Commercial Relationships: Jared E. Knickelbein, None; Susan
Hannes, None; Baoying Liu, None; Anush Arakelyan, None; Jean
Charles Grivel, None; Arvydas Maminishkis, None; H Nida Sen,
None; Sheldon S. Miller, None; Leonid Margolis, None; Robert B.
Nussenblatt, None
Support: Funds from the Intramural Research Program of the
National Eye Institute, National Institutes of Health
Infection model of the human retinal pigment epithelium cell
line ARPE-19 by Mycobacterium tuberculosis complex bacillus
Calmette-Guérin.
Victor Llorens1, Blanca Molins1, Marina Mesquida1, Maite Sainz
De La Maza1, Javier Zarranz-Ventura1, Anna Sala-Puigdollers1,
Julian Gonzalez-Martin2, Alfredo Adan Civera1. 1Institut Clínic
d’Oftalmologia (ICOF), Hospital Clínic de Barcelona, Barcelona,
Spain; 2Clinical Microbiology & Parasitology, Hospital Clínic de
Barcelona, Barcelona, Spain.
Purpose: To establish and characterize a novel infection model
of human retinal pigment epithelium (RPE) by M. bovis bacillus
Calmette-Guérin (BCG).
Methods: BCG strain Pasteur 1173 P2 was cultured in LowensteinJensen medium. Isolated colonies were suspended in culture media
and disaggregated. Suspensions were adjusted to Mc Farland 1.0.
ARPE-19 cells were grown until 90% confluence and bacterial
inoculum was dispensed at a MOI 100:1 for 3 h. Control and infected
ARPE-19 cells were trypsinized and seeded at a concentration
of 100 cells/ml. Cytotoxicity and cytoproliferation assays were
determined by WST-1 reagent and crystal violet dye elution (CVDE),
respectively 3, 24, 48, 72 and 96 h post infection. To determine
infection rates, ARPE-19 cells were trypsinized and counted by
trypan blue. A cell suspension of 50 μl was lysed, serially diluted
and cultured. Colony forming units (CFU) were counted and the
infection rate was determined as CFU/cells. In parallel, 50ml of cell
suspension were stained with modified Ziehl-Neelsen and infection
rate determined by modified Crowle method. Control and infected
epithelium were cultured for 2 months and analyzed for cytobacteriological interaction by optical microscopy.
Results: Infection rate peaked at 48 h. We observed a substantial
difference in infection rate between the two methods (16%±5.7% by
CFU count and 40%±7.7% by microscopy, p=0.058). Correlation
between both methods was moderate (r=0.538). Microscopy
examination of infected cells showed adhesion, phagocytosis,
intracellular proliferation and cytolysis with extracellular invasion
by BCG bacteria (Fig.1, a to e, respectively). Interestingly, some
giant infected cells were also observed at early infection stages. In
late infected epithelium (1 month), we observed exosome release in
those cells around BCG colonies (Fig.2). Cytotoxicity in infected
cells was minimal and did not differ significantly from uninfected
cells. However, cytoproliferation was significantly enhanced by BCG
infection at 48 h (p=0.042).
Conclusions: BCG can infect ARPE-19 cells with little toxicity.
Exosome release from infected cells could be on the bases of a
sustained intraocular immune response in tuberculosis-related uveitis
(TRU). This reproducible biosafe model provides new opportunities
for the understanding of the pathogenesis of TRU.
Figure 1.
Figure 2.
Commercial Relationships: Victor Llorens, None; Blanca Molins,
None; Marina Mesquida, None; Maite Sainz De La Maza, None;
Javier Zarranz-Ventura, None; Anna Sala-Puigdollers, None;
Julian Gonzalez-Martin, None; Alfredo Adan Civera, None
Shedding of Endomucin by Endothelial Cells under
Inflammatory Conditions: Involvement of Matrix
Metalloproteases
Jinling Yang1, Magali Saint-Geniez1, Yin Shan Eric Ng1, Marsha A.
Moses3, 4, Patricia A. D’Amore1, 2. 1Department of Ophthalmology,
The Schepens Eye Research Institute, Massachusetts Eye and Ear
Infirmary, Harvard Medical School, Boston, MA; 2Department of
Pathology, Harvard Medical School, Boston, MA; 3Vascular Biology
Program, Boston Children’s Hospital, Boston, MA, Boston, MA;
4
Department of Surgery, Harvard Medical School, Boston, MA.
Purpose: Interactions between inflammatory cells and endothelial
cells (ECs) are critical to vascular inflammation, which has been
heavily implicated in many ocular diseases. We have previously
shown that endomucin (EMCN), an apically localized, EC-specific
transmembrane mucin, blocks adhesion of inflammatory cells to
ECs under non-inflammatory conditions and is downregulated by
TNF-α. Furthermore, EMCN overexpression blocks TNF-α-induced
inflammatory cell adhesion to EC in vitro and inflammatory cell
infiltration in vivo. Activation of matrix metalloproteases (MMPs)
that occurs during inflammation mediates protein cleavage. This
study investigated the role of MMPs in the regulation of EMCN
under inflammatory conditions.
Methods: Human umbilical vein ECs (HUVECs) or human retinal
ECs (HRECs) were treated with TNF-α, IL-1β and IFN-γ for 24
hrs or pervanadate (50 mM, 30 min), a strong oxidant that mimics
inflammatory conditions. The role of MMPs was examined by
pretreatment of HUVECs with MMP inhibitors (BB94 5 mM or
GM6001 10 mM). Total and cell surface EMCN protein levels were
evaluated by immunoblotting alone or following biotinylation.
EMCN localization was revealed by immunocytochemistry.
Results: TNF-α, IL-1β or IFN-γ downregulated total EMCN protein
in HUVECs in a dose-dependent manner; at 1 ng/ml, total EMCN
protein was reduced to 33.2±1.1% (P<0.001), 27.8±5.4% (P<0.01),
59.0±2.6% (P<0.01) of controls, respectively. In HREC, 1 ng/ml
TNF-α also reduced total EMCN protein to 37.9±0.8% (P<0.001).
TNF-α-induced reduction in total EMCN protein in HUVECs
was partially blocked by BB94 (59.5±4.2% P<0.001) or GM6001
(70.7±14.2% P<0.05). BB94 led to a significant preservation of
cell surface EMCN (31.2±10.3% vs. 65.6±5.4%, P<0.01) in TNFα-treated HUVECs. Immunocytochemical localization confirmed
that BB94 partially prevented EMCN loss from the cell surface and
cell margins. Pervanadate treatment reduced total EMCN protein
to 59.3±4.3% of controls (P<0.001) and this was nearly totally
normalized (92.0±12.9% P<0.01) by BB94. A C-terminal EMCN
antibody reacted with a low molecular weight fragment (~15kD) in
lysates from pervanadate-treated cells, which was absent in BB94- or
vehicle-treated groups.
Conclusions: These results indicate a role for MMPs in the shedding
of EMCN under inflammatory conditions and suggest a novel
therapeutic target for inflammatory ocular diseases.
Commercial Relationships: Jinling Yang, None; Magali SaintGeniez, None; Yin Shan Eric Ng, None; Marsha A. Moses, None;
Patricia A. D’Amore, AGTC (C), Eleven Biotherapeutics (S)
Support: EY05318
In Vitro Infection of Human Retinal Pigment Epithelium with
Chlamydia trachomatis
Ernest Boiko1, Dmitrii Maltsev1, Alexei Pozniak4, Alevtina
Savicheva2, Kira Shalepo2, Igor Kvetnoi3, Victoria Polyakova3, Alexei
Suetov1, Irina Nuralova1. 1Department of Ophthalmology, Military
Medical Academy, St. Petersburg, Russian Federation; 2Laboratory of
Microbiology, Ott Research Institute of Obstetrics and Gynaecology,
St Petersburg, Russian Federation; 3Pathology Department, Ott
Research Institute of Obstetrics and Gynaecology, St Petersburg,
Russian Federation; 4Scientific and Research Institute of Children’s
Infections of Federal Medical and Biological Agency of Russia, St
Petersburg, Russian Federation.
Purpose: Little is known about the susceptibility of posterior
segment tissues, particularly the human retinal pigment epithelium
(hRPE), to bacterial infections. The purpose of the study was to
investigate the possibility of infecting the hRPE with Chlamydia
trachomatis (CT), and to examine both the tropism of the pathogen
for hRPE cells and expression of collagen and growth factors in
response to the infection.
Methods: Cultured hRPE and McCoy cells were inoculated with
five CT clinical isolates (serovars D-K). To detect CT, samples were
stained immunohistochemically with anti-MOMP antibodies at 24h,
48h, and 72h postinoculation (PI). The samples inoculated with
the clinical isolate that had exhibited the highest infectivity were
examined immunohistochemically for changes in the expression of
collagen IV, collagen I, basic fibroblast growth factor (bFGF) and
transforming growth factor β (TGF-β) of hRPE cells. Statistical
significance was determined by a one-way ANOVA-test with
Bonferroni post hoc test.
Results: All clinical isolates showed a higher infectivity for hRPE
cell culture than for McCoy cell culture. At 24 h PI, the percentage
of cells with inclusions varied from 1.5±0.5% to 14.6±3.3% in
hRPE cell culture against 0.4±0.1 to 8.9±0.2% in McCoy cell culture
(P<0.05). In each phase of CT life cycle, the percentage of cells with
inclusions in the hRPE culture was larger than that in the McCoy
culture. The number of intracellular inclusions in both culture types
decreased progressively from 24 h to 72 h postinoculation, with the
cycle completed by the release of elementary bodies. Collagen IV,
collagen I, bFGF and TGF-β expression at 48 h PI were significantly
increased, by 1.3-, 2.1-, 1.5-, and 1.5-fold, respectively, in the
CT-infected compared with control hRPE cell culture specimens
(P<0.05).
Conclusions: This study, for the first time, proved the possibility of
infecting hRPE cultured cells with CT, which leads to proproliferative
and proinflammatory changes in the expression of signaling
molecules and extracellular matrix components. Furthermore,
we demonstrated (1) increased susceptibility of RPE cells to CT
infection, and (2) variability in infectivity among different clinical
CT isolates. These results may be of importance in studying the
vitreoretinal pathology.
Commercial Relationships: Ernest Boiko, None; Dmitrii Maltsev,
None; Alexei Pozniak, None; Alevtina Savicheva, None; Kira
Shalepo, None; Igor Kvetnoi, None; Victoria Polyakova, None;
Alexei Suetov, None; Irina Nuralova, None
Therapeutic adjuvant effect of a trans-resveratrol formulation on
inflammatory macular edema. Randomized, perspective, double
arms study.
Pia Allegri, Ugo Murialdo, Silvia Compiano, Antonio Mastromarino.
Ocular Inflammatory Referral Center, Rapallo (Genova) Hospital,
Genova, Italy.
Purpose:
Resveratrol, a natural polyphenol present in a wide variety of plants,
showed its efficacy against eye diseases related to oxidative stressinduced cell damage. Recent studies pointed out its anti-proliferative
and anti-inflammatory efficacy too.
The purpose of the present randomized, open label, parallel arms,
treated vs. untreated-control group study was to investigate the
efficacy and tolerability of trans-resveratrol containing tablets
(RESVEGA®)(Laboratoires Théa –FR) in adult patients suffering
from autoimmune uveitic macular edema.
Methods:
59 eyes of 32 adult patients (22 females and 10 males), mean age
54y (range 37-65), affected by inflammatory not-infectious macular
edema, were enrolled in the study. Sixteen subjects (29 eyes) were
randomly assigned to receive RESVEGA® tablets TID continuously
during a 6 month period follow-up; the remaining 16 enrolled
subjects (30 eyes) were not added with this supplement; systemic
therapy was unchanged during the follow-up period for all the
enrolled patients. All the subjects completed the visits at baseline, 3
and 6 months. Primary endpoints were: ETDRS visual acuity testing
and Heidelberg Spectralis II OCT examination for central foveal
thickness (CFT) and central macular volume (CMV) reduction. A
specific questionnaire recorded subjective symptoms and tolerability.
Results:
After 6 months CFT and CMV showed a significant (p < 0.05)
reduction and VA a significant (p < 0.001) improvement from
baseline when compared to the control group in which no significant
improvement was seen. Less than 15% eyes in the RESVEGA®
group and more than 60% in the control group got worsen. No
adverse reaction or intolerance was reported.
Conclusions:
Trans-resveratrol containing tablets treatment in comparison with
control group and in addition to standard local and systemic therapy,
is associated with a significant improvement in visual acuity and in
CFT and CMV reduction at the 6-month follow-up visit, in subjects
affected by autoimmune uveitic ME, although not all the eyes showed
a complete resolution of ME probably due to vitreo-macular traction
or foveal ischemia.
Commercial Relationships: Pia Allegri, None; Ugo Murialdo,
None; Silvia Compiano, None; Antonio Mastromarino, None
Therapeutic Outcomes of a Standardized Protocol Using
Immunosuppressive Agents Early Since The Diagnosis of VogtKoyanagi-Harada Syndrome
Carlos J. Fernandez1, Juan C. Almodovar3, Rosa A. Lozada2, Carmen
Santos1, Armando Oliver1. 1OPHTHALMOLOGY, University Of
Puerto Rico, San Juan; 2Universidad Central del Caribe, San Juan;
3
VA Caribbean Healthcare System, Mayaguez.
Purpose: The use of immunosuppressive therapy, along with
systemic corticosteroids, improves the clinical outcomes of patients
with Vogt-Koyanagi-Harada Syndrome. The most efficient means
to administer immunosuppressive therapy to these patients remains
unestablished. We evaluated a retrospective series of patients
treated with a standardized protocol in which immunosuppressive
medications where started within the first month after the diagnosis of
Vogt-Koyanagi-Harada Syndrome.
Methods: We performed a retrospective chart review of all the
patients treated with a standardized treatment protocol (see Fig. 1)
at a tertiary ophthalmology referral center. We analyzed data from
demographics, clinical presentation features, visual acuity outcomes
and steroid sparing success.
Results: Sixteen eyes of eight patients were treated using the
protocol. Eighty-one and fifty percent of eyes had visual acuity worse
than 20/50 and 20/200, respectively upon presentation. Eighty-six
percent of patients completed one year of follow-up; after which,
100% percent of eyes had a visual acuity of 20/200 or better while
67% of eyes had a visual acuity of 20/30 or better. At one year,
100% of patients had achieved steroid sparing success to a dose
10mgs of prednisone or less and 43% had achieved a dose of 5mgs
of prednisone or less. The most common immunosuppressive agent
were azathioprine and mofetil mycophenolate; which were used in
63% of patients.
Conclusions: A standardized treatment protocol in which steroid
sparing agents are introduced for the treatment of Vogt-KoyanagiHarada Syndrome since early in the onset of the condition appears
to be effective in providing patients with good visual outcomes and
helps them achieve steroid sparing success in the vast majority of
cases.
Support: NIH Grants U10EY014655, U10EY014660, and
U10EY014656
Commercial Relationships: Carlos J. Fernandez, None; Juan C.
Almodovar, None; Rosa A. Lozada, None; Carmen Santos, None;
Armando Oliver, None
Support: None in the Support field below.
Dissociations of fluocinolone acetonide implants in the MUST
study
Jennifer E. Thorne1, 2, Elizabeth Sugar2, Alyce Burke2, Albert T.
Vitale3, Douglas A. Jabs4, 2, Janet T. Holbrook2. 1Ophthalmology,
Johns Hopkins Wilmer Eye Inst, Baltimore, MD; 2Johns Hopkins
School of Public Health, Baltimore, MD; 3Ophthalmology, Moran
Eye Institute, Salt Lake City, UT; 4Ophthalmology, Icahn School of
Medicine at Mount Sinai, New York City, NY.
Purpose: To describe the types and risks of fluocinolone acetonide
(FA) implant dissociation in the Multicenter Uveitis Steroid
Treatment (MUST) Trial and Follow-up Study.
Methods: We reviewed data from MUST Trial and Follow-up Study
collected from December 2005 through November 2014 on the first
implant for eyes that received FA implants as treatment for noninfectious uveitis in MUST. Dissociation was defined as FA pellet
separation from its strut. Each case of dissociation was reviewed by
the MUST Safety Officer.
Results: A total of 250 eyes (146 patients) had at least one FA
implant placed; the median follow-up from implantation was 5.8
years (interquartile range: 4.4-6.4). Twenty dissociations were
reported for 17 individuals, three had dissociations in both eyes. Nine
dissociations occurred spontaneously (identified by patient symptoms
or clinical examination) and all involved a dislocated pellet. The
5-year cumulative risk of a spontaneous dissociation was 0.6% (95%
confidence interval: 0-1.7%) with the earliest event occurring 4.8
years after placement. Eleven dissociations were related to an implant
removal surgery (noted or occurred during surgery without prior
symptoms). 31% of 36 eyes undergoing removal surgeries for the first
implant had dissociated drug pellets. Time from placement to surgery
was higher for the surgeries associated with dissociation as compared
those not associated to dissociation (5.4 vs 3.4 years, p < 0.001).
Conclusions: Dissociation is a known risk factor for FA implants.
In MUST, spontaneous dissociations occurred infrequently, with a
cumulative 5-year risk less than 1%. The fraction of surgeries with an
associated dissociation was lower in MUST than previously observed
(31% vs 40%1), with implant duration again a risk factor.
Commercial Relationships: Jennifer E. Thorne, AbbVie (C),
XOMA (C); Elizabeth Sugar, None; Alyce Burke, None; Albert T.
Vitale, None; Douglas A. Jabs, Applied Genetics Technologies Corp
(S), Novartis (S), Santen Pharmaceutical (C); Janet T. Holbrook,
None
Duration of Immunomodulator Therapy on Five-Year Uveitis
Remission Rates
Yijie Lin, Emile Sharifi, David Mostafavi, Danielle Rome, Michael
Tang, Tiffany Truong, Vicente Diaz, Sanjay Kedhar, John Mauro, C
M. Samson. Ophthalmology, New York Eye and Ear Infirmary, New
York, NY.
Purpose: To evaluate the relationship between duration of
immunomodulator therapy (IMT) and uveitis remission rates five
years after discontinuation of the IMT
Methods: 1424 charts were retrospectively reviewed from patients
with anterior, intermediate, posterior and pan-uveitis seen in the
uveitis service at New York Eye and Ear Infirmary between 2004 and
2007. Of these, 240 were identified as patients who had been on IMT
with either methotrexate and/or mycophenolate mofetil. Only the
63 patients who had a minimum five-year follow-up period after the
cessation of the IMT were included in the study. These patients were
then divided into four groups based on the duration of time they were
continuously treated with IMT: 0 to <1 year, 1 to <2 years, 2 to <3
years and greater than 3 years. Each patient’s record was reviewed to
determine if he/she remained in remission for the entirety of the five
year period following cessation of IMT.
Results: Of the patients included in the study, 21 were on
mycophenolate mofetil (33%) and 42 (67%) were on methotrexate. In
the 0-1 year IMT duration, three of thirteen patients (23%) remained
in remission during the five years following cessation of IMT, and
in the 1-2 year duration, six of fifteen patients (40%) remained in
remission. Patients on longer periods of IMT achieved somewhat
higher rates of remission after five years with seven out of sixteen
(44%) within the 2-3 year group and nine of nineteen (47%) in
the greater than 3 year group. This difference is not statistically
significant (chi-square 2.0778, p=0.56).
Conclusions: This pilot study could not show a statistical difference
in the length of remission after discontinuation of immunomodulatory
therapy in uveitis patients whether the patients had been on treatment
for less than one, one to two, two to three, or greater than three
years. This current study is, however, limited by the small number of
patients, and a more definitive answer to this question would require
a prospective study with a larger sample size.
Commercial Relationships: Yijie Lin, None; Emile Sharifi, None;
David Mostafavi, None; Danielle Rome, None; Michael Tang,
None; Tiffany Truong, None; Vicente Diaz, None; Sanjay Kedhar,
None; John Mauro, None; C M. Samson, None
Evaluation of suprachoroidal CLS-TA and oral prednisone in a
porcine model of uveitis
Glenn Noronha1, Kristin Blackwell1, Brian C. Gilger2, Jennifer
Kissner1, Samirkumar R. Patel1, Kaitlyn T. Walsh2. 1R&D, Clearside
Biomedical, Inc, Atlanta, GA; 2Department of Clinical Sciences,
North Carolina State University, Raleigh, NC.
Purpose: To compare the anti-inflammatory effects of a
suprachoroidal injection of CLS-TA, triamcinolone acetonide
injectable suspension with clinically relevant doses of oral prednisone
in a porcine model of acute uveitis.
Methods: Twenty-four hours after the induction of acute posterior
uveitis by intraocular lipopolysaccharide (LPS) injection (Day 0)
into the vitreous, 50 mL of balanced salt solution (BSS, Group 1) or
CLS-TA (2 mg, Group 3) was injected into the suprachoroidal space
(SCS). In Groups 2 and 4, oral prednisone (1 mg/kg/day, Group 2
or 0.1 mg/kg/day, Group 4) was dosed on Day 0, and repeated every
24 hours until euthanasia on day 3. The doses chosen for this study
reflect the doses typically used to treat patients with uveitis, for initial
dose (1 mg/kg/day) and maintenance dose (0.1 mg/kg/day). Only the
right eye of each animal was used in the study and the left eye was
unaltered (n=4/group). Eyes were examined daily, which included
measuring inflammation scores (modified Hackett-McDonald) and
intraocular pressure (IOP). Safety assessments and histopathology
were performed on all eyes.
Results: Following induction with LPS, mean inflammation scores
for all groups increased significantly compared with pre-induction
scores. After treatment, mean inflammation scores decreased in all
groups. On Days 1 and 2 only, Group 3 (suprachoroidal CLS-TA) had
mean cumulative inflammation scores significantly lower than Group
1 (p≤0.04), the BSS, or untreated control group. By day 3, Group 2
(high dose oral prednisone) and Group 3 (CLS-TA) had significantly
lower mean cumulative inflammation scores than Group 1 (BSS)
treated eyes (p<0.034). In Group 4 (low dose oral prednisone), mean
cumulative inflammation scores were not significantly different than
BSS treated eyes at any treatment time (p>0.05). Mean histologic
inflammation scores of the anterior and posterior segment with CLSTA (Group 3) were significantly lower than eyes treated with BSS
(Group 1).
Conclusions: These results suggest that suprachoroidal injection of
CLS-TA resulted in more rapid anti-inflammatory effect than oral
prednisone (Day 1 vs Day 3), was as effective as high dose oral
prednisone (Day 3), and was superior to low dose prednisone in its
anti-inflammatory effect in this porcine model of acute uveitis.
Commercial Relationships: Glenn Noronha, Clearside Biomedical,
Inc (E); Kristin Blackwell, Clearside Biomedical, Inc (E); Brian
C. Gilger, None; Jennifer Kissner, Clearside Biomedical, Inc (E);
Samirkumar R. Patel, Clearside Biomedical, Inc (E); Kaitlyn T.
Walsh, None
Spontaneous separation of fluocinolone acetonide implant
Amina Chaudhry, Yosuke Harada, Debra A. Goldstein.
Ophthalmology, Northwestern University, Chicago, IL.
Purpose: Retisert (Bausch & Lomb, Rochester, NY) is a long-acting
sutured fluocinolone acetonide intravitreal implant (FAI) approved
for treatment of chronic non-infectious uveitis. Complications include
increased intraocular pressure, cataract, intraocular hemorrhage,
endophthalmitis. Separation of drug pellet from sutured strut has been
reported during surgical manipulation (1) and, rarely, spontaneously;
typically 3 - 7 years after placement (2,3). We report a series of
patients with very late spontaneous FAI strut-pellet separation.
Methods: Chart review of patients with FAI separation diagnosed by
the uveitis service at Northwestern University July 2012 - September
2014. Data collected included patient demographics, ocular surgical
history, symptoms, visual acuity and complications.
Results: 5 eyes of 5 patients with spontaneous FAI separation
presented during the 2 year period. 4 were female, mean age was 50
years (35 - 68). Implants were placed from 2002 to 2009. Median
time from insertion to separation was 135 months (range 52 -140). 4
patients presented with a new onset large floater, 1 was discovered on
routine exam. 3 eyes had intraocular surgery following FAI including
cataract surgery, glaucoma surgery and vitrectomy, all done within 1
year of FAI. Median time from last surgery to pellet separation was
60 months (range 48 -120). No patient had a history of ocular trauma.
4 eyes underwent removal of the pellet. In 3 cases it was for a mobile
pellet in the vitreous. In 1 case the pellet appeared immobile at the
vitreous base so was left in place, but was removed 7 months later
when a small retinoschisis developed adjacent to the pellet. There
were no surgical complications of implant removal. Visual acuity did
not decrease after implant removal.
Conclusions: Spontaneous dissociation of FAI pellet from strut is
rare. We report the largest series of spontaneous FAI strut-pellet
separation. All presented to a single center in a 2 year period. There
have only been 2 previous reports of spontaneous pellet separation,
for a total of 6 patients, all occurring within 7 years of implantation
(2,3). 3 of our patients presented 11 years after implantation,
suggesting that very late dislocation may be a cause of new
symptoms in this population.
1. Am J Ophthalmol 2012;154(6):969-73.
2. Ocul Immunol Inflamm. 2013;21:87-89.
3. Ocul Immunol Inflamm. 2014; 11:1-4
Commercial Relationships: Amina Chaudhry, None; Yosuke
Harada, None; Debra A. Goldstein, Bausch and Lomb (C)
Long-term treatment with Tocilizumab for non-infectious uveitis
Marina Mesquida, Blanca Molins, Victor Llorens, Anna SalaPuigdollers, Jessica Matas, Javier Zarranz-Ventura, Maite Sainz De
La Maza, Alfredo Adan Civera. Ophthalmology, Hospital Clinic de
Barcelona, Barcelona, Spain.
Purpose: To report the long-term efficacy and safety of the IL-6
receptor antagonist tocilizumab (TCZ) for refractory uveitis and its
related macular edema (ME).
Methods: Data were obtained by standardized chart review. Main
outcome measures: central foveal thickness (CFT) measured by
optical coherence tomography, anterior chamber cell grade, vitreous
haze, and best-corrected visual acuity (logarithm of the minimum
angle of resolution [log-MAR]) were recorded during TCZ therapy at
months 1, 3, 6, 12, 18, and 24.
Results: Seventeen eyes from 12 patients (10 females) were included.
Mean age was 38.6 years. Mean duration of ME was 12.3 years.
Mean follow-up with TCZ therapy was 21.8 months (range, 6-24).
Before TCZ, all patients failed conventional immunosuppressive
therapy and one or more biologic agents. Uveitis diagnoses were:
juvenile idiopathic arthritis-associated-uveitis (n=5), birdshot
chorioretinopathy (n=3), idiopathic panuveitis (n=2), sympathetic
ophthalmia (n=1), and ankylosing spondylitis (n=1). Mean CFT (95%
confidence interval) was 530 ± 194 μm in baseline, 370 ± 95 μm at
month 1 (p= 0.004), 303 ±78 μm at month 3 (p=0.0009), 275 ± 72
μm at month 6 (p= 0.000025), 288 ± 107 at month 12 (p=0.002), and
297 ± 99 at month 24 (p=0.015) of follow-up. TCZ’s major efficacy
on CFT can be observed at months 6 to 9 (nadir), reaching a plateau
from month 12 of follow-up onwards.
Mean log-MAR best-corrected visual acuity improved from 0.72
± 0.65 in baseline to 0.54 ± 0.62 at month 6 (p = 0.0019), 0.54 ±
0.65 at month 12 (p = 0.007), and 0.6 ± 0.76 at month 24 (p = 0.02).
TCZ therapy was withdrawn in 2 patients due to sustained remission
at month 12. In both patients, ME relapsed 3 months after TCZ
withdrawal. Reinitiation of TCZ therapy led to good uveitis control
and ME resolution. With regards to safety, one patient developed mild
neutropenia and another patient showed increased liver enzymes,
none of which required TCZ withdrawal.
Conclusions: TCZ may be safe and effective for uveitis and its
associated ME in otherwise refractory cases.
course of uveitis was chronic anterior (n=3), acute anterior (n=1), and
intermediate (n=1). In addition, all patients suffered from systemic
rheumatologic disease, either related or not related with uveitis
(related: juvenile idiopathic arthritis, n=2; ankylosing spondylitis,
n=1; not related: rheumatoid arthritis, n=2). Ineffective pre-treatment
consisted of systemic prednisolone, at least one immunosuppressive
drug, and at least one biologic drug in all patients. Mean follow-up
was calculated for 16.2 months (4-35 months), mean duration of
tocilizumab treatment for 14.6 months (4-35 months).
Before start of tocilizumab, mean CFT was measured 738 mm (640960 mm) in right and 576 mm (530-660 mm) in left eyes. At 3 months,
a response of ME (at least 25% reduction in CFT) could be observed
in both patients with unilateral ME, in both eyes in 1 patient and in
1 eye each in 2 patients with bilateral ME. In 2 eyes, ME did not
respond to tocilizumab. During follow-up, complete absence of ME
could be observed in both patients with unilateral ME, in both eyes
in 1 patient and in 1 eye in another patient with bilateral ME. In 3
eyes, ME did not disappear. At the end of follow-up, mean CFT was
382 mm (150-820 mm) in right and 373 mm (190-630 mm) in left eyes.
During follow-up, treatment with tocilizumab has to be discontinued
in 1 patient with bilateral ME due to reactivation of the uveitis.
Improvement of visual acuity of at least 0.3 logMAR in at least 1 eye
could be observed in 3 patients (60%) during follow-up. Tocilizumab
was well tolerated, no relevant side effects occurred.
Conclusions: Treatment with tocilizumab can be considered in
patients with chronic uveitic ME, even then if previous therapies with
immunosuppressive and biologic drugs have failed.
Commercial Relationships: Christoph M. Deuter, None; Manfred
Zierhut, None; Annette Igney-Oertel, None; Theodoros Xenitidis,
None; Alexandra Feidt, None; Deshka Doycheva, None
Commercial Relationships: Marina Mesquida, None; Blanca
Molins, None; Victor Llorens, None; Anna Sala-Puigdollers,
None; Jessica Matas, None; Javier Zarranz-Ventura, None; Maite
Sainz De La Maza, None; Alfredo Adan Civera, None
Tocilizumab in uveitic macular edema refractory to previous
immunomodulatory treatment
Christoph M. Deuter1, Manfred Zierhut1, Annette Igney-Oertel2,
Theodoros Xenitidis2, Alexandra Feidt1, Deshka Doycheva1. 1Centre
for Ophthalmology, University of Tuebingen, Tuebingen, Germany;
2
Department of Internal Medicine II, University of Tuebingen,
Tuebingen, Germany.
Purpose: To analyze the efficacy of tocilizumab, a monoclonal
antibody against IL-6 receptor, in patients with chronic uveitic
macular edema resistant to various immunomodulatory drugs.
Methods: Retrospective analysis of an interventional case series.
Patients received tocilizumab (RoActemra, Roche/Chugai Pharma)
at a dose of 8 mg/kg bodyweight every 4 weeks intravenously.
Central foveal thickness (CFT) was assessed by optical coherence
tomography (OCT).
Results: We included 5 patients (3 female, 2 male; mean age 42.6
years, 23-57 years) with 8 affected eyes (5 right eyes, 3 left eyes).
ME was unilateral in 2 and bilateral in 3 patients. Localization/
Pharmacokinetics of Intravitreal Sirolimus in Non-infectious
Uveitis (NIU) of the Posterior Segment: Results from a Subset of
SAKURA Study 1 Subjects
Daniel Rosberger1, Yang Yang2, Masaaki Kageyama3, Hidetoshi
Mano3, Hitomi Takenaga3, Kenji Ueda3, Lisa Lawrence-Miyasaki2.
1
MaculaCare, New York, NY; 2Santen, Inc., Emeryville, CA; 3Santen
Ltd., Osaka, Japan.
Purpose: Optimally, an intraocular medication for NIU of the
posterior segment should have minimal systemic concentrations
to avoid systemic adverse effects, with no drug accumulation.
Intravitreal sirolimus is a locally delivered mTOR inhibitor
that is currently being studied in the Phase III SAKURA trial
as monotherapy for the treatment of active NIU of the posterior
segment. Here, we describe the whole-blood pharmacokinetics of
intravitreal sirolimus in the subset of subjects from Japan (n=14) who
participated in SAKURA Study 1.
Methods: In the 6-month, double-masked phase of SAKURA Study
1, subjects received intravitreal sirolimus injections of 44, 440, or
880 μg at Months 0, 2, and 4. Blood samples were taken at baseline
and on Days 1, 3, 14, 30, 60 (before and after the second injection),
62, 73, 90, 120 (after the third injection), 122, 133, and 150, and at
Month 6. Blood sirolimus concentrations were determined by liquid
chromatography/tandem mass spectrometry.
Results: Following the first injection, blood sirolimus concentrations
rapidly increased, reaching a Cmax of 0.337, 1.97, and 3.06 ng/
mL at a mean Tmax of 0.69, 1.3, and 4.2 days for the 44, 440, and
880 mg doses, respectively. Sirolimus concentrations gradually
declined toward the limit of quantitation level within 30 days after
the injection. The corresponding mean AUC0-60d at doses of 44, 440,
and 880 mg were 1.5, 15.0, and 30.5 ngday/mL, respectively. The
same pattern was observed after the second and third injections (see
Figure 1). The mean Cmax and AUC0-60d did not change between the
first and the third administration with any dose, suggesting that there
was no systemic accumulation of sirolimus after repeated intravitreal
injections.
Conclusions: In this subset of Japanese subjects from SAKURA
Study 1, mean sirolimus concentrations remained well below the
5-15 ng/mL considered necessary for systemic immunosuppression
throughout the entire study period, indicating that systemic exposure
to sirolimus after repeated intravitreal injections is negligible. This
finding, together with the low incidence of systemic adverse events in
SAKURA Study 1, indicates that the effects of intravitreal sirolimus
are confined to the eye.
The primary endpoint was time to treatment failure (TF) in ≥1 eye.
TF was defined as ≥1 of the following: new, active, inflammatory
vascular lesions; worsening of best-corrected visual acuity (BCVA)
by ≥15 letters at or after week 6; inability to achieve ≤0.5+ AC cell
grade or ≤0.5+ VH grade (at week 6); or 2-step increase in AC cell
grade or VH grade (after week 6). Ranked secondary endpoints
included change in AC cell grade, VH grade, and BCVA from the best
state achieved before week 6 to the final visit. Adverse events were
monitored.
Results: 217 patients were enrolled (female, 57%; mean age, 42.7
y; mean duration of uveitis, 46 months); 22% had intermediate, 33%
had posterior, and 45% had panuveitis. Patients who received ADA
were less likely to have TF (hazard ratio=0.5; 95% CI, 0.36–0.70;
P<0.001). Median time to TF was 13 weeks for placebo and 24
weeks for ADA (Figure). Patients who received ADA had fewer
causes of failure. Worsening of AC cell grade, VH grade, and BCVA
from best state achieved was reduced with ADA compared with
placebo (Table). The rates of adverse events were similar in ADA and
placebo groups (1046 vs 952 events/100 patient y, respectively).
Conclusions: In patients with active, non-infectious intermediate,
pan, or posterior uveitis (ie, uveitis involving the posterior segment)
uncontrolled on prednisone ≥10 mg daily, ADA significantly lowered
the risk for uveitic flare or BCVA loss. The safety profile was
consistent with the known safety profile across the approved ADA
indications.
Figure 1. Mean±SD sirolimus concentrations in human blood
following bimonthly intravitreal sirolimus injections.
Commercial Relationships: Daniel Rosberger, None; Yang Yang,
Santen, Inc. (E); Masaaki Kageyama, Santen Ltd. (E); Hidetoshi
Mano, Santen Ltd. (E); Hitomi Takenaga, Santen Ltd. (E); Kenji
Ueda, Santen Ltd. (E); Lisa Lawrence-Miyasaki, Santen, Inc. (E)
Adalimumab in Patients With Active, Non-infectious Uveitis
Requiring High-dose Corticosteroids: the VISUAL-1 Trial
Glenn J. Jaffe1, Jennifer E. Thorne2, david scales3, Pablo Franco4,
Samir R. Tari5, Anne Camez5, Alexandra P. Song5, Martina Kron5,
Talin Barisani-Asenbauer6, Andrew D. Dick7, 8. 1Ophthalmology,
Duke University Eye Center, Durham, NC; 2Johns Hopkins,
Baltimore, MD; 3University of Texas Health Science Center, San
Antonio, TX; 4Organización Medica de Investigation, Buenos Aires,
Argentina; 5Abbvie Inc, North Chicago, IL; 6Laura Bassi Centre of
Expertise Ocuvac, Vienna, Austria; 7University of Bristol, Bristol
Eye Hospital, Bristol, United Kingdom; 8National Institute for Health
Research, London, United Kingdom.
Purpose: To assess adalimumab (ADA) efficacy and safety in
patients with active, non-infectious uveitis requiring high-dose
corticosteroid therapy.
Methods: This multinational, double-masked trial included
patients aged ≥18 years with active, non-infectious intermediate,
posterior, or panuveitis despite ≥2 weeks of prednisone (≥10 mg/d
to ≤60 mg/d). Patients with active uveitis had ≥1 of the following:
active, inflammatory chorioretinal or retinal vascular lesion;
anterior chamber (AC) cell grade ≥2+; or vitreous haze (VH) grade
≥2+. Patients were randomized 1:1 to receive placebo or ADA
subcutaneously. The ADA group received an 80 mg baseline loading
dose and 40 mg every other week for up to 80 weeks; all patients
received prednisone 60 mg/day that was tapered to 0 mg by week 15.
who are treated with systemic steroids (as single therapy or with
methotrexate). Efficacy and safety are assessed at each visit. The
study primary endpoints are reduction from baseline in vitreous haze
and systemic-steroid sparing effects, both measured at week 16.
Other key endpoints assessed at week 16 include the change from
baseline in: central retinal thickness, best-corrected visual acuity,
and retinal vessel leakage on fluorescein angiography. A graphical
representation of the study design is presented in the Figure below.
Results: The study is ongoing. As of 29 October 2014, 33 of
57 patients have been randomized and treated; 19 subjects have
completed the Principal Treatment Period (Part A). Baseline
demographic and disease characteristics are presented in the Table
below.
Conclusions: The SATURN study may help clarify the role of IL-6
in the pathogenesis of NIU and the potential for IL-6 inhibition in the
management of posterior segment NIU.
SATURN study: Graphical Study Design
Commercial Relationships: Glenn J. Jaffe, Abbvie (C); Jennifer
E. Thorne, Abbvie (C), Allergan (F), Gilead (C), Xoma (C); david
scales, Abbvie (C); Pablo Franco, None; Samir R. Tari, Abbvie (E);
Anne Camez, Abbvie Inc (E); Alexandra P. Song, Abbvie Inc (E);
Martina Kron, Abbvie Inc (E); Talin Barisani-Asenbauer, None;
Andrew D. Dick, Abbvie Inc (C)
Support: Abbvie Inc
The SATURN Study (SARIL-NIU): Sarilumab for the Treatment
of Posterior Segment Non-Infectious Uveitis (NIU)
Quan Dong Nguyen1, Preethi A. Sundaram2, Kristine Erickson3,
Rafael varona2, David Drouot2, Valerie Corp-dit-Genti2, Husain
Kazmi3, Robert Vitti3, Marc D. de Smet4. 1Stanley M. Truhlsen Eye
Institute, University of Nebraska Medical Center, Omaha, NE;
2
Sanofi, Paris, France; 3Regeneron Pharmaceuticals, Tarrytown, NY;
4
MIOS, Lausanne, Switzerland.
Purpose: Interleukin-6 (IL-6) and/or its soluble receptor are detected
in the vitreous and aqueous humors of patients with uveitis. Inhibition
of IL-6 signaling in a murine model of experimental autoimmune
uveitis suppresses the development of uveitis. We designed an
exploratory study to evaluate the efficacy and safety of sarilumab, a
fully human monoclonal antibody directed against the alpha subunit
of the IL-6 receptor complex in the management of posterior segment
NIU.
Methods: SATURN is a 52 week multicenter, double-masked,
placebo-controlled, parallel arm, randomized trial to evaluate
the efficacy and safety of sarilumab (200 mg) administered
subcutaneously every 2 weeks in patients with posterior NIU,
Commercial Relationships: Quan Dong Nguyen, Genentech
(F), Psivida (F), Regeneron (F), Sanofi (F), XOMA (F); Preethi
A. Sundaram, Sanofi (E); Kristine Erickson, Regeneron
Pharmaceuticals (E); Rafael varona, Sanofi (E); David Drouot,
Sanofi (E); Valerie Corp-dit-Genti, Sanofi (E); Husain Kazmi,
Regeneron Pharmaceuticals (E); Robert Vitti, Regeneron
Pharmaceuticals (E); Marc D. de Smet, Regeneron Pharmaceuticals
(C), Sanofi (F)
Support: Sanofi, Inc. and Regeneron, Inc.
Medical treatment in patients with Vogt Koyanagi Harada
Syndrome in a tertiary university center in Argentina
Bernardo A. Schlaen1, Juan P. Fernandez1, Matias Portela1, Mariana
Ingolotti1, Cristobal A. Couto1, 2, Anahi Lupinacci1, Mario J. Saravia1.
1
Ophthalmology, Hospital Universitario Austral, Ciudad Autonoma
de Buenos Aires, Argentina; 2Ophthalmology, University of Buenos
Aires, Buenos Aires, Argentina.
Purpose: Purpose: To describe medical treatment received by
patients with diagnosis of Vogt Koyanagi Harada Syndrome in a
tertiary university center.
Methods: Patients with diagnosis of Vogt Koyanagi Harada
Syndrome who were seen at Hospital Universitario Austral
between June 2009 and October 2014 were included. Data recorded
included, age, sex, category of diagnosis (complete, incomplete or
probable), treatment regime (early with high dose of corticosteroids,
within 15 days of symptoms presentation; late with high dose of
corticosteroid, after 15 to 30 days of presentation; other treatment
regimen), regional treatment (subtenon triamcinolone acetonide,
intravitreal triamcinolone acetonide, intravitreal metotrexate),
immunosuppressive therapy, and complications.
Statistical analysis was carried out using excel 2012. Chi square was
used as appropriate.
Results: Thirty five patients (28 females, 7 males) with diagnosis of
Vogt Koyanagi Harada Syndrome were included. Average age was
36 ±14.38 years. Incomplete Vogt Koyanagi Harada Syndrome was
seen in 18 patients, complete in 8 cases, and probable in 9 patients.
High dose oral corticosteroids was administered to 33 patients (early
high: 12 patients; late high: 18 patients; other: 5 patients). Subtenon
triamcinolone acetonide was performed in 8 patients, intravitreal
triamcinolone acetonide in 2 patients, and intravitreal methotrexate
in 2 patients. Immunosuppressive treatment was administered to 24
patients (68.57%). Among 12 patients who underwent early high
regime 5 patients needed immunosuppressive treatment, while 17
of 18 patients with late high treatment required immunosuppressive
treatment (Chi square, P < 0.01). Seven patients (20%) underwent
cataract surgery, 5 patients were treated for ocular hypertension
(14%), 2 patients had epiretinal membrane, 2 patients had macular
edema, and 1 patient had choroidal neovascularization. None of
the 12 patients with early high dose of corticosteroid treatment
developed complications, 9 out of 18 patients with late high dose
of corticosteroid regime developed complications, and 4 out of 5
patients with other regime developed complications (Chi square, P
<0.01).
Conclusions: Treatment of patients with Vogt Koyanagi Harada
Syndrome within 15 days of symptoms presentation with high
dose corticosteroid regime reduces the need of immunosuppressive
treatment and the number of complications.
Commercial Relationships: Bernardo A. Schlaen, None; Juan
P. Fernandez, None; Matias Portela, None; Mariana Ingolotti,
None; Cristobal A. Couto, None; Anahi Lupinacci, None; Mario J.
Saravia, None
Dexamethasone intravitreal implant in the treatment of recurrent
uveitic macular edema in the post-operative cataract setting: a
case series
Marissa G. Bucci, Mark Dacey, Jesse M. Smith. Ophthalmology,
University of Colorado, Aurora, CO.
Purpose: Cystoid macular edema (CME) is an important cause of
decreased vision after cataract surgery and can prove especially
challenging in patients with known uveitis and prior CME. We
performed a retrospective, clinical study to examine the efficacy of
the dexamethasone (DEX) intravitreal implant (Ozurdex®; Allergan,
Inc., Irvine, CA) in the treatment of recurrent uveitic macular edema
in the postoperative period following cataract surgery (post-CE/IOL).
Methods: We performed a retrospective chart review of patients >18
years of age with known uveitic macular edema previously treated
with DEX. Eight patients (8 eyes) were included who underwent
cataract surgery within 1-4 months from their last DEX. These
patients then developed post-operative macular edema and were
subsequently treated with a repeat DEX implant. The main outcome
assessed was the central macular thickness (CMT) measured by
optical coherence tomography (OCT) in the pre-operative, postoperative and post-DEX period. All patients who received the DEX
implantation prior to cataract surgery met FDA-approved indications
for Ozurdex® for treatment of non-infectious intermediate or posterior
uveitis and had no active inflammation at the time of surgery.
Results: Eight eyes were included in the study. There was a
statistically significant increase in mean CMT from pre-operative
(mean, 270 ± 45 μm) to post-CE/IOL (mean, 352 ± 87 μm) (p =
0.01). There was subsequently a statistically significant reduction in
mean CMT after treatment with DEX in the post-operative period
(mean, 264 ± 53 μm) (p = 0.03). Although not statistically significant,
there was a trend towards less increase in CMT post-CE/IOL in eyes
that received DEX closer to time of surgery in the pre-operative
period.
Conclusions: Intravitreal dexamethasone implant was shown to
be effective in treating cystoid macular edema in the post-cataract
surgery setting in patients with uveitis and prior uveitic CME. It may
be beneficial to treat uveitics with a history of CME with DEX in
the immediate pre-operative period to prevent or control CME after
cataract surgery, although further studies would be needed to confirm
this.
Commercial Relationships: Marissa G. Bucci, None; Mark Dacey,
Allergan Pharmaceutics (S); Jesse M. Smith, None
Adalimumab Therapy for Refractory Birdshot
Chorioretinopathy
Freekje Van Asten1, Paulien HuisinhetVeld1, Robert W. Kuijpers2,
Aniki Rothova2, Eiko de Jong1, Carel C. Hoyng1. 1Ophthalmology,
Radboud umc, Nijmegen, Netherlands; 2Ophthalmology, Erasmus
MC, Rotterdam, Netherlands.
Purpose: To report the outcomes of adalimumab (Humira®) in
the treatment of birdshot chorioretinopathy (BSCR) refractory to
conventional immunomodulatory therapy.
Methods: In this retrospective non-comparative case series we
included nineteen patients (38 eyes) with HLA-A29 positive BSCR
who received adalimumab treatment, identified by chart review,
at ophthalmology departments of the Radboud university medical
center, Nijmegen, the Netherlands, and the Erasmus university
medical center, Rotterdam, the Netherlands. All patients received
40mg adalimumab subcutaneously at 2-week intervals during the
study period, in addition to previously initiated immunosuppressive
medication. Data regarding previous immunosuppressive therapy
were recorded. The main outcome measures included change in
visual acuity (VA), inflammatory signs on fluorescein angiographic
(FA) and optical coherence tomography (OCT), concomitant
immunosuppressive requirements, and side effects. Results were
assessed at six months and one year follow up and compared to the
situation six months and one year before the start of adalimumab.
Results: From the start of adalimumab treatment, over a follow up
of one year, the mean (± SD) Snellen VA improved from 0.47 (± 28
letters) to 0.71 (± 10 letters) (p=0.030). The trend of a decreasing
VA in the year prior to adalimumab treatment reversed to a trend of
increasing VA after the start of adalimumab, to finally equal the VA
of two years before. The effect of adalimumab on the pathologic
features on FA and OCT was less evident, with only two patients
fully free of inflammatory signs at the end of follow up. After one
year, 53% of patients were able to decrease the use of concomitant
immunosuppressive medications. Compared to start, more patients
received adalimumab monotherapy at one year follow up (53% at one
year versus 21% at start, p 0.047). Three out of 19 patients perceived
relatively mild possible treatment-related side effects, two of which
discontinued treatment within the year.
Conclusions: The data of this study suggest that adalimumab is
effective and mostly well tolerated during the first year of treatment
in otherwise treatment-refractory cases of BSCR. However, complete
remission of the inflammatory process is rarely achieved.
Commercial Relationships: Freekje Van Asten, None; Paulien
HuisinhetVeld, None; Robert W. Kuijpers, None; Aniki Rothova,
None; Eiko de Jong, None; Carel C. Hoyng, None
The intravitreal dexamethasone implant treatment of patients
with cystoid macular edema (Ozurdex®) from Birdshot
retinochoroidopathy in a tertiary center
Stéphanie Hayek, Margaux Guillard, Antoine P. Brezin, Dominique
Monnet. Ophtalmology, Hopital Cochin-Univ Paris Descartes, Paris,
France.
Purpose: To analyze the efficacy of dexamethasone intravitreal
implant 0.7 mg (Ozurdex®) in the treatment of patients with cystoid
macular edema in the context of Birdshot retinochoroidopathy.
Methods: A retrospective charts review of patients with cystoid
macular edema (ME) secondary to birdshot retinochoroidopathy
treated with dexamethasone intravitreal implant in a tertiary referral
center. The main outcome measures were the reduction in central
macular thickness (CMT) and improvement in best corrected visual
acuity (BCVA) from baseline to study end. CMT was measured by
spectral domain OCT. The following data were recorded : BCVA,
CMT, hyalitis, retinal vasculitis on fluorescein angiogram, presence
of cataract and ocular hypertonia at baseline and 1 month, 3 months,
6 months, one year after intravitreal implantation.
Results: Twenty six eyes of 19 patients were included. 15 eyes were
injected once, the others had multiple injections. In patient with only
one injection, increase of BCVA was significant at 3 months postimplant with a mean increase of 0,25 ± 0,30 logMAR(p=0,0001).
Reduction in central retinal thickness was significant in patients with
cystoid macular edema at 1 month and 3 month post-implant with a
mean decrease of respectively 108,87 ± 129,75 micron (p=0,0001)
and 148,57 ± 196,65 micron (p=0,0001). In patients with more than
one injection, reduction in central retinal thickness was significant
1 month after the first implantation with a mean decrease of 198,73
± 209,67 micron (p=0,0049). Increase of BCVA was significant at 1
month with a mean increase of 0.16 ± 0.16 logMAR à (p= 0,0039)
and 3 month with a mean increase of 0.18 ± 0.13 logMAR (p=0,002).
Intraocular inflammation was present in 65.4% at baseline, and then
26,9%, 16%, 14.3% and 10.5% of patients at 1 month, 3 months, 6
months and 1 year after receiving the implant, respectively. Adverse
events included increased intraocular pressure (23.1%) which
required medical treatment only (no surgical procedures were needed)
and cataract formation (50%).
Conclusions: The data suggest that intravitreal dexamethasone
implant 0,7mg (Ozurdex) is an important help to control macular
edema in patient with birdshot retinochoroidopathy. Moreover,
our study confirm the association of this treatment with ocular
hypertension requiring medical treatment and cataract formation.
Commercial Relationships: Stéphanie Hayek, None; Margaux
Guillard, None; Antoine P. Brezin, None; Dominique Monnet,
None
Do Demographic Factors Influence Uveitis Patients’
Understanding of Uveitis?
efrosini papagiannuli, Matthew R. Edmunds, Sue Southworth, Philip
I. Murray. Academic Unit of Ophthalmology, Birmingham and
MIdland Eye Centre, Birmingham, United Kingdom.
Purpose: There is a paucity of information concerning the level of
uveitis patients’ understanding of their disease. The purpose of this
study was to establish how much uveitis patients know about their
condition and to investigate the contribution of demographic factors
to that knowledge.
Methods: A self-designed questionnaire, comprising 20 questions
about uveitis, was distributed to 200 consecutive patients attending
a UK tertiary referral Uveitis clinic. It was initially trialled by four
uveitis patient groups and modified according to their comments.
The questionnaire requested demographic details (age range, gender,
ethnicity, residence postcode) and required responses to uveitisspecific questions (definition, epidemiology, causes, symptoms,
complications, treatment) using a three point Likert scale. Postcode
was used to determine level of social deprivation using Index of
Multiple Deprivation 2007 (IMD 2007) and participants divided
into IMD 2007 score quintiles. Univariate analyses (Mann-Whitney
and Kruskal-Wallis tests) and multivariable logistic regression were
performed
Results: Of the 200 patients, 165 (83%) provided sufficient data to be
included in the analysis. Of all respondents, 62% were female, 71%
aged >40 years and 67% of white ethnic origin, with 41% having
been under the care of a uveitis specialist for >10 years and 72%
attending ≥3 clinic appointments in the preceding 12 months. Median
questionnaire score (out of 60) was 27 (interquartile range [IQR] 15).
No patients answered all questions correctly; only 39.5% got more
than 10 questions correct but 80.5% knew the meaning of uveitis.
Using univariate analyses females scored significantly higher than
males (30 [15] versus 24 [15] p=0.001), but there was no difference
according to age, ethnicity or social deprivation quintile, nor the
duration patients had been under ophthalmic review or number of
clinic attendances in the preceding 12 months. Multivariate analyses
determined no independent influence of any of the factors on uveitis
questionnaire score.
Conclusions: Uveitis patients’ understanding of their condition
is poor. No independent influence of any demographic factor,
or historical or recent clinic attendance experience, on uveitis
questionnaire score was observed. Strategies must be devised
to augment uveitis patient understanding in order to increase
satisfaction, improve treatment adherence and subsequently enhance
clinical outcomes.
Commercial Relationships: efrosini papagiannuli, None; Matthew
R. Edmunds, None; Sue Southworth, None; Philip I. Murray,
None
Uveitis Demographics in an Indigent Population
Meghan Berkenstock, Eileen Chang, Jessica M. Ackert.
Ophthalmology, Drexel University College of Medicine,
Philadlephia, PA.
Purpose: Patients with uveitis require treatment with topical and oral
medications to adequately resolve inflammation. The side-effects of
these medications require frequent lab studies and long-term followup. For patients with no insurance or minimal coverage, suboptimal
compliance may result. There have been no studies examining
the types of uveitis and visual outcomes at large hospitals serving
indigent populations in need of low-cost care. By understanding the
types of uveitis and populations we are serving, the study allows us to
focus on obtaining resources for these patients.
Methods: A database of uveitis-related ICD9 codes from 9/2011
through 9/2014 at Drexel Eye Physicians was created. Charts of
647 patients were retrospectively reviewed. Primary endpoints were
determining the anatomic location of the uveitis and demographic
factors, such as race and gender.
Results: Results: Based on diagnosis codes, 647 charts were
reviewed and 249 patients were included. Patients were excluded if
there was no documentation of endogenous anterior, intermediate,
posterior, or panuveitis. Anatomic location and activity was graded
according to SUN. The most common anatomic location was
anterior (76.3%), followed by panuveitis (14.9%), posterior (6.8%),
and intermediate (2%). In affected patients, the most commonly
represented ethnic groups were African-American (65.5%),
Caucasian (16.9%), and Hispanic (10%). Patients with anterior
uveitis did well with the majority achieving 20/20 vision. Patients
with pan or posterior uveitis had a higher rate of vision loss. The
most common disease entities were post-op inflammation (13.65%),
sarcoid (6.4%), toxoplasmosis and HZO (2% each). In cases of
anterior uveitis, 144 cases were acute (75.79%) and 46 chronic
(24,21%). Unilateral was more common than bilateral, 62.25% versus
37.75%. There were more females, 65.5%, than males, 34.5%. The
prevalence rate was based on a total of 23,290 office visits, which
totaled 0.77% of patients.
Conclusions: The diverse population seen in Philadelphia allows for
a wide variety of conditions presenting at our uveitis specialty clinic.
The study has allowed us to identify the entities that are encountered
in our primarily indigent population and assist them in affording long
term treatment. The majority of patients are African-American with
anterior uveitis who had good visual outcome. Further evaluation is
needed to determine what barriers to care can be removed to improve
outcome.
Commercial Relationships: Meghan Berkenstock, None; Eileen
Chang, None; Jessica M. Ackert, None
Diagnostic ability of a Bayesian-Network-based algorithm for the
differential diagnosis of anterior uveitis
Julio J. González-López2, 1, Mark C. Westcott2, Diana SánchezPonce3, Pedro Beneyto4, Nelida Muñoz Sanz2, Noa Fernandez
Ledo2, Maria Pefkianaki2. 1Tennent Institute of Ophthalmology,
Glasgow, United Kingdom; 2Medical Retina and Uveitis,
Moorfields Eye Hospital NHS Foundation Trust, London, United
Kingdom; 3Universidad Complutense de Madrid, Madrid, Spain;
4
Ophthalmology, Complejo Hospitalario de Toledo, Toledo, Spain.
Purpose: The differential diagnosis of anterior uveitis is often
complex, as it is based on the presence of different signs, symptoms,
epidemiologic characteristics and ancillary tests results. The purpose
of this study is to measure the diagnostic ability of a Bayesian
Network-based algorithm for the etiologic diagnosis of 11 common
causes of anterior uveitis (idiopathic, ankylosing spondylitis,
psoriasic arthritis, reactive arthritis, inflammatory bowel diseases,
sarcoidosis, tuberculosis, Behçet, Posner-Schlossman syndrome,
juvenile idiopathic arthritis -JIA- and Fuchs heterochromic cyclitis).
Methods: A retrospective validation of a diagnostic test was
performed. A sample of 200 patients was randomly selected among
all the patients that received a diagnosis of anterior uveitis during
2013 in a tertiary-care, hospital in London. Epidemiologic data,
clinical signs and symptoms and results of ancillary tests were
obtained from the patients’ clinical notes. The data were entered in
the algorithm, and diagnostic results were compared to the senior
clinician diagnosis (gold standard). In order to improve the estimation
of the most uncommon diagnosis, the sample was enriched with 3
cases of JIA, 3 psoriasic arthritis and 4 Behçet. Robustness analysis
was performed using a second algorithm with equalized pre-test
probabilities for the 11 etiologies.
Results: In 134 of 210 patients (63.8%) the most probable etiology
by the algorithm matched the senior clinician diagnosis. In 169 of
210 patients (80.5%) the clinician diagnosis matched the first or
second most probable results by the algorithm. Taking into account
only the most probable diagnosis by the algorithm, sensitivities
for each etiology ranged from 100% (7 of 7 patients with reactive
arthritis and 5 of 5 with Behçet correctly classified) to 51.2% (43 of
84 patients with idiopathic anterior uveitis). Specificities ranged from
88.8% for sarcoidosis to 99.5% for Posner. A second model with
equal pre-test probabilities for all 11 etiologies correctly classified
86 of 210 (41.0%) for the most probable etiology, and 108 of 210
(51.4%) for first or second most probable etiologies.
Conclusions: The Bayesian network developed may help
clinicians with the differential diagnosis of anterior uveitis. Pre-test
probabilities need to be adjusted to the local population. Sensitivities
and specificities of the algorithm differ among different etiologies.
Commercial Relationships: Julio J. González-López, Bayer (C);
Mark C. Westcott, None; Diana Sánchez-Ponce, None; Pedro
Beneyto, None; Nelida Muñoz Sanz, None; Noa Fernandez Ledo,
None; Maria Pefkianaki, None
Epidemiological, angiographic and etiological features of retinal
vasculitis: retrospective study of 77 patients
Pauline Beaujeux, Fanny Tréchot, Benjamine Batta, Véronique
Cloché, Jean-Baptiste Conart, Karine ANGIOI. Ophthalmology,
CHU Nancy, France, Nancy, France.
Purpose: Retinal vasculitis (RV) are a significant cause of chronic
and severe intraocular inflammation, a topic on which few studies
have been published. This retrospective and observational study
aims at determining the clinical, angiographic and etiological
characteristics of a RV cohort.
Methods: Between May 2012 and May 2014 we retrospectively
included from an angiographic database77 patients with RV, with at
least a 6 month follow-up, who all had a record in Internal Medicine.
Results: Mean age at diagnosis was 35.1 years and mean followup was 29.1 months. The average time between the onset of
symptoms and the first examination was 4.3 months and 68.9%
of patients consulted for decreased visual acuity. At first visit we
observed pan-uveitis in 44% of cases and the disease was bilateral
in 75% of cases. For 64.7% of patients, RV were only diagnosed
by angiography and were therefore not reported in the examination
of the fundus. On fluorescein angiography, venous lesions and
ischemic forms accounted respectively for 90.7% and 34.8% of
cases. The most common complication was macular edema (65.3%).
After etiological record, 44% of RV remained idiopathic, 32%
were related to local ophthalmic pathologies, 9.3% were due to
systemic vasculitis, and 9.3% to systemic inflammation. The most
common etiology was Birdshot chorioretinopathy (9.3%). We treated
with systemic corticosteroids 85.9% of patients, with addition of
immunosuppressive therapy for 53.7% of them. Unilateral or bilateral
visual impairment defined by a visual acuity lower or equal to 20/70
concerned 23.9% of patients at the last visit.
Conclusions: Few series describe the epidemiology of RV. In our
study, inclusion of patients based on an angiographic criterion is
original, and the fact that no RV was clinically visible in two thirds
of patients confirms its relevance. Our study compares to other
published data regarding clinical and angiographic characteristics of
RV and differs by the high proportion of idiopathic RV and scarcity
of infectious RV. In our series, RV affect mostly young people
and have potentially serious functional consequences. RV remain
idiopathic in almost half of our cases. Early diagnosis of RV requires
the realisation of an angiography in any intermediate or posterior
uveitis.
Commercial Relationships: Pauline Beaujeux, None; Fanny
Tréchot, None; Benjamine Batta, None; Véronique Cloché, None;
Jean-Baptiste Conart, None; Karine ANGIOI, None
Spectral-domain optical coherence tomography findings in 506
consecutive patients with uveitis
Rafael S. Grajewski, Anna C. Boelke, Sina Meyer, Bernd Kirchhof,
Claus Cursiefen, Ludwig M. Heindl. Ophthalmology, University Eye
Clinic Cologne, Cologne, Germany.
Purpose: To analyze the macular structure in a large series of
consecutive patients with different types of uveitis using spectraldomain optical coherence tomography (SD-OCT).
Methods: 1012 eyes of 506 consecutive patients with anterior,
intermediate, posterior and panuveitis underwent standardized
macular examination using SD-OCT (Heidelberg Engineering,
Germany). Central retinal thickness (CRT), macular volume (MV),
and presence of cystoid macular edema (CME), diffuse macular
edema (DME), serous retinal detachment (SRM), epiretinal
membrane with (ERM+) and without (ERM-) retinal surface
wrinkling were determined.
Results: In dependence of the anatomic location of inflammation,
mean CRT was 327.72 mm (+ 106.29) in anterior uveitis, 356.80 mm
(+ 123.40) in intermediate uveitis, 310.15 mm (+ 122.47) in posterior
uveitis and 363.08 mm (+ 143.10) in panuveitis. The average MV
was 3.35 mm3 (+ 0.51) in anterior uveitis, 3.55 mm3 (+ 0.68) in
intermediate uveitis, 3.25 mm3 (+ 0.70) in posterior uveitis and 3.54
mm3 (+ 0.65) in panuveitis. Statistically, the differences between
the anatomic location of inflammation regarding CRT and MV were
significant (p<0.001, respectively). CME was seen in 20% of all
uveitic eyes, DME in 10%, SRD in 6%, ERM+ in 14% and ERM- in
12%. In dependence of the location, CME was detected in 18% in
anterior uveitis, 28% in intermediate uveitis, 14% in posterior uveitis,
and 27% in panuveitis.
Conclusions: Standardized spectral-domain optical coherence
tomography of the macula is recommended for all uveitis patients.
CRT, MV and the incidence of CME were different regarding the
anatomic location of inflammation, and were highest in intermediate
and panuveitis.
Commercial Relationships: Rafael S. Grajewski, None; Anna C.
Boelke, None; Sina Meyer, None; Bernd Kirchhof, None; Claus
Cursiefen, None; Ludwig M. Heindl, None
Support: German Research Foundation DFG Grant Gr 2647/4-1
Change of scleral architecture in Chronic Vogt-Koyanagi-Harada
disease
Yosuke Harada1, Pooja Bhat2, Amina Chaudhry1, Debra A.
Goldstein1. 1ophthalmology, Northwestern university, Chicago, IL;
2
ophthalmology, University of Illinois Chicago, Chicago, IL.
Purpose: Complications of Vogt-Koyanagi-Harada disease (VKH)
include cataract, glaucoma, choriodal neovascularization, subretinal
fibrosis, and optic atrophy. While chronic disease has typically been
described as affecting only the anterior segment (AJO. 131: 599-606,
2001), progressive choroidal thinning in chronic VKH patients has
been reported (Graefes Arch Clin Exp Ophthalmol. 250: 1089-95,
2012). We have seen VKH patients with apparent changes in scleral
architecture as well as retinal atrophy and choroidal thinning. The
purpose of this study is to evaluate whether chronic VKH disease
affects the sclera.
Methods: Chart review of patients with VKH seen by the uveitis
service at Northwestern University July 2012 - September 2014.
Change of scleral architecture was defined as 1) progressive
stapyloma-like posterior bowing on optic coherence tomography
(OCT), 2) progressive increase in axial length, or 3) change of
refraction of more than -1.0 D not explicable by other etiologies.
Results: 16 patients (28 eyes) with VKH were included. 4 eyes of 4
patients were excluded for lack of follow up imaging. 8 eyes (28.6%)
of 5 patients showed progressive scleral architectural changes. 5 eyes
of 3 patients developed posterior architectural changes on OCT, 1
eye had posterior bowing on OCT with documented progression of
axial length, 2 eyes of a bilaterally pseudophakic patient developed
refractive change more than -1.5D with documented increase in axial
length. All of these eyes had choroidal thinning.
Conclusions: Recently, choroidal thinning has been reported in
chronic VKH patients. Our results indicate that scleral structure may
also change in these patients, via a mechanisms as yet unknown.
While choroidal thinning of VKH patients is reported to develop
within 12 months of onset (Graefes Arch Clin Exp Ophthalmol. 250:
1089-95, 2012), scleral changes in this study continued to develop
for years after onset. This suggests that scleral change may develop
after choroidal thinning. The choroid is reported to play a role in
regulating scleral remodeling (Progress in Retinal and Eye Research.
29: 144-68, 2010), and scleral change may be caused by impairment
of scleral remodeling resulting from choroidal atrophy. Progressive
scleral architectural change may be a complication of chronic VKH.
Commercial Relationships: Yosuke Harada, None; Pooja Bhat,
None; Amina Chaudhry, None; Debra A. Goldstein, None
Seasonality of the white dot syndromes
Yasser Elshatory, Mahajan Vinit, James C. Folk. Ophthalmology,
University of Iowa, Iowa City, IA.
Purpose: While seasonality is a well-known attribute of human viral
infections, especially enteric and respiratory viruses, it is unknown
whether there is a seasonality component to the white dot syndromes,
which are frequently hypothesized to have a viral etiology. To
evaluate this, we assessed the timing of signs and symptoms
associated with these conditions to evaluate for seasonality patterns.
We performed a retrospective review of patients with white dot
syndromes to assess the month of the year they initially experienced
symptoms.
Methods: A retrospective chart review was performed of patients
presenting to the vitreoretinal service at a tertiary referral center
within the Midwest. Patients had various white dot syndromes. A
photography database was used to identify diagnosis-specific cases,
and those cases were evaluated for clearly identifiable dates of
symptom onset.
Results: Eighty seven charts of patients with acute multifocal placoid
pigment epitheliopathy, punctate inner choroidopathy, serpiginous
choroidopathy, acute zonal occult outer retinopathy, or multifocal
choroiditis were reviewed; of which, forty five cases had clearly
identifiable dates of onset. The majority of these cases presented
during the fall (n=30), followed by the summer (n=21). Fewer cases
presented during the spring (n=12), and winter (n=6). The seasonal
index (the number of cases in a month divided by the average number
of cases across all months) varied from1.87 in November to 0.27 in
January. A test for seasonality supported a strong seasonal component
to the white dot syndromes (p<0.0001).
Conclusions: White dot syndrome have a strong seasonal
predilection towards fall and summer months over spring and
winter months. This finding may suggest viral pathogens that more
frequently present during fall and summer months may underlie these
conditions. Reproducing this finding in prospective series of white
dot syndromes would further support such as conclusion.
Commercial Relationships: Yasser Elshatory, None; Mahajan
Vinit, None; James C. Folk, None
Birdshot Retinochoroidopathy Lesions on Indocyanine Green
Angiography as an Indicator of Disease Activity
Jennifer Cao1, 3, Sukhum Silpa-Archa1, 3, Clovis Freitas1, 3, C Stephen
Foster1, 2. 1Massachusetts Eye Research and Surgery Institute,
Cambridge, MA; 2Ophthalmology, Harvard Medical School,
Cambridge, MA; 3Ocular Immunology and Uveitis Foundation,
Cambridge, MA.
Purpose: While the presence of classic lesions seen on indocyanine
green angiography (ICG) has been well described in the diagnosis
of birdshot retinochoroidopathy, limited information exists on the
natural history of these lesions, with or without treatment. This is a
retrospective case series that objectively describes the evolution of
these lesions between times of disease activity and quiescence.
Methods: A retrospective review was performed on all patients with
birdshot retinochoroidopathy who had at least one ICG performed
during disease activity and disease quiescence. For each patient,
a single photograph of a single eye centered on the posterior pole
during the intermediate phase (~10min) of the ICG was selected for
analysis during time of disease activity and quiescence. Photoshop
was used to mark the lesions. The mean total number of spots and
total lesional area was compared between disease activity and
quiescence using a paired ratio t-test.
Results: A total of 26 patients (26 eyes) were analyzed. There
was a mean of 75.27 total spots encompassing a mean total area
of 24,525.42 pixels during disease activity. There was a mean of
28.35 total spots encompassing a mean total area of 7,411.00 pixels
during disease quiescence. There was a statistically significant
decrease in both the mean total number of spots (-62.34%, p<0.01)
and total lesion area (-69.78%, p<0.01) between disease activity
and quiescence. In 24 patients, resolution of spots correlated with
treatment-induced disease quiescence. In 2 patients with a relative
absence of ICG spots during treatment-induced remission, subsequent
reappearance of spots correlated with a flare of disease activity.
Conclusions: There was a statistically significant decrease in the
mean number of lesions and lesion area between time of disease
activity and disease quiescence. Our results suggest that ICG
has a role not only in diagnosis, but also in monitoring treatment
effectiveness. ICG lesions can resolve with treatment, and their
reappearance may be indicator of disease relapse.
Commercial Relationships: Jennifer Cao, None; Sukhum SilpaArcha, None; Clovis Freitas, None; C Stephen Foster, None
Complication in Uveitis
Lucia Comastri, matilde lopez, erika hurtado, Mercedes Frick,
Cristobal A. Couto. Ophthalmology, Hospital de Clinicas, Ciudad
Autonoma de Buenos Aires, Argentina.
Purpose: To describe the complications associated in cases of uveitis
and scleritis of patients who are trated at the department of uveitis at
the Hospital de Clínicas Jose de San Martin. University of Buenos
Aires
Methods: A 650 medical records of patients data base was
performed, from people that had been served between January 2006
and September 2014. Data collected was: medical record number,
name and last name, date of birth, diagnosis, progression of the
illness, laterality, antaomic location, aspect, and complications.
Results: From the 650 patients, 59 didn’t had neither uveitis nor
scleritis. From the 591 patients left, 322 didn’t show complications in
them medical record, whereas the other 269 did.
Cataracts was the most common complication we found in 99 cases,
followed by: synechiae/pupilar seclusion (54), glaucoma (47),
macular edema (32), retinal detachtment (22), epirretinal membrane
(20), iris atrophy (14), hypotony (12), calcic kerathopathy (9),
choroidal thickening/ptisis bulbi (7), pigment epithelium atrophy and
choroidal neovascular membrane (4), iridis rubeosis(3), choroidal
detachtment (2) and iridodyalisis and hemovitreus in 1 case.
Patients with cataracts, synechiae/pupilar seclusion, glaucoma,
retinal detachtment, iris atrophy, hipotony, thickening/ptisis
bulbi, epithelium atrophy/retinochoroidal atrophy iridodyalisis
and choroidal detachtment as complication had vogt koyanagui
harada (VKH) disease assosiated most frequently; then, those with
macular edema were most frequently associated with pars planitis,
those with epirretinal membrane with toxoplasmosis, those with
calcic keratopathy with juvenile rheumatoid arthritis, patients with
choroidal neovascular membrane with multifocal choroiditis and
those with rubeosis were associated with unknown causes.
Conclusions: The most common complication found in all cases
was cataracts, and the disease most frequently associated with any
complication was VKH.
Commercial Relationships: Lucia Comastri, None; matilde lopez,
None; erika hurtado, None; Mercedes Frick, None; Cristobal A.
Couto, None
Combined systemic and intravitreal antiviral treatment in acute
retinal necrosis
Emile Sharifi1, Masako Chen2, Diaz Vicente1, John Mauro1, C M.
Samson1, Sanjay Kedhar1. 1Ophthalmology, New York Eye and Ear
Infirmary, New York, NY; 2New York Medical College, New York,
NY.
Purpose: To determine the outcomes at our institution for treating
acute retinal necrosis (ARN) with combined systemic and intravitreal
antiviral agents.
Methods: A retrospective chart review was undertaken of all
patients seen at the largest tertiary eyecare hospital in New York City
from 2011 onward. Patients were identified by polymerase chain
reaction (PCR) confirmation of intraocular fluid for viral Herpes
Simplex Virus (HSV1 or 2) and Varicella Zoster virus (VZV) and
exam findings consistent with ARN. All patients received combined
intravitreal and systemic antiviral treatment. Patients who were
immunocompromised (AIDS, systemic malignancy, systemic
immunosuppresion) or presented with no light perception in the
affected eye were excluded. We report the outcomes by best corrected
visual acuity (BCVA) at 6 months and development of retinal
detachment (RD).
Results: Twelve patients and thirteen eyes met the criteria with
median follow-up time of 11 months (range 6-45 months) and with
a median age of 53 years at presentation (range 26-82 years). The
majority (8 of 12) of patients were male. At presentation, 2 eyes
(15.4%) had best corrected visual acuity better than 20/40 and 5 eyes
(38.5%) had best corrected visual acuity better than 20/200. Seven
(58.3%) patients had VZV, three had HSV2 (25%), and two had
HSV1 (16.7%). Eight eyes (61.5%) developed retinal detachments.
By viral etiology, six of eight eyes (75%) with VZV developed
RD compared to 2 of 5 (40%) eyes with HSV, which was not a
statistically significant difference (P= 0.29). Three eyes (23.0%) had
best corrected visual acuity better than 20/40, and 8 eyes (61.5%) had
best corrected visual acuity better than 20/200. Excluding the eye that
presented with 20/25 vision, the best corrected visual acuity of 6 eyes
(50%) improved by at least 2 lines, was stable in 4 eyes (33.3%), and
was worse in 2 eyes (16.7%). One patient developed contralateral
ocular involvement.
Conclusions: Combined systemic and intravitreal therapy yields
results in our patient population that are at least comparable to
other retrospective outcomes studies using just systemic antivitreal
treatment. Notably, our inclusion criterion of PCR positive viral
disease may bias to more severe disease. A larger, prospective,
randomized study is needed to answer the question if there is
additional therapeutic benefit to intravitreal antivirals being added to
systemic treatment.
Commercial Relationships: Emile Sharifi, None; Masako Chen,
None; Diaz Vicente, None; John Mauro, None; C M. Samson,
None; Sanjay Kedhar, None
Gene Expression Levels in Sarcoidosis Patients with Uveitis
John A. Gonzales1, Nisha Acharya1, Erica Browne1, Laura Koth2.
1
F.I. Proctor Foundation, F.I. Proctor Foundation, San Francisco, CA;
2
University of California, San Francisco, San Francisco, CA.
Purpose: The diagnosis of sarcoid uveitis remains challenging as
intraocular tissues are not typically amenable to biopsy, which is the
current gold standard for diagnosing this granulomatous disease.
Recent studies have shown that specific genes are not only important
in pulmonary sarcoidosis activity and progression, but also allow for
the differentiation of pulmonary sarcoidosis from other inflammatory
lung diseases. Our objective was to compare the expression levels
for genes previously described to be discriminative of pulmonary
sarcoidosis in patients with and without uveitis in the setting of
pulmonary sarcoidosis. We hypothesized that patients with uveitis in
the setting of pulmonary sarcoidosis would exhibit similar expression
levels for genes implicated in sarcoid activity as well as genes
discriminative for sarcoid.
Methods: We reviewed the gene expression levels for subjects with
biopsy proven pulmonary sarcoidosis previously enrolled into a
longitudinal study in which peripheral blood was drawn for gene
expression levels. Expression levels had been previously assessed
via quantitative PCR, being expressed as log2 cycle threshold values
relative to mean healthy control cycle threshold values. Patients
were categorized as having uveitis (based on a health-assessment
questionnaire) or not having uveitis. We performed random effects
modeling for each candidate gene comparing patients with uveitis to
those without uveitis and controlled for FEV1/FVC ratio. Bonferroni
correction was also performed.
Results: Gene expression levels were available for 167 subjects and
12 of these subjects had uveitis.
We found no significant difference in expression levels sarcoiddiscriminative genes (p values > 0.05) between patients with uveitis
and pulmonary sarcoidosis and sarcoid patients without uveitis.
Additionally, there were no significant differences in expression
levels between the two groups of patients for 45 other genes
important in granulomatous inflammation.
Conclusions: The gene expression profile between pulmonary
sarcoidosis patients with and without uveitis appears to be
comparable. While no significant differences in gene expression
levels between the two groups of patients was identified, we plan on
further correlating changes of gene expression levels longitudinally
with uveitis activity.
Commercial Relationships: John A. Gonzales, None; Nisha
Acharya, None; Erica Browne, None; Laura Koth, None
Support: That Man May See
A new index of advanced medication need in Vogt-KoyanagiHarada disease
Kazuichi Maruyama, Ai Shimizu, Yukihiro Shiga, Koji Nishiguchi,
Hiroshi Kunikata, Toru Nakazawa. Ophthalmology, Tohoku
University Graduate School of Medicine, Sendai, Japan.
Purpose: To investigate a new index of advanced medication need in
Vogt-Koyanagi-Harada disease (VKH), we measured choroidal blood
flow levels with laser speckle flowgraphy (LSFG) and choroidal
thickness with swept-source optical coherence tomography (SSOCT).
Methods: Twelve eyes of 24 patients (14 female, 10 male) were
included in this study. Patients needing additional treatments,
such as immune suppressive drugs, due to recurrent or choroidal
neovascularization were classified as the advanced group. Patients
needing only steroid treatment were classified as the normal
treatment group. A blood flow analysis was performed with LSFG
measurements of mean blur rate (MBR), taken in the choroidal
vessel area, on days 0, 14, 30 and 60. Choroidal thickness (CT) was
measured with SS-OCT, using a 12-radial-scan protocol, on days 0,
14 and 30.
Results: The advanced group exhibited low MBR throughout
the study period. On day 60, MBR in the advanced group was
significantly lower than in the steroid treatment group (p < 0.05).
CT in the advanced group was significantly lower than in the steroid
treatment group at the initial examination, but the groups approached
a similar level on days 14 and 30.
Conclusions: We found that advanced VKH patients exhibited low
MBR throughout the study period. In addition, we found that CT was
lower in the advanced group than in the steroid treatment group, but
not throughout the entire study period. Even though the pathological
background of VKH can vary, LSFG may be useful in both making
VKH prognoses and choosing therapeutic options.
Commercial Relationships: Kazuichi Maruyama, None; Ai
Shimizu, None; Yukihiro Shiga, None; Koji Nishiguchi, None;
Hiroshi Kunikata, None; Toru Nakazawa, None
Toxoplasmosis Antibody Titers as a Supportive Tool in the
Diagnosis of Ocular Toxoplasmosis
Miin Roh, Heeyoon Cho, Cagla Yasa, Laura Nicholson, Eduardo
Uchiyama, Lucy H. Young, Marlene Durand, Ann-Marie Lobo,
George Papaliodis, Lucia Sobrin. Ophthalmology, Massachusettes
Eye and Ear Infirmary, Boston, MA.
Purpose: Ocular toxoplasmosis is the most frequent cause of
infectious posterior uveitis. Although the classic teaching is that the
anti-toxoplasmosis antibody titer is not particularly informative in the
diagnosis, there are very few studies that have examined the role of
serologic testing. The purpose of this study is to determine whether
a correlation between toxoplasmosis antibody titers and ocular
toxoplasmosis presence and activity exists.
Methods: We retrospectively reviewed charts of patients who had
positive serologic testing for Toxoplasmosis gondii IgG antibodies
using an enzyme linked immunosorbent assay. A cross-sectional
study was performed using analysis of variance to compare IgG
levels among patients who had active toxoplasmosis chorioretinitis
(TC), inactive TC, and non-toxoplasmosis uveitis patients. A
longitudinal investigation to compare toxoplasmosis IgG titers in
subset of patients who had serial serologic testing at the time of
active and inactive TC was performed using the paired t test. All
analyses were performed in StataIC 12 (Stata, College Station, TX).
Results: 81 patients with active TC, 31 patients with inactive TC,
and 54 patients nontoxoplasmosis uveitis were identified. In the
cross-sectional analysis, mean toxoplasmosis IgG titers were not
significantly different between active TC (164.1 ± 97.1 IU/ml) vs.
inactive TC (145.3 ± 108.9 IU/ml) ( P=0.10). There was, however, a
significantly lower level (90.4 ± 86.6 IU/ml) in non-toxoplasmosis
uveitis patients (P< 0.001). In the longitudinal analysis of the subset
of 22 patients with ocular toxoplasmosis, there was no significant
difference in the mean level of toxoplasmosis IgG: 160.1 IU/ml
during active disease vs. 191.3 IU/ml once the disease was inactive
(P=0.19).
Conclusions: Toxoplasmosis IgG titers were higher in patients with
ocular toxoplasmosis, whether the disease was active or inactive, as
compared to non-toxoplasmosis uveitis patients. Both cross-sectional
and longitudinal data failed to show any difference in toxoplasmosis
IgG titers in active vs. inactive ocular toxoplasmosis. Future studies
are needed to confirm whether the degree of toxoplasmosis IgG
titer elevation might be useful in distinguishing whether atypical
presentations of chorioretinitis are due to toxoplasmosis.
Commercial Relationships: Miin Roh, None; Heeyoon Cho, None;
Cagla Yasa, None; Laura Nicholson, None; Eduardo Uchiyama,
None; Lucy H. Young, None; Marlene Durand, None; Ann-Marie
Lobo, None; George Papaliodis, None; Lucia Sobrin, None
A Resurgence in Ocular Syphilis: A Case Series
Thomas Kandl, Ronald J. Rescigno, David S. Chu. Ophthalmology,
Rutgers University New Jersey Medical School, Jersey City, NJ.
Purpose: According to the CDC, the rate of syphilis reported in the
United States decreased through the 1990’s,was at its lowest ever
reported in 2000, and then increased from 2001-2009. We presented
a series at ARVO in 2000 highlighting the relatively high incidence
of ocular syphilis seen at our institution during the 1990’s. This was
followed by a lack of cases encountered between 2000 and 2013.
However, 4 new cases of ocular syphilis have presented to our facility
over the past year. We present these 4 cases and examine the potential
implications in the recent rise in case volume.
Methods: To do a retrospective analysis of the patients at our
institution with ocular syphilis from 2013-2014
Results: All four of our patients presented with complaints of
decreased vision and had clinical findings consistent with panuveitis
with optic nerve involvement. One of the four patients was HIV
positive and was diagnosed with AIDS, not undergoing treatment
at the time of diagnosis. One patient was initially treated with oral
corticosteroids for presumed sarcoidosis resulting in a significant
worsening of symptoms and visual acuity prior to treatment for
tertiary syphilis. One patient received local administration of
corticosteroid for panuveitis by a private ophthalmologist prior
to presenting to our facility. All four patients underwent thorough
systemic work up with an Infectious Disease consult. All four patients
responded to appropriate treatment with penicillin with improvement
in clinical findings and visual acuity. Two of the patient’s final visual
acuity returned to normal in both eyes. Two patient’s visual acuity
returned to normal in one of the eyes with only mild improvement in
the other eye.
Conclusions: Ocular syphilis often takes several years to manifest
after initial infection. The case volume of ocular syphilis at our
institution parallels national trends of syphilis infection. The paucity
of cases during the early 2000’s corresponds with a decline in syphilis
cases during the 1990’s, and the rise in ocular cases over the past year
corresponds with the increasing rate of syphilis encountered from
2001-2009. We predict a rise in ocular syphilis cases, and though still
a relatively rare entity, syphilis should remain high on the differential
diagnosis when encountering a patient with severe uveitis as prompt
diagnosis and treatment can avoid preventable, permanent visual loss.
Commercial Relationships: Thomas Kandl, None; Ronald J.
Rescigno, None; David S. Chu, None
Clinical Patterns of Uveitis in Children in Two Ophthalmological
Centers in Bogota, Colombia
Alejandra De-La-Torre1, 5, Marcela Lonngi2, 3, Camila Aguilar2, 3,
Hernán Ríos3, Cristian Aristizábal-Duque4, Francisco Rodríguez6,
7 1
. Immunology and Uveitis, Universidad del Rosario, Bogotá,
Colombia; 2Ophthalmology, Universidad del Rosario, Bogotá,
Colombia; 3Ophthalmology, Fundación Oftalmológica Nacional,
Bogotá, Colombia; 4Universidad del Quindío, Armenia, Colombia;
5
Immunology and Uveitis, Fundación Oftalmológica Nacional,
Bogotá, Colombia; 6Retina and Vitreous, Universidad del Rosario,
Bogotá, Colombia; 7Retina and Vitreous, Fundación Oftalmológica
Nacional, Bogotá, Colombia.
Purpose: To describe the clinical features of uveitis in pediatric
patients treated in two ophthalmological centers in Bogotá,
Colombia, in a 13 year-period between January 2000 and July 2013.
Methods: Retrospective observational and descriptive study.
Systematic review of the clinical records of pediatric patients with
diagnosis of uveitis at the Fundación Oftalmológica Nacional and
at the Uveitis Clinic from Universidad del Rosario in Bogotá,
Colombia. Data was analyzed and compared to previous reports.
Results: Uveitis was found in 311 children, of which 161 were
female (51.8%) and 150 were male (48.2%). Mean age of
presentation was 10.1 years. Posterior uveitis was the most common
type of uveitis (58.8%), more frequently of insidious onset (77.8%)
and chronic course (25.1%). The most common etiology was
infectious (58.2%); being toxoplasmosis the most frequent cause
(44.6%), followed in by toxocariasis (17.7%). Idiopathic uveitis
(33%) was found to be the second one in frequency. Less frequent
causes were trauma (3.5%), juvenile idiopathic arthritis (1.9%),
Vogt–Koyanagi–Harada syndrome (1%), spondyloarthropathies
(0.3%), sarcoidosis (0.3%), and Eales disease (0.3%). There was a
statistically significant difference in visual acuity between anterior
(20/67) and intermediate uveitis (20/69), compared to posterior
uveitis (20/417) and panuveitis (20/209) (p <0,05).
Conclusions: This work is the first report of clinical features of
uveitis in children in Colombia. Infectious etiologies, especially
parasitic diseases, are the main cause of uveitis in pediatric
population in our country. This study will improve awareness and
knowledge of pediatric uveitis in under-development countries, and
it would contribute to the improvement of public health policies of
pediatric visual-health.
Commercial Relationships: Alejandra De-La-Torre, None;
Marcela Lonngi, None; Camila Aguilar, None; Hernán Ríos,
None; Cristian Aristizábal-Duque, None; Francisco Rodríguez,
None
Occult multifocal choroiditis
Niloofar Piri, Henry J. Kaplan. Ophthalmology and Visual Sciences,
University of Louisville, Louisville, KY.
Purpose: To report occult multifocal choroiditis as an
underdiagnosed entity.
Methods: Retrospective chart review of diagnostic testing in four
patients with idiopathic uveitis
Results: Four patients with idiopathic uveitis (3 anterior uveitis,
one vitritis) demonstrated multiple hypofluorescent (dark) spots
on ICG angiography without clinical evidence of choroiditis on
funduscopy or SD-OCT. All had increased serum IgG titers to herpes
viruses (HSV1, 2; CMV; EBV; HHV6 and VZV), with two showing
markedly elevated IgM titers to HSV 2.Three patients showed very
high IgG titers to EBV, HHV6 and VZV. The patient who presented
with vitritis, responded to Acyclovir treatment with complete
resolution of vitritis; 3 months later she developed acute anterior
uveitis with diffuse stellate KPs and raised intraocular pressure.
Anterior chamber paracenthesis was performed and PCR was done
which was positive for HSV 2 and confirmed the prior diagnosis. She
responded clinically to another course of Acyclovir treatment.
Conclusions: In patients with idiopathic uveitis non-responsive to
corticosteroid treatment or unexplained decreased vision, occult
multifocal choroiditis should be considered.ICG angiography may be
essential in detecting occult multifocal choroiditis, and can lead to the
investigation of unexpected herpesvirus infection.
Commercial Relationships: Niloofar Piri, None; Henry J. Kaplan,
None
Ocular manifestations of syphilis: an eight-year restrospective
study in southern France
Benjamin Butet, Stéphanie Baillif. Service d’Ophtalmologie, CHU
Nice Hopital Saint-Roch, Nice, France.
Purpose: Over the last decade, there is a trend for increasing
frequency of syphilis in developed countries. Prompt recognition of
syphilitic uveitis is essential. The purpose of this study is to describe
the ocular manifestations of syphilis and its outcome.
Methods: A retrospective, observational, non comparative review
of clinical records was performed. Every treponemal and non
treponemal serologies performed at the Nice University Hospital
from november 2005 to october 2013 were collected. Cases with
documented syphilis infection and ocular involvement were reviewed
over that period. Collected data included clinical ophthalmologic
features (visual acuity, slit lamp and fundus), complementary
examinations, treatment and follow-up.
Results: 51 patients had active syphilis infection over that period.
Eleven patients were diagnosed with syphilitic uveitis (21%). Ocular
inflammation led to the diagnosis of syphilis in 9 patients (81.8%).
Clinical features were bilateral for 9/11 patients (81.8%) and
characterized by papillitis (10 patients ; 90.9%) which was isolated
in 3 cases, and part of a panuveitis for 7 patients (63.6%). Panuveitis
exhibited vitreous inflammation (100%), venous vasculitis (57.1%),
posterior placoid choroiditis (42.8%) multifocal choroiditis (14.2%),
vitreous hemorrhage (28.5%), associated with inflammation of the
anterior segment (71.4%).
Isolated anterior granulomatous uveitis was found in one patient
(9%). Initial mean visual acuity was 20/40 (range: 20/20 to “light
perception”) and 20/25 at last visit. HIV co-infection was diagnosed
in 3 patients (27.2%). A lumbar puncture found a central nervous
system infection in 7 patients (63.6%).
Conclusions: Posterior bilateral uveitis was the most common
ophthalmologic manifestation of syphilis. Anatomical and functional
ocular prognosis was good after appropriate antibiotic therapy.
Syphilis should still be considered in the differential diagnosis of
ocular inflammation. An HIV co-infection should be searched for.
Commercial Relationships: Benjamin Butet, None; Stéphanie
Baillif, None
Hyperreflective Dots in Spectral Domain Optical Coherence
Tomography as Phenotypic Marker in Uveitis-Associated Cystoid
Macular Edema
Alexandre Sellam, Nathalie Massamba, Audrey Fel, Phuc Lehoang,
Bahram Bodaghi. Ophthalmology, Hôpital Pitié Salpétrière, Paris,
France.
Purpose: To evaluate agreement between hyperreflective dots (HRD)
in retina layers by spectral domain optical coherence tomography
(SD-OCT), laser flare photometry associated with best-corrected
visual acuity (BCVA) results in patients with uveitis-associated
cystoid macular edema (CME). Thus, the aim of the study was to
demonstrate that the HRD in SD-OCT could be a phenotypic marker
in CME-associated posterior uveitis.
Methods: All patients referred to our tertiary care center for posterior
uveitis had full examination including: BCVA, laser flare photometry,
SD-OCT, fluorescein and infracyanine angiography. Causes of
uveitis were multiple: infectious, inflammatory and unknown. The
characteristics of the HRD were evaluated in all SD-OCT scans. HRD
were defined as small focal hyperreflective material scattered mainly
in outer retinal layers but also spreading to all retinal layers observed
in at least one available scan. Two different ophthalmologists counted
HRD using a horizontal B scan cross section through the fovea.
The eyes were divided into three groups according to the location
of HRD on SD-OCT : RNFL + ILM, INL + OPL and ONL. They
were classified on the basis of quantity (i.e. absent, few if less than
10, moderate if between 10 and 20, or numerous if more than 20).
Central macular thickness (CMT) was also evaluated.
Results: Thirty eyes of 22 patients were included. Twelve men and
10 women with posterior uveitis were examined. HRD were present
in all patients with CME in the outer and inner retinal layers. Overall,
22 HRD were found in the RNFL and ILM, 24 in INL and OPL and
13 in ONL.
Mean flare measurement was 13 +/- 11.5. Mean BCVA was 20/200.
Mean central macular thickness was 420 μm.
Two subgroups were identified. Nineteen eyes presented decreased
BCVA, normal or elevated Flare, numerous HRD and increased
CMT, corresponding to active posterior uveitis. Other 11 eyes
presented decreased BCVA, increased CMT, normal laser Flare
photometry, those results corresponding to inactive posterior uveitis.
The difference with the two groups was numerically significant.
Conclusions: HRD are correlated to CMT and BCVA but not to the
Flare. Flare is not predictive of inflammation for posterior uveitis.
HRD in posterior uveitis associated with CME could be an early
marker of inflammation corresponding to the activation of microglial
cells.
Commercial Relationships: Alexandre Sellam, None; Nathalie
Massamba, None; Audrey Fel, None; Phuc Lehoang, None;
Bahram Bodaghi, None
“Spotless” Birdshot Chorioretinopathy: Improved Diagnostic
Sensitivity with Indocyanine Green Angiography
Christopher R. Henry1, Ashvini K. Reddy2, Marco A. Gonzalez1,
Steven Yeh3, Thomas A. Albini1. 1Bascom Palmer Eye Institute, Miami
Beach, FL; 2Ophthalmology, University of Virginia, Charlottesville,
VA; 3Ophthalmology, Emory University, Atlanta, GA.
Purpose: To describe a cohort of patients with birdshot
chorioretinopathy (BSCR) who did not manifest characteristic
birdshot lesions on clinical examination or fluorescein angiography
(FA) but had retinal vasculitis, low-grade to moderate vitritis and
hypofluorescent lesions on indocyanine green angiography (ICGA).
Methods: Retrospective chart review.
Results: We present five patients from the Bascom Palmer Eye
Institute, the University of Virginia, and Emory University evaluated
from 2007 to 2014, with mild-moderate vitritis and retinal vasculitis
without evidence of characteristic birdshot lesions on clinical
examination. All patients were positive for HLA-A29 and had
hypofluorescent lesions visible on ICGA that were not detectable on
FA.
Average age at presentation was 49.8 years (range 40-62). Three
patients were female and two were male. All patients presented with
bilateral eye disease. Best-corrected presenting visual acuity ranged
from 20/20-20/80 OD (median 20/30) and 20/20-20/80 OS (median
20/25). Three patients were successfully managed with steroidsparing immunosuppressive therapy and one patient was successfully
treated using bilateral 0.7mg dexamethasone implant (Ozurdex)
injections. A final patient with very mild vitritis declined medical
therapy and remained asymptomatic with 20/20 visual acuity through
9 months follow up.
Characteristic imaging findings of a 46 year old female patient are
shown in Figure 1. This patient presented with a six-month history
of iridocyclitis unresponsive to topical steroids. BCVA was 20/30
OD and 20/25 OS. Examination was remarkable for rare anterior
chamber cell, mild-moderate vitritis, disc edema, and retinal
vasculitis OU. Macular edema was absent on clinical examination
and OCT. FA demonstrated retinal vasculitis. ICGA confirmed diffuse
hypofluorescent lesions consistent with BSCR. HLA A29 testing was
subsequently positive. The patient began therapy with mycophenolate
mofetil and cyclosporine A, with which her symptoms improved.
Retinal vasculitis and vitritis have fully resolved.
Conclusions: Patients with retinal vasculitis and low grade vitritis
with or without macular edema may have BSCR evident on ICGA
before lesions are visible on clinical examination or FA. Expanding
BSCR diagnostic criteria to include the presence of hypocyanescent
lesions on ICGA could potentially improve the sensitivity of
diagnosis.
Commercial Relationships: Christopher R. Henry, None; Ashvini
K. Reddy, None; Marco A. Gonzalez, None; Steven Yeh, None;
Thomas A. Albini, None
Clinical features of PIOL with optic nerve head involvement
Kazunobu Asao, Noriyasu Hashida, Kei Nakai, Kohji Nishida.
Ophthalmology, Osaka University Medical School, Osaka, Japan.
Purpose: To investigate the clinical features of cases of primary
intraocular lymphoma (PIOL) with optic nerve head involvement
Methods: We retrospectively studied 5 patients (two women, three
men, the mean age 61.8±11.1 years) with optic nerve head invasion
of 42 patients with a diagnosis of PIOL at our hospital between
January 2010 and October 2014. After the onset of optic nerve head
involvement, we examined the clinical features, invasion, changes of
visual acuity, effects of local and systemic treatment and prognosis.
Results: The period from the time of onset to the optic nerve invasion
was 16±14.5 months (4-40months). Histological examination of
biopsy sample revealed that the types of lymphoma were diffuse large
B-cell type; 4 cases, follicular lymphoma; 1case. Visual acuity (VA)
measurement revealed the following results. At the time of invasion,
VA in the patients was 2/20 or less in three cases and the rest of two
cases was 10/20 or more. VA was maintained during the followup period. Clinical features of optic disc involvement were optic
disc swelling, edema, serous retinal detachment and retinal artery
occlusion. Concerning the treatment, systemic chemotherapies were
performed in all cases and whole brain irradiation was performed in
two cases. For the local treatment, intravitreal methotrexate (IVM)
was performed. Finally the times of IVM were 4.6 times (1-7 times).
During the follow-up, contrast enhanced MRI was performed every
three months to detect central nervous system involvement. Brain
involvement was detected in one case before the invasion and finally
increased to 3 cases during the follow-up period. Four cases had good
prognosis with survival and one case had died.
Conclusions: Malignant lymphoma showed different clinical
findings. Optic nerve head invasion could be a form of relapse.
During the follow-up, VA did not get worse and there is a possibility
that we can maintain the VA with IVM in some cases. Since optic
nerve head involvement is likely to cause brain involvement,
systemic medical treatment and careful observation are needed.
Commercial Relationships: Kazunobu Asao, None; Noriyasu
Hashida, None; Kei Nakai, None; Kohji Nishida, None
Human Papilloma virus detection in squamous cell carcinoma of
the conjunctiva
Daniel R. Capiz, Guadalupe M. Tejeda-rojas, Hector Perez Cano.
Orbit and oculoplastics, Hospital de la Luz, Df, Mexico.
Purpose: To evaluate the presence of human papilloma virus (HPV)
in squamous cell carcinoma of the conjunctiva.
Methods: Archival paraffin embedded tissue from squamous cell
carcinoma with different degrees of differentiation from the years
1992-1995, 2003-2004, 2010-2012 were analyzed for the presence of
(HPV) by polymerase chain reaction.
Results: There were 44 samples processed, the median age of
presentation was 60.2 years, male gender was mostly affected
by 58%, the nasal localization was present in 88.63%, one of the
samples was taken from orbital exenteration and the rest were
retrieved by excisional biopsy. Poorly differentiated carcinoma was
reported in 12 samples, 18 as moderate differentiated, 14 samples
as well differentiated. HPV was present in 3 samples (6.86%), well
differentiated carcinoma was reported in all 3 cases.
Conclusions: The presence of HPV DNA was low. There is
insufficient data to correlate the presence of HPV and the grade of
differentiation.
Commercial Relationships: Daniel R. Capiz, None; Guadalupe M.
Tejeda-rojas, None; Hector Perez Cano, None
The added value of undiluted vitreous biopsy samples processed
by the Cellient® tissue processor (Hologic) in intermediate or
posterior uveitis
Joachim Van Calster, Rita Van Ginderdeuren. Ophthalmology,
University Hospitals Leuven, Leuven, Belgium.
Purpose: In this prospective study, the added value of undiluted
vitreous biopsy samples in the diagnosis of intermediate and
posterior uveitis was evaluated. Vitreous biopsies are difficult to
handle because of the paucity of cells and the gelatinous structure
of the vitreous. Histopathological analysis of the vitreous is useful
in difficult uveitis cases to differentiate uveitis from lymphoma or
infection and to define the type of cellular reaction.
Methods: 97 consecutive undiluted vitreous samples were isolated
in patients with unsolved intermediate or posterior uveitis. A 1.52.5cc sample was taken through a single 23G port using the EVA
vitrectomy platform (DORC) with a twin duty cycle high speed
cutter. The samples were analysed with the Cellient® tissue processor
(Hologic). This machine is a fully automated processor starting from
a specified container with PreservCyt® (fixative fluid) with cells to
paraffin. Routine histochemical and immunostainings were evaluated.
Results: In 94.8% of the cases, sufficient material was found to
provide an added value in the diagnostic workup. In 34%, a Cytolyt®
mucolytic wash was necessary to prevent clotting of the tubes in the
Cellient® tissue processor due to the viscosity of the sample. In 7%
the diagnosis was an acute inflammation (presence of granulocytes),
in 42% chronic active inflammation (presence of T-lymphocytes),
in 36% low-grade inflammation (presence of CD68 cells, with <5%
T-lymphocytes); and in 9% a malignant process (lymphoma). In 5%
no diagnosis was found. In the chronic active inflammation group
39% was a granulomatous inflammatory process.
Conclusions: This standardized protocol for sampling and handling
undiluted vitreous biopsies gives a superior result in morphology,
number of cells, and possibility of immuno-histochemical stainings.
The diagnosis can be established or confirmed in 94.8% of cases.
Commercial Relationships: Joachim Van Calster, Allergan (C),
Allergan (R), Bausch&Lomb (F), Bayer (C), Bayer (R), DORC (C),
DORC (R), MSD (C), Novartis (C), Novartis (F), Novartis (R); Rita
Van Ginderdeuren, None
336 Uveitis: Posterior
Tuesday, May 05, 2015 11:00 AM–12:45 PM
Deficiency of complement anaphylatoxin receptors leads to
ameliorated experimental autoimmune uveitis
Lingjun Zhang1, 3, Brent A. Bell1, Chi-Chao Chan2, Rachel R.
Caspi2, john Fung1, Xiaomin Zhang3, Feng Lin1. 1Cleveland Clinic,
Cleveland, OH; 2National Eye Institute, Bethesda, MD; 3Tianjing
Med University, Tianjing, China.
Purpose: Previous reports suggest that complement anaphylatoxin
receptors (C3aR/C5aR) could regulate T cell responses through
different mechansims, however, the role of these complement
receptors in the pathogenesis of experimental autoimmune uveitis
(EAU) remains elusive. This study is to clarify the importance of
these complement receptors in the development of EAU.
Methods: WT and C3aR/C5aR knockout (KO) mice (C57BL/6J
background) were immunized with an interphotoreceptor retinoid
binding protein (IRBP) peptide to induce EAU following a newly
established protocol which induces more severe EAU in B6 mice
than the tranditional method. After immunization, the development
of EAU was monitored by indirect ophthalmoscopy, and clinical
scores were assigned according to previously published criteria. Two
weeks after immunization, mice were also examined by scanning
laser ophthalmoscopy (SLO) and spectral-domain optical coherence
tomography (OCT), followed by histopathological analysis of the
eyes and antigen-specific Th1/Th17 recall assays using splenocytes
from the mice. In addition, the same number of in vitro activated T
cells from immunized WT or KO donors were adoptively transferred
to naïve WT recipients and the development of EAU was assessed by
regular indirect ophthalmoscopy.
Results: C3aR/C5aR KO mice developed significantly milder EAU
than WT controls as judged by clinical scores, retinal histopathology
scores, and SLO and OCT analyses. Consistent with the imaging
results, C3aR/C5aR KO mice also demonstrated reduced IRBP-
specific Th1 and Th17 responses compared with WT mice. In
addition, adoptive transfer of activated T cells from immunized
C3aR/C5aR KO donors induced less severe EAU in naïve recipients
than the same numbers of cells from WT immunized donors.
Conclusions: Complement anaphylatoxin receptors C3aR and C5aR
are required for the development of EAU, suggesting that targeting
these receptors could be a valid approach for treating patients with
autoimmune uveitis.
Commercial Relationships: Lingjun Zhang, None; Brent A. Bell,
None; Chi-Chao Chan, None; Rachel R. Caspi, None; john Fung,
None; Xiaomin Zhang, None; Feng Lin, None
Suppression of uveitis with melanocortin receptor specific
agonists
Andrew W. Taylor, David Yee, Darren J. Lee. Ophthalmology, Boston
Univ School of Medicine, Boston, MA.
Purpose: Injections of the neuropeptide alpha-melanocyte
stimulating hormone (α-MSH) suppress experimental autoimmune
uveitis (EAU). There are several melanocortin receptors (MCr)
for α-MSH with each signaling for different functions in different
immune cells. Therefore, using MCr specific agonists it is possible to
define the anti-inflammatory activity of α-MSH in treating EAU.
Methods: EAU was induced in C57BL/6 mice immunized with a
peptide of interphotoreceptor retinoid-binding protein (IRBP) in
complete Freunds adjuvant followed by pertussis toxin injections.
The retinas were examined by slit lamp microscopy every 2 - 3
days and scored for inflammation. When the mice showed the first
symptoms of uveitis they were injected with either the MC1r agonist
PNT-107, or the MC5r agonist PG901. Examination of the eyes was
continued. When the EAU of the treated mice resolved, their spleen
cells were assayed for IRBP stimulated cytokines (IL-2, IL-6, IL-10,
IL-17, IFN-γ). For the PG901 treated mice their spleen cells were
adoptively transferred to mice immunized for EAU to see whether the
cells suppressed EAU. The retinas were examined histologically.
Results: In mice treated with PNT-107 or PG901 there was a
significant acceleration in resolving EAU. It was resolved within
15 days after treatment compared to the >80 days of untreated EAU
mice. The IRBP-stimulated response by the spleen cells was different
between the EAU mice treated with PNT-107 and PG901. The
PNT-107 treated mice had a significantly suppressed T cell response.
The PG901 treated EAU mice still had an IRBP-stimulated effector
Th1/Th17 response; however, adoptive transfer of the stimulated
cells suppressed EAU in recipient mice. The retinas of eyes treated
with either agonist were structurally intact with no evidence of
photoreceptor loss.
Conclusions: The treatment of EAU with α-MSH peptide results
in suppression of inflammatory immunity with the induction of
regulatory T cells. These actions of α-MSH are through two different
melanocortin receptors with MC1r being mostly anti-inflammatory,
and MC5r mediating regulatory immunity. While anti-inflammatory
mechanisms, and prevention of future reactivation of uveitis may be
dependent on the specific melanocortin receptors, both agonists were
highly effective in rapidly suppressing uveitis.
Commercial Relationships: Andrew W. Taylor, Mallinckrodt
(Questcor) Pharmaceuticals (C), Mallinckrodt (Questcor)
Pharmaceuticals (R), Palatin Technologies (C), Palatin Technologies
(F), Palatin Technologies (R); David Yee, None; Darren J. Lee,
None
Support: Massachusetts Lions Research Foundation, Palatin
Technologies
SOCS3 deletion in myeloid cells worsens retinal inflammation
and increases angiogenesis in experimental autoimmune
uveoretinitis
Jiawu Zhao1, Mei Chen1, Adrien Kissenpfennig2, Heping Xu1. 1Centre
for Experimental Medicine, Queen’s University Belfast, Belfast,
United Kingdom; 2Centre for Infection and Immunity, Queen’s
University Belfast, Belfast, United Kingdom.
Purpose: Myeloid-derived cells, including neutrophils and
macrophages, are critically involved in retinal damage and
angiogenesis in experimental autoimmune uveoretinitis (EAU).
Suppressors of cytokine signalling (SOCS) proteins are negativefeedback regulators of the JAK/STAT pathway. The aim of this study
is to investigate the effect of SOCS3 deletion in myeloid cells to
EAU development and its associated angiogenesis.
Methods: EAU was induced in C57BL/6 (WT) and LysM-CreSOCS3fl/fl mice. Retinal inflammation was evaluated clinically
using the topical endoscopic fundus imaging (TEFI) system and
optical coherence tomography (OCT), and pathologically by light
microscopy. Retinal vascular leakage was examined by fluorescein
fundus angiography (FFA) and angiogenesis was studied by confocal
microscopy of retinal flatmounts. Real-time RT-PCR and Western
blot were used to explore factors that were critically involved in
inflammatory and angiogenic processes.
Results: TEFI and OCT investigations revealed more severe
inflammation and tissue damage in LysM-Cre-SOCS3fl/fl EAU mice
compared to WT EAU mice. FFA and flatmount staining showed
more angiogenic membrane in LysM-Cre-SOCS3fl/fl EAU mice.
In addition, the expression of IL-1β, CCL2 and VEGF-A in the
retina was increased in LysM-Cre-SOCS3fl/fl EAU mice, which was
accompanied by enhanced pSTAT3 and pErk1/2 expression.
Conclusions: The deletion of SOCS3 in myeloid cells worsens
retinal inflammation and increases retinal angiogenesis in EAU.
SOCS3 may be a potential therapeutic target in autoimmune retinal
inflammation and inflammation-induced angiogenesis.
Commercial Relationships: Jiawu Zhao, None; Mei Chen, None;
Adrien Kissenpfennig, None; Heping Xu, None
NK-DC crosstalk controls the autopathogenic Th17 response and
suppresses uveitis through an innate IFN-γ/IL-27 axis
Rachel R. Caspi1, Nicholas van Panhuys2, Jun Chen1, Phyllis Silver1,
Yingyos Jittayasothorn1, Ronald N. Germain2, Wai Po Chong1.
1
Laboratory of Immunology, National Eye Inst/NIH, Bethesda, MD;
2
National Institute of Allergy and Infectious Diseases/NIH, Bethesda,
MD.
Purpose: IFN-γ is a pathogenic cytokine involved in inflammation.
Paradoxically, its deficiency exacerbates experimental autoimmune
uveitis (EAU), encephalomyelitis, and arthritis. We have previously
demonstrated that unlike adaptive IFN-γ produced by Th1 cells
within the target tissue, innate IFN-γ produced systemically during
the first days after immunization is protective. However, its cellular
source and cellular target were unknown.
Methods: IFN-γ-/- mice have an amplified Th17 response and are
highly susceptible to EAU. We used a reductionist system of IFN-γ-/mice repleted with IFN-γ+/+ NK cells to study the role of NK cellderived IFN-γ on EAU induced by uveitogenic immunization with
IRBP. Fundoscopic examination, immunological assays and in vivo
2-photon imaging were used to examine disease development and the
associated immune responses.
Results: Following immunization for EAU, DCs recruited IFN-γproducing NK cells to the draining lymph node and interacted with
them in a CXCR3-dependent fashion. The interaction caused DCs to
produce IL27, which in turn enhanced IFN-γ production by NK cells,
forming a self-amplifying positive feedback loop. IL-10, produced
by the interacting cells themselves, was able to limit this process.
The NK-DC-dependent IL-27 inhibited development of the adaptive
pathogenic IL-17 response and induced IL-10-producing Tr1-like
cells, which ameliorated disease in an IL-10-dependent manner.
Conclusions: We demonstrate that innate production of IFN-γ
from NK cells is necessary and sufficient to trigger an endogenous
regulatory circuit driven by an NK-DC interaction, which controls
the adaptive Th17 response and limits tissue-specific autoimmunity
though an innate IFN-γ/IL-27 axis.
Commercial Relationships: Rachel R. Caspi, None; Nicholas van
Panhuys, None; Jun Chen, None; Phyllis Silver, None; Yingyos
Jittayasothorn, None; Ronald N. Germain, None; Wai Po Chong,
None
Support: NIH/NEI intramural funding, project # EY000184
The application of exosomes derived from mesenchymal stem
cells in experimental autoimmune uveitis (EAU)
Xiaomin Zhang1, Lingling Bai1, Hui Shao2, Xiaorong Li3. 1Uveitis
& Ocular Immunology, Tianjin Med University Eye Hospital,
Tianjin, China; 2Department of Ophthalmology and Visual Sciences,
Kentucky Lions Eye Center, University of Louisville, Louisville, KY;
3
Retina, Tianjin Medical University Eye Hospital & Eye Institute,
Tianjin, China.
Purpose: We previously showed that mesenchymal stem cells
(MSCs) can ameliorate EAU and reduced tissue impairment. This
study tested the hypothesis that the exosomes derived from MSCs
could reduce the severity of EAU as their parent cells.
Methods: EAU was induced by immunization with
interphotoreceptor retinoid-binding protein 1177-1191 (R16) in
Lewis rat. Exosomes were purified from supernatants of human
umbilical cord MSCs by multistep gradient centrifugations and
ultrafiltrations. The animals immunized with R16 were then treated
with MSC-derived exosomes by periocular injection starting from the
onset of ocular inflammation (day 9) to day 14 post-immunization.
Clinical signs were daily examined by slit-lamp, and ocular
histology and electrophysiology (ERG) were evaluated at different
time points. The infiltration of T cell subsets and macrophage were
determined using flow cytometry and immunochemistry respectively.
Splenocytes were collected from both naïve and EAU rats, and the
chemotactic suppression of exosomes on NKs, NKTs, neutrophils,
and macrophages was examined by a transwell system.
Results: Exosome therapy significantly reduced the inflammatory
intensity of ongoing EAU. Both clinical and histological scores were
significantly lower in treatment group than those in control group
(P0.05) with reduced infiltrating Th1, Th17 cells and macrophages
in the former. In addition, ERG in the treated group was remarkable
ameliorated compared to the control group. Chemotaxis assay
showed that MSC derived exosomes suppressed the chemoattractive
effects of CCL2 and CCL-21 on NK, NKT, Gr-1+, and CD68+cells
(P0.05).
Conclusions: Like their parent cells, exosomes derived from MSCs
can reduce intraocular inflammation when administered during the
onset of EAU. Because of the unique features of exosomes, they
might replace their cells for therapeutic intervention in uveitis.
Further experiments are undergoing to explore the possible inhibitory
mechanisms of MSC derived exosomes on EAU.
Commercial Relationships: Xiaomin Zhang, None; Lingling Bai,
None; Hui Shao, None; Xiaorong Li, None
Support: NSFC grant 81371005
Benefits of Systemic Anti-inflammatory Therapy Versus
Fluocinolone Acetonide Intraocular Implant for Intermediate,
Posterior and Panuveitis: 54 month results of The Multicenter
Uveitis Steroid Treatment Trial and Follow-up Study
John H. Kempen1, Michael M. Altaweel2, Lea T. Drye4, Janet T.
Holbrook4, Douglas A. Jabs3, Elizabeth A. Sugar4, Jennifer E.
Thorne5. 1Ophthal-Biostatistics & Epidemiol, Scheie Eye Inst/Univ
of Penn, Philadelphia, PA; 2Ophthalmology and Vision Sciences,
University of Wisconsin, Madison, WI; 3Ophthalmology, Icahn
School of Medicine at Mt. Sinai, New York, NY; 4Department of
Biostatistics, Bloomberg Sch Public Hlth-JHU, Baltimore, MD;
5
Johns Hopkins Wilmer Eye Inst, Baltimore, MD.
Purpose: To compare the benefits of fluocinolone acetonide implant
therapy versus systemic corticosteroid therapy supplemented with
immunosuppression when indicated for intermediate, posterior, and
panuveitis.
Methods: 255 subjects with intermediate, posterior, or panuveitis
previously randomized to implant or systemic therapy were followed
through 54 months from original randomization, 79.2% completing
the 54 month visit. Masked best-corrected visual acuity, visual field
mean deviation, activity of uveitis, and presence of macular edema
per reading center grading were ascertained prospectively.
Results: The average trajectory of visual function in uveitic eyes
was similar over 54 months in both groups, with best-corrected
visual acuity improving from baseline by 2.4 and 3.1 letters at 54
months (p=0.73) in the implant and systemic group respectively.
The automated perimetry mean deviation score remained similar
to baseline throughout 48 months’ follow-up. Overall control of
inflammation was superior in the implant group at all time points
assessed (p<0.016), although most eyes in the systemic therapy arm
also had substantial inflammatory improvement, achieving control
or low levels of inflammation. While macular edema improved
significantly more often with implant treatment within the first six
months, the systemic group gradually improved over time thereafter
such that the proportions with macular edema converged in the two
groups by 36 months and were overlapping thereafter (p=0.41 at 48
months).
Conclusions: Visual outcomes of fluocinolone acetonide implant
and systemic treatment for intermediate, posterior, and panuveitis are
similarly favorable through 54 months. The implant maintains a clear
advantage in controlling inflammation through 54 months. However,
with systemic therapy a large majority of patients also experienced
greatly improved inflammatory status and macular edema improved
equally with longer follow-up. Based on cost-effectiveness and
side effect considerations, systemic therapy seems indicated as the
initial treatment for most bilateral uveitis cases. Implant therapy is a
reasonable alternative approach, especially for unilateral cases, and in
situations where systemic therapy is not feasible or is not successful.
Commercial Relationships: John H. Kempen, Bausch & Lomb
(F); Michael M. Altaweel, None; Lea T. Drye, None; Janet T.
Holbrook, None; Douglas A. Jabs, None; Elizabeth A. Sugar,
None; Jennifer E. Thorne, None
U10EY014656
Novel anti-TNF-α antibody mimetic to treat ocular inflammation
David A. Copland1, 2, Hanieh Khalili3, 2, Richard W. Lee1, 2, Peng
T. Khaw2, Steve Brocchini3, 2, Andrew D. Dick1, 2. 1Ophthalmology,
University of Bristol, Bristol, United Kingdom; 2UCL Institute
of Ophthalmology, London, United Kingdom; 3UCL School of
Pharmacy, London, United Kingdom.
Purpose: Systemic administration of Infliximab, a chimeric mAb that
neutralizes TNF-α, is used off-label for the treatment of autoimmune
ocular inflammatory diseases such as uveitis. Our aim is to develop
a Fab-PEG-Fab (FpF) molecule using Fab fragments derived from
Infliximab, with the expectation that FpFs will subvert Fc-mediated
inflammation and permit intravitreal administration.
Methods: To test the in vivo efficacy of Infliximab, EAU was
induced in B10.RIII mice and disease severity determined by clinical
assessment and flow cytometric analysis of retinal infiltrate. Groups
of mice were treated following onset of clinical disease with a
single intravitreal dose (2.5μl, 15μg/eye) of Infliximab or control
vehicle alone. Retinal infiltrate was examined and enumerated by
flow cytometry, 4 days following treatment. The FpF-infliximab was
prepared using the Fab from the proteolytic digestion of infliximab
and a PEG di(bis-sulfone) reagent which site specifically underwent
conjugation with the accessible disulphide on two Fabs to yield the
FpF which was then purified by ion exchange chromatography (IEX).
Affinity and binding analyses were performed using BIAcore NTA
chip.
Results: The assessment of retinal infiltrate in mice receiving a
single intravitreal administration of Infliximab reveals a significant
reduction in CD45+ cells with reduced CD11b+, Ly6G+, CD4+ &
CD8+ populations. Clinical assessment demonstrates that Infliximab
suppresses disease progression, with some eyes maintaining only the
initial signs of retinal inflammation. Using the PEG di(bis-sulfone)
which has the bis-alkylating moiety on each terminus to link two FabInfliximab, the FpF molecule was successfully prepared and purity
evaluated by SDS-PAGE analysis. Purified FpF-Infliximab maintains
binding affinity for TNF-α, and kinetic studies demonstrate a lower
dissociation rate constant compared to whole Infliximab, suggesting a
tighter TNF-αinteraction with FpF-Infliximab.
Conclusions: The chimeric Infliximab mAb successfully suppresses
ocular infiltrate with reduced clinical disease severity in EAU. FpFInfliximab molecules can be synthesized, and importantly possess
a similar binding affinity and lower dissociation rate constant as
the parent IgG. Together the data supports ongoing experiments
to evaluate FpF-infliximab’s potential as a novel therapeutic for
treatment of ocular inflammation.
Commercial Relationships: David A. Copland, None; Hanieh
Khalili, None; Richard W. Lee, None; Peng T. Khaw, None; Steve
Brocchini, None; Andrew D. Dick, None
Support: UCL Research Innovation Fund 2013
348 Ocular disease, inflammation, and cytokines
Tuesday, May 05, 2015 11:00 AM–12:45 PM
Contributing Section(s): Anatomy/Pathology, Biochemistry/
Molecular Biology, Clinical/Epidemiologic Research, Cornea,
Glaucoma, Lens, Physiology/Pharmacology, Retinal Cell Biology,
Retina
Cytokine Profile TH1 / TH2 / TH17 in aqueous humor of patients
with Primary Open Angle Glaucoma (POAG) and primary
angle closure glaucoma (PACG) compared with patients Healthy
controls.
Ana P. Lopez-Venegas, Maria E. Mendoza-Mendieta, Victor M.
Bautista. Retina, Instituto de Oftalmologia - Conde de Valenciana,
Mexico City, Mexico.
Purpose: To compare the profile of TH1 / TH2 / TH17 in Aqueous
Humor of patients with POAG and PACG compared with healthy
controls.
Methods: Aqueous humor of patients with POAG and PACG
trabeculectomy operated and healthy controls operated for cataract
surgery (10 patients in each group) was obteined and was measured
by flow cytometry levels of IL2, IL4, IL6, IL-10, TNF, INFG, and
IL17. Data analysis was performed with FCAP Array software. The
interpretation of results was performed with the Prism program using
the Bonferroni multiple comparison test.
Results: An increase of IL-17 in aqueous humor of patients with
GPAC compared with healthy controls was found with p <0.05,
likewise a trend towards lower IL-6 was found with p> 0.05.
Conclusions: The present study shows that the primary glaucoma
may be associated with changes in the profile of cytokines in
aqueous humor being, different cytokine profile of POAG and
PACG, the latter with a TH17 response. This is the first study that
found a significant elevation of IL-17 in aqueous humor of patients
with glaucoma, which suggests that in these patients a chronic
inflammatory process most likely related to the mechanism of
apposition iridotrabecular unlike POAG patients in which there is no
such mechanism.
Commercial Relationships: Ana P. Lopez-Venegas, None; Maria
E. Mendoza-Mendieta, None; Victor M. Bautista, None
Cytokines and chemokines profile in aqueous humor of PACG
eyes
Roopam Duvesh5, Krishnadas SR1, George Puthuran1, Sharmila
Rajendrababu1, Rengaraj Venkatesh1, Srinivasan Kavitha1, Pradeep
Y. Ramulu2, Robert Wojciechowski3, 4, Periasamy Sundaresan5.
1
Glaucoma Clinic, Aravind Eye Hospital, Madurai, India; 2Glaucoma
Division, Johns Hopkins Hospital, Baltimore, MD; 3Department of
Epidemiology, Johns Hopkins Bloomberg School of Public Health,
Baltimore, MD; 4Inherited Disease Research Branch, National
Human Genome Research Institute, Baltimore, MD; 5Department of
Genetics, Aravind Medical Research Foundation, Madurai, India.
Purpose: To determine cytokine and chemokine levels in the aqueous
humor of Primary angle closure glaucoma (PACG) and cataract
patients.
Methods: Aqueous humor samples were collected from 19 subjects
with PACG and 19 cataract patients as controls. The levels of
27 cytokines, chemokines and growth factors were measured in
the aqueous humor samples of study subjects using multiplex
bead immunoassay (Bio-Rad). Data on patient demographics
and preoperative intraocular pressure (IOP) were also collected.
Differences in cytokine concentration between PACG cases and
cataract controls were compared using a Wilcoxon rank sum test
and a t test comparing log2-transformed data. A 2-sided Bonferroniadjusted threshold of p=0.002 was used for statistical significance.
Results: The aqueous concentrations of 27 cytokines, chemokines
and growth factors were simultaneously measured for both PACG
group and cataract group. PACG group showed higher levels of
Macrophage Inflammatory Proteins (MIP)-1β (Median= 51.29 pg/ml)
as compared to cataract controls (Median= 23.24 pg/ml; p=0.0002).
Seven cytokines (IL-2, IL-4, IL-15, G-CSF, IFN-γ, PDGF-BB
and RANTES) were below the limits of detection in few samples.
IL-5,IL-6,IL-7,IL-8,IL-9,IL-10,IL-12,IL-13,IL-17,Eotaxin, basicFGF,GM-CSF,IP-10,MCP-1,MIP-1α,MIP-1β,TNF-α and VEGF
were detected in all the study samples.
Conclusions: Concentration of the MIP-1β is significantly higher
in the aqueous humor of PACG patients when compared to cataract
patients indicating MIP-1β might have possible role in angle closure
glaucoma.
Commercial Relationships: Roopam Duvesh, None; Krishnadas
SR, None; George Puthuran, None; Sharmila Rajendrababu,
None; Rengaraj Venkatesh, None; Srinivasan Kavitha, None;
Pradeep Y. Ramulu, None; Robert Wojciechowski, None;
Periasamy Sundaresan, None
Latanoprost eye drops increases interleukine-33 in mice eyes
Alexis G. Matos, Jhimmy Talbot, Raphael Peres, Larissa
Domenegueti Ferreira, Jose C. Filho, Jayter S. Paula. Ribeirão Preto
Medical School, University of Sao Paulo, Ribeirao Preto, Brazil.
Purpose: Long-term use of topical antiglaucoma drugs is
associated with ocular surface inflammation and is a risk factor
for trabeculectomy failure. Interleukine-33 (IL-33) is a member
of the IL-1 family of cytokines and has been associated with the
perpetuation of pro-inflammatory processes.
Methods: Latanoprost (0.05 mg/ml, 4 μl; N=12) or phosphate
buffered saline (PBS, 4 μl, N=12) eye drops were applied to the
right eye of C57BL/6 mice once a day for 10 days. After euthanasia,
samples of cornea and conjunctiva were collected, at 5th and 10th
day, for ELISA for IL-6 and IL-33. IL-33, IL-6 and TNF-alpha were
also measured with ELISA in samples from mice 3T3 fibroblasts
cultured in DMEM. These cells were treated, after reaching 80%
confluence at third passage, with HBSS-Hanks added or not to
latanoprost 0.05 mg/ml, for 60 min and were analyzed after 24 hours.
Data were presented as mean±SEM and compared using the Student
t test.
Results: In vivo treatment with latanoprost increased IL-6 levels
(91.3±4.9 pg/mg vs 140.9±14.7 pg/mg; p=0.0123) and decreased IL33 levels (159.4 ±4.1 pg/mg vs 102.7 ± 17.9 pg/mg; p= 0.004) at 5th
day. However, at 10th day, both IL-33 and IL-6 levels were increased,
but not significantly. 3T3 fibroblasts treated with latanoprost showed
significant higher levels of IL-33 (0.03 ±0.01 pg/mg vs 0.67 ±0.36
pg/mg; p=0.019) and TNF-alpha (0.03 ±0.01 pg/mg vs 1.66 ±0.46;
p=0.024) than controls. No significant difference was observed in the
comparison of IL-6 levels in vitro.
Conclusions: The present findings confirmed the expression of proinflammatory mediators in the ocular surface due to the prostaglandin
analog exposure and demonstrated for the first time the increased
expression of IL-33 with latanoprost treatment. Although IL-33producing fibroblasts might play a central role in the cicatrization
process, the exact effects of IL-33 on the conjunctival healing process
and the initial decrease in IL-33 levels deserve further explanation.
Grafic 1. Determination by ELISA for levels of IL-6, TNF-alpha and
IL-33 after 6 and 24 hours in culture samples fibroblast 3T3 cells,
treated or not with PG analogues.
Therefore, we here studied whether and how RGCs are affected by a
severe LPS-induced retinal inflammation.
Methods: Inflammation was induced by administration of bacterial
lipopolysaccharide (LPS) to organotypic cultured mouse retina. At 24
h, 48 h and 5 days after LPS- administration these parameters were
studied: i) microglial activation using Iba-1, ED1- and galectin-3
immunohistochemistry (IHC), ii) cytokine release profile, iii) gliosis
(GFAP-IHC), iv) gross morphology and v) apoptosis and RGC
(NeuN-IHC) death in the ganglion cell layer (gcl). Non-LPS exposed
retina was used as control.
Results: We demonstrate a LPS-induced retinal inflammation model,
characterized by a severe inflammatory response, exemplified by
a significant increase in: 1) microglia proliferation, 2) numbers of
activated cell profiles, 3) expression of markers for a late and severe
inflammatory response (i.e. ED1, galectin-3) and 4) inflammatory
cytokines. TNF-α, IL-2, IL-6 and, especially the cytokine KC/
GRO, was found in high levels. In addition, tissue damage after LPS
exposure was further demonstrated by a glia response. An increase in
apoptotic cells in the gcl was found, but not a loss in RGCs.
Conclusions: There is an urgent need for neuroprotective treatments
for neurodegenerations, including glaucoma. Knowledge on key
events in the inflammatory process described to accompany many
neurodegeneration, may lead to proposals of novel treatment
paradigms. Using the present LPS-induced inflammation protocol, a
significant inflammatory response was demonstrated, proven by the
expression of ED1 and galectin-3. However, although an increase in
apoptotic cells in the gcl was found, it was not followed by a loss of
cells in the GCL, including RGC, in the time-interval studied. This
may be explained by the high expression of the cytokine KC/GRO,
reported to have a neuroprotective effect in other neurodegenerative
diseases. Further studies on KC/GRO’s role inflammation is needed
for exploring its potential as a neuroprotective candidate.
Commercial Relationships: Patrik M. Bauer, None; Marina Zalis,
None; Fredrik Johansson, None; Tomas Deierborg, None; Ulrica
Englund Johansson, None
Grafic 2 Determination by ELISA of IL-6 levels and IL-33 after 5 and
10 days, cornea and conjunctiva samples of mice treated or not with
PG.
Commercial Relationships: Alexis G. Matos, None; Jhimmy
Talbot, None; Raphael Peres, None; Larissa Domenegueti
Ferreira, None; Jose C. Filho, None; Jayter S. Paula, None
Retinal ganglion cells are not affected by severe retinal
inflammation demonstrated by microglia proliferation and
increased expression of ED1 and galectin-3
Patrik M. Bauer1, Marina Zalis2, Fredrik Johansson1, Tomas
Deierborg3, Ulrica Englund Johansson2. 1Biology, Dept Functional
Zoology, Lund University, Lund, Sweden; 2Ophthamology, BMC,
Lund, Sweden; 3Experimental Neuroinflammation, BMC, Lund,
Sweden.
Purpose: In glaucoma, inflammation contributes to the disease
progression; as a direct contributor to the pathology or a result of the
degeneration. In-depth knowledge is needed on the mechanisms by
which inflammation contributes to retinal ganglion cell (RGC) death,
before an efficient anti-inflammatory treatment may be proposed.
Preventing glaucoma by blocking activation of the NLRP3
inflammasome in the optic nerve head
Fei Fei2, Anitha Krishnan2, Ann Marshak Rothstein1, Bruce R.
Ksander2, Meredith S. Gregory-Ksander2. 1Medicine, University of
Massachusetts Medical School, Worcester, MA; 2Ophthalmology,
Schepens Eye Research Institute / Massachusetts Eye & Ear
Infirmary/ Harvard Medical School, Boston, MA.
Purpose: There is considerable evidence that the initial injury in
glaucoma occurs in the optic nerve head (ONH), where activated
astrocytes produce mediators that damage axons and trigger
inflammatory cell recruitment. We discovered that ONH astrocytes
in human and mouse eyes constitutively express high levels of a
critical regulator of inflammation called NLRP3, which is part of
a multi-protein complex that contains the adapter protein ASC and
activates caspase-1 to produce IL-1β and IL-18. We hypothesize that
activation of the NLRP3 inflammasome in ONH astrocytes initiates
inflammation and axonal damage in glaucoma.
Methods: NLRP3 expression was assessed by immunofluorescence
and PCR of human and mouse ONH. Intracameral microbead
injection was used to elevate IOP in WT C57BL/6J mice and
different B6 KO mice (NLRP3 KO, ASC KO, IL-1R KO, and IL-18
KO). Controls were uninjected and saline-only injected eyes. IOP
was monitored by rebound tonometry. At designated time points,
mice were euthanized and the ONH was processed for: (ii) RGC
counts (ß-III tubulin), (iii) axon counts (PPD), (iv) immunostaining
(GFAP, IL-1β, Iba1), and (v) RT-PCR.
Results: Normal human and mouse ONH possessed astrocytes with
high levels of NLRP3 but no evidence of inflammasome activation.
Mice with elevated IOP displayed additional increases in NLRP3
and ASC, plus activated caspase-1. To demonstrate a functional role
of the activated inflammasome in glaucoma, microbead-induced
elevated IOP was induced in a series of KO mice that targeted
inflammasome components (NLRP3 and ASC), or products (IL-1β
and IL-18). All WT and KO mice displayed a similar increase in
IOP for 21 days as compared with controls. The absence of NLRP3
or ASC resulted in a significant reduction in RGC and axon loss, as
compared to WT controls. In addition, loss of IL-1R or IL-18 also
produced a significant reduction in RGC and axon loss. The reduced
RGC loss in NLRP3 KO mice correlated with a reduced infiltration
of Iba1+ cells into the ONH as compared to WT controls.
Conclusions: The components of the NLRP3 inflammasome are
constitutively expressed in normal ONH astrocytes but remain
inactive. Elevated IOP triggers activation of the inflammasome
and production of IL-1β / IL-18, which are critical mediators of
inflammation and necessary for development of glaucoma. Blocking
inflammasome activation leads to neuroprotection in the presence of
elevated IOP.
Commercial Relationships: Fei Fei, None; Anitha Krishnan,
None; Ann Marshak Rothstein, None; Bruce R. Ksander, None;
Meredith S. Gregory-Ksander, None
Support: Massachusetts Lions Eye Research Fund
Immune mediators in aqueous humor from patients with normaltension glaucoma
Katsuhiko Maruyama, Shunichiro Ueda, Yoshihiko Usui, Takeshi
Kezuka, Hiroshi Goto. ophthalmology, Tokyo Medical University,
Shinjuku, Japan.
Purpose: Several studies have demonstrated that the levels of some
immune mediators in aqueous humor are elevated in exfoliation
glaucoma, pseudophakic glaucoma, primary open-angle glaucoma,
and angle-closure glaucoma. However, the concentrations of immune
mediators in normal-tension glaucoma (NTG) patients remain
unclear. Therefore, we determined the immune mediator profile in
aqueous humor samples obtained from medically treated eyes with
NTG.
Methods: Twenty-six patients (26 eyes) with NTG undergoing
trabeculectomy (NTG group) and 18 patients (18 eyes) with cataract
undergoing phacoemulsification and intraocular lens implantation
(control group) were enrolled in this cross-sectional study. Patients
with a history of intraocular surgeries including laser treatment, or
other ocular diseases were excluded. The ages in NTG group and
control group were 52.4 +/- 6.4 (37–65) and 72.2 +/- 9.3 (51–90)
years [mean +/- standard deviation (range)], respectively (p<0.001,
independent t-test). Undiluted aqueous humor samples were collected
at the start of surgery, and cytometric beads array kits were used
to determine the concentrations of 28 immune mediators including
angiogenin, interleukin (IL)-6, IL-8, vascular endothelium growth
factor (VEGF), monokine induced by interferon gamma (Mig),
interferon gamma inducible protein-10 (IP-10), and monocyte
chemoattractant protein (MCP)-1. The levels of each immune
mediator were compared between both groups using by multiple
regression analysis. P <0.05 was considered significant.
Results: Angiogenin, IL-8, IP-10, Mig, and MCP-1 were detected in
majority of cases. In NTG group and control group, the angiogenin
levels were 5552.0 (1161.1-27316.4) and 5322.0 (3666.4-8245.0)
pg/mL [median (range)], the IL-8 levels were 1.8 (0-20.0) and
2.0 (0-10.6) pg/mL, the IP-10 levels were 40.2 (0-269.4) and 66.9
(8.0-417.2) pg/mL, the Mig levels were 9.5 (0-104.0) and 29.8 (0173.6) pg/mL, and the MCP-1 levels were 233.7 (136.8-806.3) and
340.1 (171.9-779.9) pg/mL, respectively. The differences were not
statistically significant.
Conclusions: Our study suggests that there is no immune mediator to
express specifically in medically treated NTG patients without history
of intraocular surgery.
Commercial Relationships: Katsuhiko Maruyama, None;
Shunichiro Ueda, None; Yoshihiko Usui, None; Takeshi Kezuka,
None; Hiroshi Goto, None
Activation of NLRP3 Inflammasome in Proliferative Diabetic
Retinopathy
Veluchamy A. Barathi5, 2, Rayne R. Lim1, Bhav H. Parikh1, Yeo S.
Wey5, Tien Yin Wong3, 1, Alessandra Mortellaro4, Shyam S. Chaurasia1,
2 1
. Retina Research Group, Singapore Eye Research Institute,
Singapore, Singapore; 2Ophthalmology, Yong Loo Lin School of
Medicine, National University of Singapore, Singapore, Singapore;
3
Vitreo-Retina, Singapore National Eye Centre, Singpaore,
Singapore; 4Singapore Immunology Network (SIgN), Agency of
Science, Technology and Research (A*STAR), Singapore, Singapore;
5
Translational Pre-Clinical Model Platform, Singapore Eye Research
Institute, Singapore, Singapore.
Purpose: Proliferative diabetic retinopathy (PDR) is a blinding
vitreoretinal disease involving inflammatory and angiogenic
components. In this study, we studied NLRP3 inflammasome in a
double transgenic mouse model, Akimba (Ins2Akita x VEGF+/-) that
combines hyperglycemia and overexpression of vascular endothelial
growth factor (VEGF) and displayed majority of the signs of PDR.
Methods: Akimba mice and its parental strains, Akita (Ins2Akita)
and Kimba (trVEGF029) mice were used in this study. Blood
glucose, fundus photography, FFA and ERG was measured in
these mice weekly for 12 weeks. Inflammatory pathway and
NLRP3 inflammasome was investigated using real time-PCR,
immunohistochemistry and western blots.
Results: We found increased levels of IL-1b in all the mouse models,
with maximum levels in Akimba retina. This increase was highly
correlated with the expression of NLRP3 inflammasome componentsASC, NLRP3, & Caspase-1 and associated with higher levels of
pro-inflammatory mediators detected. Results indicated increase
in activated macrophages that upregulated NLRP3 inflammasome,
possibly in all the retinal layers, which released active IL-1β in the
ganglion cell and nerve fiber layer, initiating the autoinflammatory
feedback loop and thereby promoting the inflammatory mediators to
elicit a severe inflammatory response in PDR.
Conclusions: This study demonstrated the effects of the interplay
between VEGF upregulation and hyperglycemia in the Akimba mice
retina. We conclude that the Akimba mouse indicates several features
of PDR and suggests an important role for NLRP3 inflammasome in
the pathogenesis of PDR.
Commercial Relationships: Veluchamy A. Barathi, None; Rayne
R. Lim, None; Bhav H. Parikh, None; Yeo S. Wey, None; Tien Yin
Wong, None; Alessandra Mortellaro, None; Shyam S. Chaurasia,
None
Support: A*Star BMRC/SPF2014/002/SIPRAD
Optimized intravitreal IL-6 antagonist for the treatment of
diabetic macular edema
Michael Schmidt, Yuri Matsumoto, Alison Tisdale, Patricia Lowden,
Joe Kovalchin, Paul Wu, Kathryn Golden, Chris Dombrowski, Blanca
Lain, Eric S. Furfine. Eleven Biotherapeutics, Cambridge, MA.
Purpose: IL-6 is a pleiotropic cytokine with established roles in
inflammation and angiogenesis. Vitreal IL-6 levels are significantly
elevated in patients with diabetic macular edema and are positively
correlated with disease severity. In animal models including laser
CNV, IL-6 blockade reduces retinal leukostasis and angiogenesis.
In humans, systemic IL-6 blockade can lower VEGF levels in
rheumatoid arthritis patients and separately reduce cystoid macular
edema in patients with uveitis. EBI-031 is an anti-IL-6 antibody
optimized for intravitreal (IVT) treatment of diabetic macular edema
(DME) and other ocular inflammatory diseases with high potency,
long intravitreal retention, and rapid systemic clearance.
Methods: EBI-031 binding to IL-6 was assessed by ELISA and SPR.
IL-6 antagonism was assessed using a HEK-BlueTM IL-6 reporter
cell line with either IL-6 (cis- signaling) or “hyper-IL-6” which is
a covalent complex with soluble IL-6 receptor (trans-signaling).
Intravitreal pharmacokinetics were determined in New Zealand White
rabbits and drug levels measured by ELISA.
Results: EBI-031 binds human IL-6 at site II, or the site that contacts
gp130, with pM affinity and blocks signaling of IL-6 and the IL-6/
sIL-6Rα complex in cellular assays >900 fold more potently than
the commercial IL-6 inhibitor tocilizumab. The antibody is thermally
stable (Tm = 76oC for its Fab fragment) and can be concentrated to
>100 mg/mL with little measurable aggregation. In rabbit PK studies,
IVT administered EBI-031 had a vitreal clearance half-time of ~10
days, longer than aflibercept (~6 days) or tocilizumab (~5 days). EBI031 also accumulated at higher levels in the retina. A single mutation
in the IgG2 Fc domain of EBI-031 reduced FcRn-mediated recycling
such that systemic levels after IVT exposure were lower than the
wild-type antibody or tocilizumab. Finally, a computational model
of IL-6 signaling suppression suggests that sufficient concentrations
of EBI-031 should be present in the vitreous to inhibit 95% of IL-6
signaling for over 100 days in humans.
Conclusions: A novel anti-human IL-6 antibody, EBI-031, potently
inhibits IL-6 cis- and trans- signaling and has excellent biophysical
properties for IVT administration. In addition, the long intravitreal
half-life and retinal residence time suggest IL-6 signaling could be
substantially blocked for 3 or more months in patients with DME.
Commercial Relationships: Michael Schmidt, Eleven
Biotherapeutics (E), Eleven Biotherapeutics (P); Yuri Matsumoto,
Eleven Biotherapeutics (E); Alison Tisdale, Eleven Biotherapeutics
(P); Patricia Lowden, Eleven Biotherapeutics (E); Joe Kovalchin,
Eleven Biotherapeutics (E), Eleven Biotherapeutics (P); Paul Wu,
Eleven Biotherapeutics (E); Kathryn Golden, Ele (E); Chris
Dombrowski, Eleven Biotherapeutics (E); Blanca Lain, Eleven
Biotherapeutics (C); Eric S. Furfine, Eleven Biotherapeutics (E),
Eleven Biotherapeutics (P)
Genetic engineering and characterization of recombinant mouse
IL-27 cytokine
Justine Yeung, Lekha Nair, Lin Sun, Charles Egwuagu. National Eye
Institute, National Institutes of Health, Bethesda, MD.
Purpose: Interleukin-27 (IL-27) is an anti-inflammatory
heterodimeric cytokine comprised of IL-27p28 and Ebi3 subunits.
It is constitutively secreted by retinal microglia and suppresses
intraocular inflammation through endogenous production of antiinflammatory proteins including IL-10, suppressors of cytokine
signaling (SOCS), and complement factor H (CFH). Therefore,
targeted delivery of IL-27 into the retina may be beneficial in the
treatment of uveitis. However, it has so far not been possible to
evaluate the therapeutic efficacy of IL-27 in uveitis or other ocular
inflammatory conditions due to the dearth of IL-27. The overall
objective of this study is therefore to produce a biologically active
IL-27 and to examine whether delivery of exogenous IL-27 can be
used to ameliorate uveitis in the mouse experimental autoimmune
uveitis model.
Methods: The mouse IL-27 (IL-27p28/Ebi3) was engineered by
linking mouse IL-27p28 and Ebi3 with an intervening 22 amino
acids linker-peptide. The cDNA construct encoding the IL-27 fusion
protein was cloned into a pMIB/V5-HIS vector and expression was
under the direction of the Baculovirus immediate-early promoter,
OpIE2, which allows for high-level, constitutive expression of the
proteins in insect cells. The expression vector was transfected into
High Five insect cells and stably transfected clones were subjected to
several cycles of Blasticidin S HCl selection.
Results: The secreted recombinant IL-27 was partially purified
by HIS-TAG affinity chromatography and size-exclusion
chromatography using a FPLC system. The recombinant IL-27 has
been characterized by Western blot analysis using antibodies against
IL-27p28, Ebi3 and the V5 protein tag. Consistent with the predicted
molecular size of IL-27, the rIL-27 fusion protein migrated on SDSPAGE at an apparent molecular size of 54kDa.
Conclusions: A 54-kDa-mouse recombinant IL-27 has successfully
been engineered and efficient secretion in insect cell cultures has
been demonstrated. Large-scale ex vivo production of the rIL-27 will
allow in-depth analysis of this fusion protein and evaluation of its
therapeutic potential in murine models of uveitis (EAU) and multiple
sclerosis (EAE).
Commercial Relationships: Justine Yeung, None; Lekha Nair,
None; Lin Sun, None; Charles Egwuagu, None
Toll-like receptor 4 is the receptor for RBP4-induced
inflammation in human retinal capillary endothelial cells
Mei Du, Ashley Martin, Krysten M. Farjo. physiology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK.
Purpose: Serum retinol-binding protein (RBP4) is the transport
protein for Vitamin A (retinol) in the blood, but RBP4 also has
retinol-independent pro-inflammatory activity that contributes to the
development of insulin resistance in type 2 diabetes. Moreover, serum
RBP4 levels are significantly increased in patients with proliferative
diabetic retinopathy compared to diabetic patients with mild or
no retinopathy. We have previously shown that elevation of RBP4
induces pro-inflammatory gene expression in human retinal capillary
endothelial cells (HRCEC) through a retinol-independent mechanism
that is dependent on NADPH oxidase and NF-kB, and thus may
contribute to the formation of vascular lesions in diabetic retinopathy.
However, the cell membrane receptor that mediates RBP4-induced
endothelial inflammation is unclear, since HRCEC do not express
the RBP4 receptor for retinol uptake, STRA6. The current study
focuses on identifying the receptor and signal transduction pathways
activated by RBP4 to induce inflammation in HRCEC.
Methods: Since previous studies have shown that RBP4 stimulates
pro-inflammatory activity in macrophages via TLR4-MAPK
signaling and inhibits insulin signaling in adipocytes via STRA6STAT5 signaling, we tested if RBP4 stimulated either of these
pathways in HRCEC. HRCEC were treated with purified recombinant
human RBP4 in the presence or absence of inhibitors for TLR4, JNK,
ERK, p38, STATs, NADPH oxidase, and NF-kB. MAPK and STAT
pathway activation was assessed by western blotting for phosphoJNK,-ERK, -p38, -STAT1, -STAT3, and -STAT5. Pro-inflammatory
gene expression was analyzed by western blotting and ELISA. In
vitro leukostasis assays were performed to measure the net effect of
inhibitors on RBP4-induced inflammation.
Results: Both a chemical TLR4 inhibitor (TAK-242) and TLR4
neutralizing antibodies significantly blocked RBP4-induced
expression of pro-inflammatory genes, including VCAM-1, ICAM1, IL-6, MCP-1, and E-selectin. We observed phospho-activation
of MAPK signaling, but not STAT signaling in response to RBP4
treatment. Similarly, MAPK inhibitors significantly reduced RBP4mediated inflammation, whereas STAT inhibition did not have a
significant effect.
Conclusions: RBP4 induces inflammation in HRCEC via TLR4/
MAPK signaling. This suggests RBP4 and TLR4 may be therapeutic
targets in diabetic retinopathy.
Commercial Relationships: Mei Du, None; Ashley Martin, None;
Krysten M. Farjo, None
Are suppressor of cytokine signaling (SOCS)1 and SOCS3
stimulated during development of murine cytomegalovirus
(MCMV) retinitis in mice immunosuppressed by corticosteroids?
Christine I. Alston1, 2, Moon K. Han1, Hsin Chien1, Richard D. Dix1,
2 1
. Department of Biology, Viral Immunology Center, Georgia State
University, Atlanta, GA; 2Department of Ophthalmology, Emory
University School of Medicine, Atlanta, GA.
Purpose: Mice with retrovirus-induced immunosuppression
(MAIDS) over a 10-week period develop systemic T-cell dysfunction,
but without loss of macrophages. We have shown previously that
SOCS1 and SOCS3 mRNAs and proteins are highly stimulated
during development of experimental retinitis within MCMVinfected eyes of C57BL/6 mice with MAIDS that are susceptible to
retinitis (frequency of 80 – 100%). Because others have shown that
development of experimental MCMV retinitis in eyes of C57BL/6
mice immunosuppressed by corticosteroid treatment results in
significant macrophage loss [Duan et al, 1994] and a reduced
frequency of retinitis (~50%) [Zhang et al, 2007], we sought to
determine whether SOCS1 and SOCS3 mRNAs are stimulated in
MCMV-infected eyes of drug-immunosuppressed mice to the same
extent as that observed for SOCS1 and SOCS3 mRNA expression in
MCMV-infected eyes of MAIDS mice.
Methods: Drug-induced immunosuppression was accomplished
in healthy C57BL/6 mice by intramuscular injection of
methylprednisolone every 3 days beginning at day -2 relative
to MCMV inoculation. The left eyes of groups of drugimmunosuppressed C57BL/6 mice were infected with MCMV
subretinally; right eyes were mock infected (controls). At 3, 6, and 10
days postinfection, eyes were collected and compared for SOCS1 and
SOCS3 mRNA production by quantitative real-time RT-PCR assay.
Results: Whereas SOCS1 and SOCS3 mRNAs were highly
stimulated within MCMV-infected eyes of mice with MAIDS at 6
days postinfection, our present study showed little or no stimulation
of SOCS1 and SOCS3 mRNAs within MCMV-infected eyes of
drug-immunosuppressed mice at day 6, although there was modest
stimulation of these molecules at day 3.
Conclusions: A comparison of MCMV-infected eyes of drugimmunosuppressed mice and our previous work with MCMVinfected eyes of mice with MAIDS revealed sharply different patterns
of SOCS1 and SOCS3 mRNA production during development
of retinitis. This surprising outcome may be due to quantitative
differences in macrophage populations observed in the two mouse
models of experimental MCMV retinitis.
Commercial Relationships: Christine I. Alston, None; Moon K.
Han, None; Hsin Chien, None; Richard D. Dix, None
Support: NIH Grant EY010568, NIH/NEI Core Grant P30/
EY006360, NIH/NEI Training Grant 5T32/EY007092-28, GSU
Department of Biology Bridge Grant, and Fight for Sight
Sitagliptin inhibits neuroinflammatory processes in retinal
microglia
António F. Ambrósio1, 2, Sandra Correia1, João Martins3, Dan
Brudzewsky1, Elisa Campos3, Ana R. Santiago2, 3. 1IBILI, Faculty
of Medicine, University of Coimbra, Portugal/AIBILI, Coimbra,
Portugal; 2Centre for Neuroscience and Cell Biology, University
of Coimbra, Coimbra, Portugal; 3Centre of Ophthalmology and
Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra,
Portugal, Coimbra, Portugal.
Purpose: The breakdown of blood-retinal barrier (BRB) is a hallmark
of diabetic retinopathy, and it has been correlated with increased
nitric oxide (NO) production, mainly via inducible nitric oxide
synthase (iNOS). Reactive microglia, which produce high amounts of
NO, have been implicated in the pathogenesis of diabetic retinopathy.
Sitagliptin is an inhibitor of dipeptidyl-peptidase-IV (DPP-IV) used
for the treatment of type 2 diabetes. However, sitagliptin is also able
to prevent the BRB breakdown in type 1 diabetic animals, likely by
exerting anti-inflammatory effects. In this study, we investigated the
potential anti-inflammatory effects of sitagliptin in retinal microglial
cells.
Methods: We used primary retinal cell cultures and retinal
organotypic cultures. Both cultures were exposed for 24h to
lipopolysaccharide (LPS) to trigger an inflammatory response. iNOS
immunoreactivity (iNOS-IR) in retinal microglia and microglia
morphology (area, perimeter, Feret’s diameter and circularity) were
evaluated by immunofluorescence. Images were acquired by confocal
microscopy at the level of the ganglion cell layer. NO production was
evaluated in primary cultures media by Griess reaction method.
Results: In primary retinal cell cultures, exposure to LPS increased
iNOS-IR in microglial cells (1011±117% of control; p=0.001) and
sitagliptin inhibited this increase (368.1±30% of control; p=0.01).
LPS also triggered and increase in NO production (5.76±0.56 mM;
p=0.001), which was inhibited by sitagliptin (3.80±0.3 mM; p=0.05).
In retinal organotypic cultures, LPS also increased iNOS-IR in
microglial cells (2960±289.3% of control; p=0.01) and this effect was
inhibited by sitagliptin (1535±137.9% of control; p=0.01). Moreover,
LPS induced alterations in microglia in several morphological
parameters (for example, circularity index significantly increased
from 0.20±0.01 mm in control conditions to 0.33±0.1 mm with LPS;
p=0.01). Sitagliptin prevented the alterations triggered by LPS in
microglia morphology.
Conclusions: These results indicate that sitagliptin has antiinflammatory effects by controlling the reactivity of retinal microglial
cells, as evidenced by its capacity to inhibit microglia morphological
changes as well as the upregulation in iNOS-IR in microglia and NO
production triggered by LPS.
FCT (PTDC/NEU-OSD/1113/2012 and PEst-C/SAU/UI3282/2013),
Portugal, and COMPETE-FEDER, and AIBILI.
Commercial Relationships: António F. Ambrósio, None; Sandra
Correia, None; João Martins, None; Dan Brudzewsky, None; Elisa
Campos, None; Ana R. Santiago, None
Support: FCT (PTDC/NEU-OSD/1113/2012 and PEst-C/SAU/
UI3282/2013), Portugal, and COMPETE-FEDER, and AIBILI.
Quantitative morphometry of microglia in retinal inflammation
Akito Shimouchi, Harumasa Yokota, Chiemi Matsumoto, Akira
Takamiya, Taiji Nagaoka, Akitoshi Yoshida. Ophthalmology,
Asahikawa Medical University, Asahikawa, Japan.
Purpose: Microglial form and function have been thought to be
inextricably linked in various retinal diseases. We have demonstrated
that computer-assisted morphometry is useful for precisely evaluating
changes in microglial form during retinal neovascularization
(Shimouchi et al, ARVO 2014). In this study, we investigated whether
microglial morphometry could assess the sequential changes in
microglial form during retinal inflammation.
Methods: Uveitis model was induced by intraperitoneal injection of
lipopolysaccharide (LPS) in C57BL/6J mice. Retinal sections were
prepared using the eyes collected at 12, 24 and 36 hours after LPS
treatment. Retinal vessels were labeled with lectin and microglia
were labeled with Iba1. Confocal images of retinal microglia were
processed with Image J to calculate form factor (FF, roundness),
branching density (BD), convexity (CON), solidity (SOL, spatial
density), and fractal dimension (DF, structural complexity).
Results: FF (Mean±SD; 0.076±0.038), BD (0.021±0.006) and
SOL (0.407±0.160) were significantly higher and DF (1.351±0.037)
was significantly lower in the retina at 12 hours compared with
control (FF=0.034±0.014, BD=0.011±0.003, SOL=0.180±0.066,
DF=1.447±0.010, control: n=24, 12 hours: n=27, p<0.0001), which
meant that microglia significantly changed their form to be amoeboid.
Although morphometric values of microglia still showed that
microglia were significantly amoeboid at 24 hours (n=30) after LPS
injection, the degree was attenuated in comparison with the retina
at 12 hours. In contrast, there was no significant difference in those
morphometric values of microglia between the retinas at 36 hours
(n=17) after LPS injection and control.
Conclusions: Computer-assisted morphometry succeeded in
depicting the sequential changes in microglial form during retinal
inflammation. According to morphometric analysis in the current
study, microglia became activated and returned to normal state by 36
hours after LPS injection. Microglial morphometry is suggested to
offer a new index of retinal inflammation that takes place in a number
of retinal diseases.
Commercial Relationships: Akito Shimouchi, None; Harumasa
Yokota, None; Chiemi Matsumoto, None; Akira Takamiya, None;
Taiji Nagaoka, None; Akitoshi Yoshida, None
High concentration of sodium chloride induces the inflammatory
cytokine production by ARPE-19 cell
Zi Ye1, Dike Zhang1, Chaokui Wang1, Aize Kijlstra2, Peizeng Yang1.
1
The First Affiliated Hospital of Chongqing Medical University,
Chongqing, China; 2University Eye Clinic Maastricht, Maastricht,
Netherlands.
Purpose: The development of certain ocular diseases is associated
with some environmental factors. Excess sodium chloride (Nacl)
uptake is one of the most common environmental risk factors in a
various diseases. In this study, we investigated whether the Nacl
could directly influence the production of inflammatory cytokines
by ARPE-19 cell, an immortalized cell line which has structural and
functional properties characteristic of human RPE cells.
Methods: ARPE-19 cells were cultured with LPS in the presence or
absence of 20nM and 40nM Nacl. Mannitol was used to exclude the
effect of osmotic pressure. The expression of IL-6, IL-8, MCP-1 and
VEGF in ARPE-19 cells were detected by ELISA. Cell proliferation
was determined by WST-8 assay using Cell Counting Kit-8. Flow
cytometry were performed to study the effect of Nacl on cell
apoptosis.
Results: The concentration of 20nM Nacl did not show any effect
on the expression of inflammatory cytokines. However, 40nM Nacl
could significantly induced the expression of IL-6, IL-8, MCP-1
and VEGF in LPS-stimulated ARPE-19 cells. The osmotic pressure
equaled to that of 40nM Nacl had no influence on the production
of these four cytokines. There was no effect of 40nM Nacl on the
proliferation and apoptosis of ARPE-19 cells.
Conclusions: A concentration of 40nM Nacl could directly induce
the expression of inflammatory cytokines in ARPE-19 cells. These
results suggest that excess Nacl uptake may play a role in regulating
RPE function and is possibly involved in the development of
inflammatory diseases in the retina or choroid.
Commercial Relationships: Zi Ye, None; Dike Zhang, None;
Chaokui Wang, None; Aize Kijlstra, None; Peizeng Yang, None
The role of HTLV-1 infected RPE cells in the pathogenesis of
HTLV-1 uveitis
Koju Kamoi, ZHAORONG GUO, Shintaro Horie, Kyoko OhnoMatsui. Ophthalmology, Tokyo Medical and Dental Univ, Tokyo,
Japan.
Purpose: Human T-cell lymphotropic virus-1 (HTLV-1) infection
affects ocular tolerance, which causes various eye diseases. Most
studies have not focused on the effect of HTLV-1 infection to ocular
tissues, although the infection has an intimate involvement in HTLV1 related eye diseases. In this study, we investigate the inflammatory
change that caused by HTLV-1 infection to the retinal epithelium
(RPE) cells.
Methods: HTLV-1 producing T cell line (MT2) and RPE cell line
(ARPE-19) were co-cultured via direct contact or indirect contact
methods. The efficiency of HTLV-1 infection was determined by realtime PCR. The production of inflammatory cytokines/ chemokines
was measured by Cytometric Beads Array.
Results: In RPE cells, HTLV-1 infection could be established even
through indirect contact with HTLV-1 producing T cells. Induction
of IL-6 and IL-8 were seen in both direct contact and indirect contact
infection, but IL-10, TNF-alpha, IL-1β, and IL-12p70 were not
secreted. Chemokines such as MCP-1 and RANTES were detected
in direct contact and indirect contact, but IL-10 production was seen
only through direct contact.
Conclusions: HTLV-1 infection to the RPE cells induces
inflammatory cytokines and chemokines, which can result in
intraocular inflammation. This mechanism may contribute to the
pathogenesis of HTLV-1 uveitis.
Commercial Relationships: Koju Kamoi, None; ZHAORONG
GUO, None; Shintaro Horie, None; Kyoko Ohno-Matsui, None
Support: Health Labour Sciences Research Grant, Grant-in-Aid for
Young Scientists (B)
Character of PMA-Stimulated THP-1 Cells under Ocular
Diabetic Condition
Shintaro Horie, Koju Kamoi, Zhaorong Guo, Kyoko Ohno-Matsui.
Department of Ophthalmology and Visual Science, Tokyo Medical
and Dental University, Tokyo, Japan.
Purpose: The purpose of this study is to investigate the
characteristics of human macrophages under ocular diabetic
condition. Since increased advanced glycation endproducts (AGEs)
are major causes of diabetic microvasculopathy, macrophages
characters induced by AGEs were analyzed and were compared to
that induced by other cytokines.
Methods: Human monocyte cell line THP-1 was stimulated by PMA
(Phorbol 12-myristate 13-acetate). PMA-stimulated THP-1 was
used as differentiated macrophages in vitro. PMA-THP-1 cells were
cultured with AGEs or various cytokines (M1 inducer: IFN-g, M2
inducer: IL-4 & IL-13, RPE secreting cytokine: PGE2). ELISA or
FACS was performed in order to measure cytokines and chemokines
in the cell supernatants. Quantitative mRNA expressions were also
examined by RT-PCR.
Results: VEGF was highly secreted by PMA-stimulated THP-1 in
the presence of AGEs or PGE2. The mRNA expression of vegf was
higher, while ccr2 was less than control respectively. Furthermore,
not only massive secretion of IL-8 but also multiple increased
productions of TNF-α, IL-1β, IL-6, IL-10, MCP-1, RANTES were
induced by AGEs.
Conclusions: Pro-angiogenic myeloid cells are presumably
induced through intraocular cytokines or AGEs directly. Together
with robust VEGF and IL-8 secretions, multiple chemokines or
cytokines released from myeloid cells may orchestrate diabetic ocular
pathology.
Commercial Relationships: Shintaro Horie, None; Koju Kamoi,
None; Zhaorong Guo, None; Kyoko Ohno-Matsui, None
Support: Ministry of Education, Culture, Sports, Science and
Technology,Japan: Grant in AID for Young Scientists (B) 26861438
Genetic engineering and characterization of Ebi3/CNTF fusokine
Lekha Nair. National Eye Institute, National Institutes of Health,
Bethesda, MD.
Purpose: Purpose: IL-27 and IL-35 are anti-inflammatory,
heterodimeric cytokines in the IL-12 family of cytokines, which
displays subunit and receptor promiscuity. IL-27 and IL-35 share
subunit Ebi3, which is hypothesized to be responsible for the antiinflammatory effects of IL-27 and IL-35. Ciliary neurotrophic factor
(CNTF) is a nerve growth factor that has been shown to prevent
neural cell damage. Though its effects on the immune system are
unknown, it has been shown that CNTF can interact with receptors of
the IL-6 family, many of which are homologous or shared by the IL12 family cytokines. Our goal in this study is to genetically engineer
recombinant CNTF and a covalently linked Ebi3 and CNTF fusokine
and investigate whether they would have immunoregulatory functions
in mouse models of CNS autoimmune diseases such as experimental
autoimmune uveitis (EAU) and experimental autoimmune
encephalomyelitis (EAE).
Methods: Method: The CNTF/Ebi3 fusokine consists of mouse Ebi3
and CNTF linked by a 22 amino acids linker-peptide between the two
subunits. The cDNA construct encoding the single chain CNTF or
CNTF/Ebi3 fusokine construct was cloned into pMIB/V5-HIS vector
with expression under the direction of the Baculovirus immediateearly promoter, OpIE2, which allows for high-level, constitutive
expression of the proteins in insect cells. The expression vector was
transfected into High Five insect cells and stably transfected clones
were subjected to several cycles of Blasticidin S HCl selection.
Results: Results: The secreted recombinant CNTF or Ebi3/CNTF
has been partially purified by HIS-TAG affinity chromatography and
size-exclusion chromatography using an FPLC system. The fusokine
has been characterized by western blot analysis using antibodies
against Ebi3 and the V5 protein tag. Consistent with the predicted
molecular size, the Ebi3/CNTF fusokine is a protein at 54kDa.
Conclusions: Conclusion: We have successfully bio-engineered
a 54 kDa Ebi3/CNTF fusokine and demonstrated its efficient
secretion in insect cell cultures. Large-scale, ex-vivo production of
the Ebi3/CNTF fusokine will undoubtedly allow in-depth analysis
of the effects of this novel fusokine on T cell differentiation and its
therapeutic potential in murine models of uveitis (EAU) and multiple
sclerosis (EAE).
Commercial Relationships: Lekha Nair, None
Aqueous humor inflammatory cytokine levels in chronic retinal
vein occlusion
Satoru Inoda1, Hidenori Takahashi1, 3, Xue Tan2, Natsuko Kakinuma3,
Yoko Nomura2, Shin-ichi Sakamoto1, Yusuke Arai1, Yujiro Fujino3, 2,
Hidetoshi Kawashima1, Yasuo Yanagi2. 1ophthalmology, Jichi Medical
University, Shimotsuke, Japan; 2ophthalmology, University of Tokyo
school of Medicine, Tokyo, Japan; 3JCHO Tokyo Shinjuku Medical
Center, Tokyo, Japan.
Purpose: To examine the aqueous humor levels of inflammatory
cytokines in chronic retinal vein occlusion (RVO).
Methods: This is an IRB-approved prospective study. From Sep.
2010 to Jun. 2013, consecutive patients were recruited at JCHO
Tokyo Shinjuku Medical Center. Aqueous humors were collected
just before intravitreal bevacizumab injections. Treatment-naive
patients whose onset was within 3 months were defined as an acute
group. Patients received injections whose RVO durations were
more than a year were defined as a chronic group. Patients who had
received periocular steroid injection within three months or retinal
photocoagulation were excluded. Concentrations of IP-10, MCP1, MMP-9, IL-6, IL-10, CXCL1, CXCL13, CCL11, and CXCL12
were measured using multiplex cytokine assay. Log-transformed
concentrations were compared to control using multiple linear
regression after adjusting for sex, age, axial length, and posterior
vitreous detachment.
Results: 10 acute branch RVO (BRVO), 6 chronic BRVO, 4 acute
central RVO (CRVO), 4 chronic CRVO, and 80 control eyes, whose
aqueous humor was collected at cataract surgery, were used for
analysis. In acute BRVO, only IL-10 was decreased (P=0.04). In
contrast, in chronic BRVO, there was no change in the cytokine
levels. In acute CRVO, IP-10 (220 pg/mL vs 73, p=0.007), IL-6 (11
vs 4.9, p=0.02), CXCL12 (570 vs 190, p=0.001) were increased. In
contrast, in chronic CRVO, MCP-1 (160 vs 500, p=0.002), CCL11
(1.7 vs7.0, p=0.04), CXCL12 (77 vs 190, p=0.049), CXCL13 (1.1 vs
5.1, p=0.01) were decreased.
Conclusions: Several inflammatory cytokines are elevated in acute
RVO as previously demonstrated; however, no cytokines were
elevated in chronic BRVO and several inflammatory cytokines are
rather suppressed in chronic CRVO.
Commercial Relationships: Satoru Inoda, None; Hidenori
Takahashi, None; Xue Tan, None; Natsuko Kakinuma, None;
Yoko Nomura, None; Shin-ichi Sakamoto, None; Yusuke Arai,
None; Yujiro Fujino, None; Hidetoshi Kawashima, None; Yasuo
Yanagi, Novartis Pharma (F), Santen pharmaceutical (F)
Activation of B and T lymphocyte attenuator inhibits Th17/Th1
cell immune response in Behcet’s disease
Peizeng Yang1, Zi Ye1, Bolin Deng1, Chaokui Wang1, Dike Zhang1,
Aize Kijlstra2. 1Ophthal, The 1st Hosp, Chongqing Medical
University, Chongqing, China; 2University Eye Clinic Maastricht,
Maastricht, Netherlands.
Purpose: Behcet’s disease (BD) is a chronic, systemic and recurrent
inflammatory disease with overreacted Th17/Th1 cell immune
response. Recent studies have shown that B and T lymphocyte
attenuator (BTLA) plays a regulatory role in the excessive immune
and inflammatory response. In this study, we investigated whether
BTLA activation could be exploited to inhibit the development of
abnormal immune responses in BD patients.
Methods: BTLA mRNA and protein expression in peripheral blood
mononuclear cells (PBMCs) and CD4+ T cells from active ocular BD
patients and normal controls was examined using RT-PCR and flow
cytometry. The production of IL-17 and IFN-gamma by BTLA+CD4+
T cells and BTLA-CD4+ T cells were detected by flow cytometry.
ELISA and flow cytometry were performed to study the effect of
BTLA on Th17/Th1 cell response and DC function using agonistic
anti-BTLA antibody.
Results: The expression of BTLA was significantly decreased in
active ocular BD patients as compared to normal controls. Decreased
expression of BTLA was associated with increased Th17/Th1
cell immune response in ocular BD patients. Activation of BTLA
inhibited the abnormal Th17/Th1 cell response and expression of IL22 by CD4+ T cells both in patients and normal controls. Addition of
the agonistic anti-BTLA antibody resulted in a decreased production
of Th17/Th1 related cytokines IL-1β, IL-6, IL-23 and IL-12p70, and
reduced expression of CD40 in DCs.
Conclusions: Decreased expression of BTLA in the ocular BD
patients may be not able to exert its negative regulation effect on
Th17 and Th1 cell immune responses and DC function and therefore
is involved in the development and recurrence of this disease.
Agonistic agents of BTLA may be a potential therapeutic approach
for BD and other inflammatory diseases mediated by Th17/Th1
immune responses.
Commercial Relationships: Peizeng Yang, None; Zi Ye, None;
Bolin Deng, None; Chaokui Wang, None; Dike Zhang, None; Aize
Kijlstra, None
Support: Natural Science Foundation Major International (Regional)
Joint Research Project 81320108009
Clinical Trial: ChiCTR-CCC-12002407
Anti-inflammatory Effect of Angiogenin in Endotoxin-induced
Uveitis Rat Model
Won Soo Kim, Soo Jin Lee, Kyoung Woo Kim, Jae Chan Kim.
Department of Ophthalmology, Chung-Ang University Hospital,
Seoul, Korea (the Republic of).
Purpose: Previously, we revealed anti-inflammatory activity of
angiogenin (ANG) in human corneal fibroblast cells inflammed
by tumor necrosis factor (TNF)-α through an inhibition of TANKbinding kinase 1 (TBK1) mediated nuclear factor-κB (NF-κB)
nuclear translocation. As a further study, we investigated the in vivo
anti-inflammatory effect of ANG using the endotoxin-induced uveitis
(EIU) rats.
Methods: EIU in Lewis rats was induced by lipopolysaccharide
(LPS) injection in the footpad. EIU rats were treated by balanced salt
solution (BSS group, 5 rats), 1% prednisolone acetate (PD group, 5
rats) or ANG (200 μg/ml; ANG group, 5 rats) 4 times a day. After
3 days, we enucleated both eyes, collected aqueous humor (AqH),
and then analyzed number of infiltrating cells, protein concentration,
inflammatory marker levels using immunodot blot assay. With
enucleated eyes, we analyzed inflammatory cytokines, toll-like
receptor (TLR)-4, myeloid differentiation factor (Myd) 88 expression
using quantitative RT-PCR and Western blot analysis.
Results: The levels of infiltrating leukocytes, protein concentration,
and inflammatory cytokines and chemokines in the AqH were
significantly elevated in the AqH of BSS group compared to
the control. Then, PD and ANG significantly lowered all those
inflammatory levels in the AqH. There was no difference between PD
and ANG group. Additionally, TLR-4, Myd 88, IL-1β, 2, 4, 6 and,
-10 in eyeball tissues were down-regulated by ANG.
Conclusions: These results suggest that ANG suppresses LPSinduced inflammatory responses in EIU rats through an inhibition of
TLR-4 and Myd 88-related signals. ANG may be a novel therapeutic
candidate in the ocular inflammatory condition.
Commercial Relationships: Won Soo Kim, None; Soo Jin Lee,
None; Kyoung Woo Kim, None; Jae Chan Kim, None
RIP3-induced NLRP3 inflammasome activation is the major
driver for P23H rhodopsin photoreceptor degeneration
Cheryl Y. Gregory-Evans, Ishaq A. Viringipurampeer, Andrew
L. Metcalfe, Emran Bashar, Zeinabsadat Mohammadi, Orson L.
Moritz, Kevin Gregory-Evans. Ophthalmology, University of British
Columbia, Vancouver, BC, Canada.
Purpose: Recent studies have suggested that a proportion of mutant
P23H protein is retained in the rod photoreceptor ER where it is
folded, but is incorrectly glycosylated and unstable. It has been
proposed that this leads to ER stress and photoreceptor cell death.
The mechanistic framework underlying photoreceptor cell death is
still not fully understood. We have previously shown that there is an
inflammatory component to retinal degeneration in P23H transgenic
rat model. We hypothesize that inflammasome activation is a major
contributor to P23H retinal cell death.
Methods: Inflammasome activation was analyzed by histology,
TUNEL staining, ELISA, immunohistochemistry and western
blotting in P23H rat retinal samples at P21, P45 and P120.
Small molecule drugs were used to target specific parts of the
inflammasome pathway in vivo and functional assessment of
the therapeutic benefit was measured by electroretinography.
Immunohistochemical images were obtained by confocal scanning
microscopy.
Results: Up-regulation of the RIP1/RIP3 complex resulted in the
translocation of phosphorylated DRP1 to the mitochondria of rod
photoreceptors, resulting in the production of reactive oxygen
species (ROS). Inflammasome components NLRP3 and activated
caspase-1 were up-regulated in genetically normal cones, whereas
their expression was absent from wildtype controls. The mature form
of two downstream pro-inflammatory cytokines, Il-1ß and Il-18,
were present in mutant retinal tissue extracts. NLRP3 was also upregulated in activated microglia that had infiltrated the mutant retina.
Treatment with either necrostatin-1s or N-acetylcysteine decreased
NLRP3 expression, reduced reactive oxygen species, inhibited IL1β expression, inhibited cone cell death, reduced the infiltration of
activated microglia and prevented attenuation of the microvasculature
in the retina. In contrast 3-aminobenzamide (an inhibitor of
caspase-independent cell death) had no effect on inhibiting retinal
degeneration.
Conclusions: Collectively, this novel data provides compelling
evidence that inflammasome activation in the neurosensory retina is a
critical component in the pathogenesis of rod and cone photoreceptor
degeneration in the P23H model of retinitis pigmentosa. Limiting the
inflammasome-driven response will be crucial in preserving retinal
integrity when designing future therapeutic options.
Commercial Relationships: Cheryl Y. Gregory-Evans, None;
Ishaq A. Viringipurampeer, None; Andrew L. Metcalfe, None;
Emran Bashar, None; Zeinabsadat Mohammadi, None; Orson L.
Moritz, None; Kevin Gregory-Evans, None
Support: CIHR Team Grant 222728
Differences in Aqueous Concentrations of Cytokines in Pediatric
and Adult patients with Coats’ disease
Jing Feng, Xiaoxue Zheng, Yanrong Jiang. Peking Univ People’s
Hospital, Beijing, China.
Purpose: To investigate the differential aqueous concentrations of
VEGF and inflammatory cytokines in pediatric and adult patients
with Coats’ disease.
Methods: A total of 19 eyes of 19 patients with Coats’ disease,
11 eyes of 11 pediatric patients, and 8 eyes of 8 adult patients, 6
patients (6 eyes) with congenital cataract and 9 patients (9 eyes)
with senile cataract as the control group were examined. Aqueous
humor samples were assessed for interleukin 6, 8, 1β (IL-6, IL-8,
IL-1β, respectively), basic fibroblast growth factor (bFGF), monocyte
chemoattractant protein 1 (MCP1), tumor necrosis factor alpha
(TNFα), and vascular endothelia growth factor (VEGF) by multiplex
bead assay.
Results: Significantly higher concentrations of VEGFTNFαIL-8IL6IL-1β were found in pediatric patients with Coats’ disease (P=0.002,
P=0.026, P=0.036, P=0.008, and P=0.010). Concentration of VEGF
in pediatric patients with 3b stage of Coats’ disease was significantly
higher than that of 3a stage (P=0.008). In adult patients with Coats’
disease, the aqueous levels of IL-8, IL-6 and IL-1β were significantly
higher than that of the controls (P=0.036P=0.008, and P=0.010). The
concentration of IL-6 was significantly associated with the extent of
exudative retinal detachment (P=0.02, R2=0.901). Compared with
pediatric patients, the VEGF level in the aqueous humor of adult
patients was significantly lower (P=0.011).
Conclusions: Besides VEGF, IL-6 as a major protein involved in
Inflammation may be associated with Coats’ disease, especially in
adult patients.
Scatterplot showing the association between the aqueous
concentration of interleukin 6 (IL-6) and the extent of exudative
retinal detachment in adult patients with Coats’ disease, with a
statistically significant correlation between both parameters (P=0.02,
R2=0.901).
Commercial Relationships: Jing Feng, None; Xiaoxue Zheng,
None; Yanrong Jiang, None
Proinflammatory status in the aqueous humor of high myopic
cataract eyes
Xiang-Jia Zhu, Ke-Ke Zhang, Wen-Wen He, Jin Yang, Yi Lu.
Ophthalmology, Eye and ENT Hospital of Fudan University,
Shanghai, China.
Purpose: To detect inflammatory cytokines expressed in the aqueous
humor (AH) of high myopic cataract (HMC) patients.
Methods: In the screening stage, AH samples from 15 age-related
cataract (ARC) patients and 15 HMC patients were collected and
assayed using a RayBio Human Cytokine Antibody Array. The
cytokines detected by the screening assays were verified using a BioPlex Suspension Array System in AH samples obtained from 35 ARC
patients and 45 HMC patients.
Results: The cytokine antibody array showed that the expression
level of interleukin-1 receptor antagonist (IL-1ra) in the AH was
higher in ARC than in HMC, whereas opposite trends were found
for monocyte chemoattractant protein-1 (MCP-1), regulated on
activation, normal T-cell expressed and presumably secreted
(RANTES), IL-8, platelet-derived growth factor-BB, and IL-6 (all P
< 0.05). In the verification assay using the suspension cytokine array,
only the expression levels of IL-1ra and MCP-1 were significantly
different between ARC and HMC groups (P =0.014 and 0.038,
respectively).
Conclusions: The expression of IL-1ra was significantly lower and
the expression of MCP-1 was significantly higher in the AH of HMC
than in ARC, indicating a proinflammatory status in the anterior
chamber of HMC eyes.
Commercial Relationships: Xiang-Jia Zhu, None; Ke-Ke Zhang,
None; Wen-Wen He, None; Jin Yang, None; Yi Lu, None
Cytokine profile in active scleritis
Maite Sainz De La Maza1, Blanca Molins1, Marina Mesquida1, Victor
Llorens1, Javier Zarranz-Ventura1, Anna Sala-Puigdollers1, Jessica
Matas1, Alfredo Adan Civera1, Charles S. Foster2. 1Instituto Clinico
Oftalmologia, Hospital Clinico Oftalmologia, Barcelona, Spain;
2
Massachusetts Eye Research and Surgery Institute, Cambridge, MA.
Purpose: Scleritis may be a chronic, painful, progressive, potentially
blinding condition often associated with systemic connective tissue or
vasculitic diseases, some of them potentially lethal. Serum cytokine
profiling may contribute to the understanding of the physiopathology
processes underlying scleral inflammation. Our purpose is to evaluate
serum cytokine profile from patients with active scleritis.
Methods: Two-center prospective case-control study. The serum of
16 active scleritis patients not treated with any local, periocular, or
systemic immunomodulatory therapy (IMT) was analyzed with the
Luminex platform (Millipore’s MILLIPLEX® Human Cytokine/
Chemokine kit) to determine the levels of 11 cytokines including
interleukin (IL)-1β, IL-6, IL-2, IFN-γ, IL-10, IL-12p40, IL-13,
IL-17A, IL-5, TNFα, and TNFβ) and with ELISA to determine
the levels of TGF-β1, IL-22, and IL-23. Serum of 17 age-matched
healthy volunteers was used as control. Non-parametric analysis
was performed using the Mann-Whitney test for comparison of
unpaired data from two groups. Cytokine profile from patients
with scleritis were correlated with type of scleritis (non-necrotizing
and necrotizing), degree of inflammation (0-4+:≤ 2+ and 〉2+), and
systemic associated disease (yes or no).
Results: Serum levels of interleukin IL-22 were elevated in active
scleritis patients naïve to IMT compared with that of controls (4,66+/0,84 pg/mL vs 1,91+/-0,39 pg/mL, P=0.008). Those levels trended
down after scleritis remission with the use of IMT (P〈0.05). The
serum levels of other cytokines were not significantly different from
control levels. No significant correlations were found with specific
cytokine profiles and type of scleritis or systemic associate disease,
probably limited by the small number of patients. Serum levels of
IL-2, IL-6 and TNFβ were significantly elevated in patients with 〉2+
inflammation (P〈0.05).
Conclusions: Serum levels of IL-22 are significantly elevated in
active scleritis patients naïve to IMT. IL-22, a T helper (Th)17
and Th22 derived cytokine, may play a critical role in the
physiopathology of scleritis and could be a promising therapeutic
target.
Commercial Relationships: Maite Sainz De La Maza, None;
Blanca Molins, None; Marina Mesquida, None; Victor Llorens,
None; Javier Zarranz-Ventura, None; Anna Sala-Puigdollers,
None; Jessica Matas, None; Alfredo Adan Civera, None; Charles
S. Foster, None
Tear film hyperosmolarity breaks down the ocular surface’s
immune tolerance: a role in initiating dry eye?
Mauricio Guzman, Irene Keitelman, Florencia G. Sabbione, Analía
Trevani, Mirta N. Giordano, Jeremias G. Galletti. Immunology,
Institute of Experimental Medicine, Buenos Aires, Argentina.
Purpose: Tear film hyperosmolarity is commonly observed in dry
eye, but its exact role in the pathogenesis of this ocular surface
disorder is unknown. As a localized form of autoimmune disease, dry
eye involves disruption of the ocular surface’s immune homeostasis
by still uncharacterized stimuli. The purpose of this work was to
evaluate whether hyperosmolar stress could affect conjunctival
immune tolerance in mice.
Methods: 8-to-12 week-old female Balb/c mice were instilled
isoosmolar (0.3 Osm) or hyperosmolar (3 Osm) saline on both eyes
3 times daily for 5 days. Ovalbumin (OVA) was also instilled or
OVA-pulsed dendritic cells (DCs) were subconjunctivally injected
at different time points. Induced T cell responses were measured
after subcutaneous immunization with OVA by the delayed-type
hypersensitivity (DTH) assay. In addition, T cells in draining lymph
nodes (day 5) were evaluated by flow cytometry. Supernatants from
Pam212 epithelial cells exposed to iso- or hyperosmolar medium
were assayed for cytokines or in a phagocytosis assay with the Raw
264.7 macrophage cell line.
Results: Compared to non-instilled immunized mice, OVA-instilled
mice developed reduced DTH responses (i.e. were tolerized), as did
their OVA+0.3Osm saline-instilled cage mates (p<0.05). By contrast
OVA+3Osm saline-instilled mice exhibited full DTH responses
(i.e. were not tolerized). In the latter, T cells in draining lymph
nodes showed increased expression of activation (CD69, CD25)
and memory (CD44) markers. Local injection of immature DCs
in 3Osm saline-instilled mice, but not in 0.3Osm saline-instilled
mice, induced increased T cell proliferation in the draining lymph
nodes. Consistently, supernatants from Pam212 cells exposed to
hyperosmolar medium for 4h, but not to control medium, had higher
interleukin-1β and -6 levels and increased the phagocytic activity of
macrophages.
Conclusions: Hyperosmolar stress is sufficient to abolish
conjunctival immune tolerance to a harmless antigen in Balb/c mice,
suggesting that this event by itself can skew the immune response at
the ocular surface. The hyperosmolarity-induced proinflammatory
mucosal environment favors local maturation of DCs and T cell
responses in the draining lymph nodes. These results altogether
suggest that hyperosmolar stress might play a role in dry eye
initiation as an immune disruptor.
Commercial Relationships: Mauricio Guzman, None; Irene
Keitelman, None; Florencia G. Sabbione, None; Analía Trevani,
None; Mirta N. Giordano, None; Jeremias G. Galletti, None
Support: PICT 2013-1436, FONCyT, Argentina
Regulation of pro-inflammatory cytokine secretion in human
meibomian gland epithelial cells
Afsun Sahin1, Didem Cosan2, Cağri Oner2, Hasan V. Gunes2,
Nilgun Yildirim1, Hikmet Basmak1. 1Department of Ophthalmology,
Eskisehir Osmangazi University Medical School, Eskisehir, Turkey;
2
Department of Biology, Eskisehir Osmangazi University Medical
School, Eskisehir, Turkey.
Purpose: Androgens are known to modulate the function
of ocular surface and adnexal epithelial cells, suppress
lipopolysaccharide(LPS)-induced proinflammatory responses in
nonocular sites. However, there is limited data if androgens show
this anti-inflammatory activity on ocular surface and adnexa. We
hypothesize that human ocular adnexal epithelial cells have the
ability to produce and release pro-inflammatory cytokine (IFNgamma, TNF-alpha, IL-10, IL-1beta, IL-2, IL-4, IL-6, IL-8,
VEGF-A) in response to lipopolysaccharide exposure. We also
hypothesize the following: (1) Lipopolisaccharide binding protein
(LBP) potentiates LPS-induced cytokine secretion by human
meibomian gland epithelial cells; (2) dihydrotestosterone (DHT), a
potent androgen, attenuates the immune effect of LPS. The objective
of this study was to test our hypotheses.
Methods: Immortalized human meibomian gland epithelial cells
(from David A.Sullivan, Schepens Eye Research Institute, Boston,
MA) were cultured in keratinocyte serum-free medium until reaching
100% confluence. Cells were then exposed to DMEM/F12 medium
with 10% BCS for 2 days, followed by an incubation in serum-free
DMEM/F12 for 1 day. After this time period, cells were treated with
vehicle or 15 ug/ml LPS (E. Coli, strain 0127:B8) for 6, 12 and 24
hours. Culture media were collected and analyzed for IFN-gamma,
TNF-alpha, IL-10, IL-1beta, IL-2, IL-4, IL-6, IL-8, VEGF-A levels
with Luminex technology.
Results: Lipopolysaccharide stimulates time-dependent secretion
of IL-1beta, IL-6, IL-8, and VEGF-A by human meibomian gland
epithelial cells. This effect is potentiated by exposure to LPS-binding
protein. This potentiation, in turn, is significantly reduced by cellular
treatment with dihydrotestosterone.
Conclusions: Human meibomian gland epithelial cells have the
ability to generate pro-inflammatory cytokine in response to LPS
exposure. This proinflammatory process is modulated by LPSbinding protein and by dihydrotestosterone. When induced by
appropriate stimuli, this cytokine production may have a role in the
generation of inflammation in ocular surface disease.
Commercial Relationships: Afsun Sahin, None; Didem Cosan,
None; Cağri Oner, None; Hasan V. Gunes, None; Nilgun Yildirim,
None; Hikmet Basmak, None
Kupffer Cells Modulate Uveal Melanoma Metastasis to the Liver
Erwin Puente, joseph R. brown, Leila Sadegh, jessamee Mellon, Jerry
Y. Niederkorn. University of Texas Southwestern, Dallas, TX.
Purpose: To explore the role of myeloid derived suppressor cells
(MDSCs) in the development of liver metastasis from intraocular
melanoma. MDSCs are immature myeloid cells that are capable
of inhibiting the immune system. In other malignancies, MDSCs
have been shown to increase tumor growth and metastatic disease.
We hypothesized that MDSCs promote liver metastasis from uveal
melanoma, and thus, inhibition of MDSCs would lead to decreased
liver metastasis.
Methods: Two models of melanoma liver metastasis have been
used to produce melanoma liver metastases in mice. Murine
B16LS9 melanoma cells produce liver metastases following either
intracameral or intrasplenic injection. In both models liver metastases
develop and display similar immunological properties, especially
in regard to innate immunity in the liver. In the present study we
injected B16LS9 melanoma cells intrasplenically into syngeneic
C57BL/6 mice as a means of inducing melanoma liver metastases.
Liver metastasis was quantitated by histopathology. In some
experiments, mice were concurrently treated with anti-Gr1 antibody
to deplete MDSCs. Mice were also treated with either anti-Ly6G to
deplete neutrophils or clodronate liposomes to deplete Kupffer cells
respectively.
Results: Although anti-Gr1 treatment produced a >70% reduction
in the Gr1+CD11b+ putative MDSC, it did not lead to decreased
liver metastases. Instead, mice treated with anti-Gr1 antibody had
significantly more liver metastases compared to isotype control
antibody-treated mice (P>0.05). Anti-Gr1 antibody can also deplete
neutrophils and Kupffer cells. Accordingly, we hypothesized that
anti-Gr1 effect on liver metastasis was mediated by neutrophils or
Kupffer cells, which are resident macrophages of the liver. Specific
inhibition of neutrophils with anti-Ly6G produced an 85% reduction
in circulating neutrophils, but did not affect the development of
liver metastasis (P < 0.05). However, depletion of Kupffer cells with
clodronate liposomes resulted in an 80% increase in the number of
liver metastases (P = 0.001).
Conclusions: Taken together, these studies suggest that neither
MDSCs nor neutrophils affect melanoma liver metastasis while
Kupffer cells play an important role in limiting melanoma metastasis
to the liver. Understanding the mechanisms of uveal melanoma
metastasis may lead to new therapies to prevent metastatic disease
and thus improve patient morbidity and mortality.
Commercial Relationships: Erwin Puente, None; joseph R.
brown, None; Leila Sadegh, None; jessamee Mellon, None; Jerry
Y. Niederkorn, None
Support: NIH grants CA030276 and EY020799; and Research to
Prevent Blindness
Monitoring of interleukine-10 and Interleukine-6 for Primary
Vitreoretinal Lymphoma
Lévêque Pierre-Maxime1, ADIL DARUGAR1, Nathalie Cassoux5,
Magali Legarf-Tavernier4, Helene Merle-Berale4, Sylvain Choquet3,
Khê Hoang-Xuan2, Bahram Bodaghi1, Phuc Lehoang1, Valérie
Touitou1. 1Ophthalmology Department, Hôpital Pitié Salpétriere,
Paris, France; 2Neuro-oncology Department, Hôpital Pitié Salpétriere,
Paris, France; 3Hematology Department, Hôpital Pitié Salpétriere,
Paris, France; 4Hemato-biology Department, Hôpital Pitié Salpétriere,
Paris, France; 5Ophthalmology Department, Institut Curie, Paris,
France.
Purpose: Molecular techniques, especially quantitative analysis
of the interleukine-10 (IL-10) and interleukine-6 (IL-6) have
contributed to dramatic improvement of the diagnostic delay of
primary Vitreoretinal Lymphoma (PVRL). If cytology remains the
gold-standard for diagnosis, cytokine analysis is very efficient for
screening purposes to identify patients who would benefit from a
diagnostic vitrectomy. Very few data are available concerning the
contribution of this cytokine analysis for the follow-up of PVRL
patients.
Methods: Medical records of patients with a cytological diagnosis
of PVRL seen in a single tertiary center between June 2009 and
April 2014 were retrospectively reviewed. Demographic and clinical
characteristics of the population at the time of the diagnosis were
studied. Levels of IL-10 and IL-6 (humour aqueous and vitreous) at
the diagnosis and during the follow-up were analyzed and correlated
to the prognosis of these patients.
Results: We studied 71 eyes from 39 patients. Aqueous humor level
of IL-10> 50pg /mL was observed in 82.7% patients, with a ratio
IL-10 /IL-6> 1 in 87.5% of cases. Vitreous level of IL-10> 400pg/
mL was observed in 62.5% of patients. Both IL-10 and IL-6 levels
decreased as clinical remission was observed. Kinetic of decrease
of IL-10 level was greater in patients responders to chemotherapy
compared with patients ultimately presenting ocular relapse.
Regardless of the therapeutic option chosen (systemic or local
treatment), the rate of intraocular cytokine decreased in the same
proportion.
Conclusions: Our results confirm the hypothesis that IL-10 level
reflects tumor burden. Repeated cytokine measurment is usefull
for the follow-up of patients with a PVRL. Ocular relapses can be
detected very promptly and patients non-responsive to chemotherapy
could also be identified early. A prospective study is on-going to
determin the frequency of measurements of these interleukines during
the treatment.
Commercial Relationships: Lévêque Pierre-Maxime, None; ADIL
DARUGAR, None; Nathalie Cassoux, None; Magali LegarfTavernier, None; Helene Merle-Berale, None; Sylvain Choquet,
None; Khê Hoang-Xuan, None; Bahram Bodaghi, None; Phuc
Lehoang, None; Valérie Touitou, None
Effect of Hormone Replacement Therapy on Lens Opacity by
Scheimpflug Densitometry and Serum Inflammatory Cytokine
and Antioxidant Levels.
Kim Eun Chul2, Minho Kim2, Man Soo Kim1. 1Seoul St. Mary’s
Hospital, College of Medicine, The Catholic University of Korea,
Seoul, Korea, Seoul, Korea (the Republic of); 2Bucheon St. Mary’s
Hospital,College of Medicine, The Catholic University of Korea,
Bucheon, Korea (the Republic of).
Purpose: Female sex hormones was reported to play an important
role in protecting against cataract progression.
To evaluate the effect of hormone replacement therapy (HRT) on
lens opacity measured by Scheimpflug densitometry and serum
inflammatory cytokines and antioxidant levels.
Methods: The control group of 128 patients (Group 1) did not
use HRT at any time after menopause. The treatment group of 136
patients (Group 2) had used HRT over 5 years after menopause.
Lens density was measured using a Scheimpflug imaging system
(Pentacam). Multiplex bead analysis was conducted with serum.
Cortical, nuclear, and posterior subcapsular density, pentacam
nucleus staging (PNS), pentacam densitometry of zone (PDZ)
measured by pentacam, and antioxidant and inflammatory cytokines
activities in serum.
Results: Nuclear and posterior subcapsular density, PNS, and
percentage of PDZ in group 1 were significantly higher than those
of group 2 (P <0.05). The serum IL-1β, IL-6, IL-8, and TGF-β
concentration in group 1 were significantly higher than those of group
2 and the serum catalase, superoxide dismutases 1 (SOD 1), and
superoxide dismutases 2 (SOD 2) fluorescence intensity in group 1
were significantly lower than those of group 2 (P <0.05).
Conclusions: Long-term use of HRT may have a protective effect
against cataract formation. HRT seem to be more effective in
decreasing inflammation and increasing antioxidant contents in the
serum of postmenopausal women.
Commercial Relationships: Kim Eun Chul, None; Minho Kim,
None; Man Soo Kim, None
Clinical Trial: ISRCTN72439325
Angiogenin mediates immune modulation and antiinflammatory
reaction attenuating protein kinase IKK-ε in
human corneal fibroblast cells
Jae Chan Kim, Seung Hoon Lee, Kyoung Woo Kim, Won Soo Kim.
Ophthalmology, Chung-Ang Univ. Hospital, Seoul, Korea (the
Republic of).
Purpose: Angiogenin (ANG), a component of tear, is involved in
the innate immune system and related with inflammatory disease.
We investigate that ANG has the immune modulatory function in
inflamed human corneal fibroblast (HCF) cells.
Methods: HCF cells cultured in normal medium were
exposed to media with tumor necrosis factor-alpha (TNF-α) or
lipopolysaccharide (LPS). Then, the cells were treated in presence
or absence of the ANG. Total RNA was isolated from cultures and
the mRNA level was measured by real-time reverse transcription
polymerase chain reaction (PCR). Protein levels were measured by
immunodot blot assay. Immunocytochemistry was used to observe
NF-κB expression in HCF cells.
Results: While ANG decreased the mRNA level of interleukin1beta (IL-1β), -6, -8, TNF-α receptor (TNFR) 1, 2, toll-like receptor
(TLR) 4, myeloid differentiation primary response gene (MYD) 88,
and complement components except for C1r and C1s, it elevated
the mRNA expression of IL-4 and -10. Signal intensity of IL-6, -8
and monocyte chemotactic protein (MCP)-1 and -2 enlarged by
was weakened by ANG treatment. ANG reduces the protein levels
of IκB kinase epsilon (IKK-ε) and TANK-binding kinase (TBK) 1
production induced by TNF-α, not LPS was decreased by ANG. The
expression of NF-κB in nuclei was decreased after ANG treatment.
Conclusions: These results demonstrate that ANG reduced
inflammatory response in HCF cells induced by TNF-α or LPS
through commonly suppression of IKK-ε mediated NF-κB
activation. We thought these findings support to targeting of immunemediated inflammatory therapeutic substance and finding out the
comprehension of immune-mediated inflammatory diseases.
Commercial Relationships: Jae Chan Kim, None; Seung Hoon
Lee, None; Kyoung Woo Kim, None; Won Soo Kim, None
Effect of Denatured Lysozyme on Human Corneal Epithelial
Cells
David J. McCanna1, Sarah Oh1, Junghee Seo1, Lakshman N.
Subbaraman1, Chantal Coles-Brennan2, Zohra Fadli2, Lyndon W.
Jones1. 1Centre for Contact Lens Research, School of Optometry
and Vision Science, University of Waterloo, Waterloo, ON, Canada;
2
Johnson and Johnson Vision Care Inc., Jacksonville, FL.
Purpose: Protein denaturation on contact lenses (CL) has been
hypothesized as one of the possible leading causes for unsuccessful
CL wear probably due to the loss in proteins functions, change in
the exposed chemical structure or to the fact that denatured proteins
can be recognized as an antigen and therefore induces an immune
response. The binding of lysozyme to some CL materials has been
shown to result in a conformational change of the protein, leading to
a loss in enzymatic activity. This investigation evaluated the effect
of denatured lysozyme on human corneal epithelial cells (HCEC) by
measuring cell viability and the release of inflammatory cytokines
after HCEC exposure to heat-inactivated lysozyme
Methods: HCEC were cultured in multi-well plates and exposed to
lysozyme that was denatured to various enzymatic activity levels.
After a 16 hr exposure to the lysozyme (1.9mg/mL in media), cell
viability was evaluated by staining the cells with the viability dyes
calcein, ethidium homodimer-1 and annexin V and imaging the
cells using a confocal microscope. The metabolic activity was also
determined using the alamarBlue assay. The media from the cell
wells were analyzed for cytokine levels using the human proinflammatory V-plex assay (IFN-γ, IL-10, IL-12 p70, IL-13, IL-1β,
IL-2, IL-4, IL-6, IL-8 and TNF-α) from MesoScale Discovery.
Results: The confocal images of stained HCEC exposed to various
levels of denatured lysozyme did not show a change in the cell
viability. However, a significant decrease in metabolic activity was
observed following exposure to different levels denatured lysozyme
when compared to native (100% active) lysozyme and the media
controls (p<0.05). Exposure of HCEC to the denatured lysozyme also
caused asignificant increase in the release of several inflammatory
cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-12 and IL-13) from the
HCEC (all p<0.05) depending on the level of denatured lysozyme in
contact with the cells.
Conclusions: The results of this study for the first time show that
denatured lysozyme can have a detrimental effect on HCEC. Both
a reduction in metabolic activity and the release of inflammatory
cytokines occurred after HCEC exposure to denatured lysozyme.
Preventing proteins such as lysozyme from denaturing due to
deposition on contact lenses may be desired to preserve corneal cell
homeostasis essential for successful contact lens wear.
Commercial Relationships: David J. McCanna, None; Sarah Oh,
None; Junghee Seo, None; Lakshman N. Subbaraman, Johnson
and Johnson Vision Care Inc. (F); Chantal Coles-Brennan, Johnson
and Johnson Vision Care Inc. (E); Zohra Fadli, Johnson and Johnson
Vision Care Inc. (E); Lyndon W. Jones, Johnson and Johnson Vision
Care Inc. (F)
Support: This research was supported by Johnson & Johnson Vision
Care Inc.
369 Retina / RPE immunobiology
Tuesday, May 05, 2015 3:45 PM–5:30 PM
Contributing Section(s): Retinal Cell Biology
The role of impaired retinal pigment epithelial function in
modulating macrophage activation
Jian Liu, David A. Copland, Sofia Theodoropoulou, Miriam Barba,
Lindsay B. Nicholson, Andrew D. Dick. Ophthalmology, School of
Clinical Sci, University of Bristol, Bristol, United Kingdom.
Purpose: Progression of age-related macular degeneration (AMD)
involves degeneration hallmarks of retinal pigment epithelium and
photoreceptor loss is accompanied by unchecked immune response
or pathological angiogenesis. Here we investigated whether RPE
cells, in presence of impaired autophagy or under oxidative stress,
differentially condition macrophage responses and thus drive further
cell death or promote angiogenesis.
Methods: Wortmannin and chloroquine were used to suppress
autophagy at upstream and downstream checkpoints, respectively,
in murine RPE cells. Rotenone was used to induce RPE autophagy.
Oxidative stress was induced by hydrogen peroxide. Beclin-1
gene silencing was performed using siRNAs, to inhibit autophagy.
RPE cytotoxicity was assessed by LDH release. An in vitro
phagocytosis assay was applied to elucidate the role of impaired
RPE in modulating inflammatory cytokine and nitric oxide (NO)
production from bone marrow derived macrophages (BMMΦ).
Macrophage caspase-1 activation was examined by western blot and
immunofluorescence microscopy. Macrophage derived angiogenesisassociated proteins were assessed by antibody array. The proangiogenic potential of RPE-macrophage conditioned medium (CM)
was assessed in an explant choroidal sprouting model. To confirm in
vivo, autophagy was inhibited by intravitreal injection of wortmannin.
RPE damage and macrophage accumulation/activation were detected
in whole-mounts of RPE/choroid.
Results: Inhibition of basal activity or rotenone-induced autophagy
in RPE cells using wortmannin or chloroquine led to increased cell
death, mediated by caspase-3 activation. Beclin-1 knockdown also
provoked RPE susceptibility to rotenone toxicity. After treatment
with damaged RPE cells caused by impaired autophagy or oxidative
stress, BMMΦ expressed markers of inflammasome activation and
secreted higher levels of IL-1β, IL-6 and NO. Comparably, in vivo
suppression of retinal autophagy led to RPE degeneration and focal
accumulation of activated macrophages. In addition, impaired RPE
cells differentially conditioned macrophage production of proteins
involved in promulgating inflammation or driving angiogenesis. CM
of macrophages treated with oxidative stressed RPE was more proangiogenic.
Conclusions: Our data suggest that the nature of RPE degeneration
will differentially regulate macrophage phenotype and function and
drive angiogenesis.
Commercial Relationships: Jian Liu, None; David A. Copland,
None; Sofia Theodoropoulou, None; Miriam Barba, None;
Lindsay B. Nicholson, None; Andrew D. Dick, None
Support: National Eye Research Centre (Grant RJ5818) and
Rosetrees Trust (Grant M418)
Vascular endothelial growth factor regulates expression of
protective complement factor H in specialized capillary beds
Lindsay Keir3, 1, Rachel Firth1, Lyndsey Aponik3, Daniel Feitelberg3,
Carli M. Wittgrove3, Peter D. Westenskow3, Yoshihiko Usui3, Anna
Richards2, Moin Saleem1, Martin Friedlander3. 1School of Clinical
Science, University of Bristol, Bristol, United Kingdom; 2Queens
Medical Institute, University of Edinburgh, Edinburgh, United
Kingdom; 3Department of Molecular and Cell Biology, The Scripps
Research Institute, La Jolla, CA.
Purpose: Local VEGF production in microvascular beds is vital
for endothelial cell function. Both the outer retina and renal
glomerulus possess specialized epithelial cells that produce VEGF
to maintain a fenestrated endothelium. In mice, ablation of retinal
pigmented epithelial (RPE) cell VEGF causes rapid degeneration
of the choriocapillaris and vision loss. Loss of glomerular VEGF
leads to thrombotic microangiopathy (TMA) and haemolytic uremic
syndrome (HUS). VEGF antagonists can cause glomerular TMA
with HUS and may accelerate progression of dry AMD to geographic
atrophy. Complement dysregulation is also a feature of atypical HUS
and AMD, with genetic alterations in factor H (CFH) implicated
in both diseases. Linking these observations, we show that VEGF
regulates local CFH expression in both the outer retina and renal
glomerulus.
Methods: Eyes from AMD patients and controls were analyzed
for CFH and VEGF expression and for evidence of complement
activation. CFH was also analyzed in human RPE and glomerular
cells before and after VEGF treatment or antagonism. RPE cells were
treated with VEGF and various inhibitors to study the mechanism of
VEGF induced CFH up regulation. CFH expression and complement
deposition was examined in RPE and podocyte-specific VEGF
knockout mice.
Results: AMD patient eyes showed reduced CFH staining in the
RPE and complement activation in the choriocapillaris. Loss or
inhibition of epithelial derived VEGF led to local reduction in CFH
expression and increased complement deposition in the retina and the
glomerulus. This was mediated by reduced VEGFR2- PKC signaling
that decreased activation of the CREB transcription factor.
To further examine this relationship, RPE specific VEGF knockout
mice are being crossed with complement C3 knockout mice to
determine if phenotypic rescue of the choriocapillaris occurs. The
AMD Y402H CFH polymorphism is being introduced into human
RPE cells using CRISPR/Cas technology to allow further functional
characterization.
Conclusions: The relationship between VEGF and CFH provides
new insights into local mechanisms that protect the microvascular
endothelium from complement-mediated injury and may provide
an explanation for the off target effects of anti-VEGF agents. This
work highlights biological similarities between the outer retina
and glomerulus which could have implications for other diseases
including diabetes.
Commercial Relationships: Lindsay Keir, None; Rachel Firth,
None; Lyndsey Aponik, None; Daniel Feitelberg, None; Carli M.
Wittgrove, None; Peter D. Westenskow, None; Yoshihiko Usui,
None; Anna Richards, Alexion (F), Glaxo Smith Kline (E); Moin
Saleem, None; Martin Friedlander, None
Support: NEI/NIH EY11254; The Lowy Medical Research
Foundation; MRC Centenary Award
Impaired alternative complement pathway function does not
protect against light-induced retinopathy in mice
Shawn M. Hanks, Joanna Vrouvlianis, Barrett Leehy, Maura Crowley,
Michael Maker, Sha-Mei Liao, Bruce D. Jaffee, Chad E. Bigelow.
Ophthalmology, Novartis Institutes for BioMedical Research,
Cambridge, MA.
Purpose: Alternative complement pathway-linked genetic
polymorphisms are associated with an increased risk of age-related
macular degeneration. The purpose of this study is to determine if
mice deficient in alternative complement pathway component factor
B are protected against light-induced retinal degeneration.
Methods: Study was performed on 9-10 week old female
complement factor B knockout (FB-/-) mice on a Balb/c background
(Leu450 RPE65 isoform) with FB+/+ wild type mice acting as
controls. Plasma and ocular FB protein levels were evaluated by
western blot. Mice were either dark adapted overnight prior to 12
hours of blue light exposure (~550 lux, 420 nm center wavelength)
or were maintained in standard cyclic housing room lighting (<100
lux, white light, 12 hour cycle). Retinal degeneration was assessed
using electroretinography (ERG) 1 day after light exposure and by
optical coherence tomography (OCT) 2 days after light exposure.
Dark-adapted visual function was evaluated by quantifying ERG
a-wave amplitude in response to a 2.7 log scotcdsm-2 flash. Retinal
thickness was quantified from linear OCT scans centered on the optic
nerve. Reported statistical comparisons are only between groups that
received blue light (one-way analysis of variance with a Tukey’s
posttest).
Results: Western blot analysis confirmed FB-/- mice to be deficient
in FB protein for both ocular tissue and plasma. Blue light exposure
resulted in similar photoreceptor dysfunction in FB+/+ and FB-/- mice
(41% and 57% reduction in ERG a-wave, respectively, P > 0.05).
OCT findings were consistent with this result, with blue light exposed
FB+/+ and FB-/- mice exhibiting photoreceptor layer thinning (29% and
42%, respectively, P < 0.05).
Conclusions: Mice deficient in alternative complement pathway
component factor B exhibit blue light-induced retinal degeneration
that is similar to that observed in mice with an intact complement
pathway. Loss of factor B does not confer protection against lightinduced retinopathy in albino mice.
Commercial Relationships: Shawn M. Hanks, Novartis Institutes
for Biomedical Research (E); Joanna Vrouvlianis, Novartis
Institutes for Biomedical Research (E); Barrett Leehy, Novartis
Institutes for Biomedical Research (E); Maura Crowley, Novartis
Institutes for Biomedical Research (E); Michael Maker, Novartis
Institutes for Biomedical Research (E); Sha-Mei Liao, Novartis
Institutes for Biomedical Research (E); Bruce D. Jaffee, Novartis
Institutes for Biomedical Research (E); Chad E. Bigelow, Novartis
Institutes for Biomedical Research (E)
Complement component 5 facilitates the influx of microglia/
macrophages into the photoreceptor layer of light damaged
mouse retina
Michael Sulewski, Delu Song, Jiantao Song, Chenguang Wang,
Esther Clark, Jacob Sterling, Joshua L. Dunaief. Scheie Eye
Institute, University of Pennsylvania, Philadelphia, PA.
Purpose: Although the precise cellular mechanism is unknown,
previous work has demonstrated that complement components 3a
and 5a (C3a and C5a) contribute to retinal damage in a light-damage
model for retinal degeneration. Because C3a and C5a are chemotactic
and macrophages express receptors for these proteins, we tested the
hypothesis that migration of retinal microglia/macrophages may be
an integral component of complement-dependent immune damage.
Methods: Wild-type (WT) as well as C3aR-/- and C5aR-/- mice, all
on a Balb/c background, were exposed to bright white fluorescent
light for 3h. At 48h following exposure, animals were sacrificed
and eyes were enucleated. The neurosensory retina was dissected
from one eye from each animal for quantification of Iba1 mRNA
expression by RT-qPCR. Contralateral eyes were cryo-sectioned
or flat-mounted and immunolabeled with Iba1 specific antibodies.
Retinal flat-mount samples were imaged with a Zeiss confocal
imaging system. Iba1 positive cells in cryo-sections were counted
by a masked observer in three zones (zone 1: ganglion layer to outer
plexiform layer, zone 2: outer nuclear layer, zone 3: inner and outer
segment layers). Statistical differences among groups were analyzed
by one-way ANOVA.
Results: Relative mRNA levels of Iba1, a marker of microglia/
macrophages, in the neurosensory retina were significantly less in
C5aR-/- mice exposed to LD (n= 3, P=0.02) but not in C3aR-/- mice
(n=4, P=0.70) when compared to WT-LD mice (n=3). Confocal
images showed fewer Iba1 positive cells in retinal flat mounts from
C5aR-/- compared to WT and C3aR-/- mice. In cryosections, the
number of Iba1 positive cells was significantly increased in WT
retinas exposed to LD compared to non-LD wild-type retinas (WTLD vs NLD, zone 1: 28.2 vs 10.3, zone 2: 24.5 vs 3.3, zone 3: 15.4
vs 4.7 P<0.001 ). Following LD, the number of Iba1 positive cells
was not significantly different in C3aR-/- retinas (zone 1: 24.7, zone
2: 21.4, zone 3: 14.0) when compared to WT-LD. In C5aR-/- mice,
the number of Iba1 positive cells were significantly reduced (zone 1:
14.1, zone 2: 11.6, zone 3: 8.4; P<0.01) when compared to WT-LD
mice.
Conclusions: C5aR but not C3aR is important for microglia/
macrophage migration to all layers of the retina following LD. The
C5aR pathway may be important in microglia-mediated retinal
degeneration including age-related macular degeneration.
Commercial Relationships: Michael Sulewski, None; Delu Song,
None; Jiantao Song, None; Chenguang Wang, None; Esther
Clark, None; Jacob Sterling, None; Joshua L. Dunaief, None
The retinal microenvironment induces microglia-like function,
morphology, and phenotype in recruited mononuclear cells
Dale S. Gregerson, Neal D. Heuss, Mark J. Pierson, Scott W.
McPherson. Ophthalmology, University of Minnesota, Minneapolis,
MN.
Purpose: Evidence has suggested that large influxes of recruited
mononuclear cells into retina resolve by mechanisms that retain the
MG once the recruited cells are gone. We have proposed that retina
contains at least two distinct niches, one occupied by MG (N1) and
one (or more) occupied by recruited cells (N2), with MG preserved
in N1 by receipt of a specific signal(s). Recruited cells occupying
N2 are proposed to lack expression of critical receptor(s) that would
retain them in retina. These studies ask if recruited cells can occupy
N1 in the absence of MG.
Methods: Transgenic mice expressing YFP and a Tamoxifen (TAM)
receptor linked to cre in the chemokine receptor CX3CR1 were
crossed with floxed diphtheria toxin A subunit so that CX3CR1+
cells suicide in the presence of TAM. These mice were crossed to
CD11c-DTR/GFP mice in which dendritic cells constitutively express
the diphtheria toxin receptor and GFP, allowing recruited cells to be
tracked, analyzed, depleted, and/or distinguished from resident MG.
Populations of retinal mononuclear cells were also sorted for analysis
by single cell RT-PCR. Mice were given an optic nerve crush (ONC)
to stimulate active recruitment of circulating mononuclear cells.
Results: Flow cytometry and RT-PCR analysis of chemokines and
receptors showed that recruited cells residing in retina become
microglia-like, while newly recruited cells have a distinct chemokine
receptor profile. Recruited mononuclear cells produced the same
neurotrophic factors associated with microglia. Total, acute depletion
of microglia led to slow, but substantial, reconstitution with recruited
mononuclear cells. A unilateral ONC after TAM depletion strongly
recruited mononuclear cells to the ipsilateral retina, with a smaller but
significant influx to the contralateral retina. Fluorescence microscopy
of whole mounts showed that reconstituting cells had a microglia-like
distribution in both normal and injured retina.
Conclusions: Recruited mononuclear cells are nearly
indistinguishable from microglia in short-term assays. A subset
of recruited cells also expressed GFP from the CD11c-DTR/GFP
transgene, similar to studies we have done in mice not depleted
of microglia. Since recruited cells cannot, by strict definition, be
called MG, the term “pseudo-microglia” may be a more accurate
designation for the GFP- population in CD11c-DTR/GFP mice.
Commercial Relationships: Dale S. Gregerson, None; Neal D.
Heuss, None; Mark J. Pierson, None; Scott W. McPherson, None
Support: Wallin Neuroscience Discovery Fund (DSG); NIH/NEI
R01EY021003 (DSG)
Mouse Model for Tamoxifen-inducible Cre Targeting of Retinal
Microglia
Senthil Selvam1, 2, Andrew Scott2, Sidath E. Liyanage1, Michael
Powner1, Marcus Fruttiger1. 1Institute of Ophthalmology, University
College London, London, United Kingdom; 2Moorfields Eye
Hospital, London, United Kingdom.
Purpose: Microglia are a specialised population of the mononuclear
phagocyte lineage. Resident microglia are distinct to invading
populations of circulating monocyte-derived macrophages in
their localisation to the central nervous system (CNS) and their
homeostatic role in the neurogeneis and angiogenesis of the
developing retina. Microglial activation is commonly seen in
inflammatory and degenerative pathologies of the retina. Resident
retinal microglia are known to specifically express the protein ionized
calcium binding adaptor molecule 1 (Iba1; also known as allograft
inflammatory factor-1, Aif1). Using the Aif1 gene we present a mouse
model of tamoxifen inducible, cre-recombinase activation that allows
the genetic targeting of resident central nervous system microglia.
Methods: A bacterial artificial chromosome (BAC), containing
the Aif1 gene was obtained and a construct coding for a tamoxifen
inducible form of Cre (CreERT2) was recombined into the open
reading frame of the Aif1 gene. The modified BAC was then used
for transgenesis by pronuclear injections and resulting offspring was
screened for founders by PCR. In vivo recombination efficiency was
tested in a ROSA-EGFP reporter strain (Gt(ROSA)26Sortm4(ACTBtdTomato,-EGFP)Luo/J) using flow cytometry in both p7 neonatal
mice and 6 week old adult mice. Using the same strain we
demonstrate the use of this tool to label Iba1 expressing resident
retinal microglia in murine models of oxygen induced retinopathy
(OIR), laser induced choroidal neovasculairsation (CNV) and
endotoxin induced uveitis (EIU).
Results: Two founders were obtained and a transgenic line was
established from one of them (Aif1-CreER). Flow cytometry of
the ROSA-EGFP reporter mice showed that recombination could
be achieved in upto 72% of resident microglia in the p7 perinatal
retina and upto 95% in the 6 week adult retina. The brain, choroid
and spleen also showed GFP expression. Immunohistochemistry
performed on retinal flat mounts of ROSA-EGFP reporter mice
undergoing OIR, laser induced CNV and EIU demonstrated colocalisation of GFP expression and Iba1 antibody labelling.
Conclusions: This model of Aif1-CreER provides a valuable research
tool to genetically target resident microglia in the central nervous
system of mice in various murine models of retinal disease.
GFP expression in resident retinal microglia.
Commercial Relationships: Senthil Selvam, Fight for Sight (F);
Andrew Scott, None; Sidath E. Liyanage, None; Michael Powner,
None; Marcus Fruttiger, Amakem (F), AstraZeneca (F), Fight for
Sight (F), Novartis (C), Novartis (F)
Support: Fight For Sight PhD Funded Studentship
A phase 1/2 open-label, safety and tolerability study of
triamcinolone acetonide administered to the suprachoroidal
space in patients with non-infectious uveitis
Debra A. Goldstein1, Sunil K. Srivastava2, Quan Nguyen3, Glenn
Noronha4, Michelle Widmann4. 1Ophthalmology, Northwestern
Med Faculty Foundation, Chicago, IL; 2Cole Eye Institute, The
Cleveland Clinic Foundation, Cleveland, OH; 3Truhlsen Eye
Institute, University of Nebraska Medical Center, Omaha, NE; 4R&D,
Clearside Biomedical, Inc, Atlanta, GA.
Purpose: To evaluate the safety and tolerability of a suprachoroidal
injection of triamcinolone acetonide (TA) in subjects with noninfectious uveitis.
Methods: A single 4 mg (100 mL) dose of TA (Triesence®, Alcon
Labs, 40 mg/mL injectable suspension) was administered into the
suprachoroidal space (SCS) of subjects with non-infectious uveitis
from 3 centers in the US, with follow up data collection scheduled
on day 1, week 1, 2, 4, 8, 12, 16, 20 and 26. To be enrolled, subjects
had to have non-infectious intermediate, posterior or panuveitis and
macular edema (ME) with central subfield thickness (CST) of ≥310
microns on SD-OCT, or a vitreous haze score of ≥ +1.5. In subjects
with bilateral disease, the more severely affected eye was enrolled.
Results: Nine eyes of 9 subjects were enrolled. Five eyes had
panuveitis, 1 had intermediate, 2 had anterior/intermediate and
1 had anterior uveitis. Seven eyes had ME at baseline. Mean age
was 56 years (range 42-78 years); 78% were female. Injection was
successfully completed in 8 of 9 eyes. The ocular adverse events
reported at the time of or immediately following suprachoroidal
injection were ocular pain and irritation. At 12 weeks, 4 of 7 eyes
with ME had ≥ 20% decrease in CST. The complete 26- week safety
and tolerability data will be reported.
Conclusions: In this open-label study, a single suprachoroidal
administration of TA was generally safe and well-tolerated in subjects
with non-infectious uveitis. This study supports the continued
development of suprachoroidal injection of TA for the treatment of
patients with non-infectious uveitis.
Commercial Relationships: Debra A. Goldstein, Clearside
Biomedical, Inc (C); Sunil K. Srivastava, Clearside Biomedical, Inc
(C); Quan Nguyen, None; Glenn Noronha, Clearside Biomedical,
Inc (E); Michelle Widmann, Clearside Biomedical, Inc (E)
409 Autophagy, aging and inflammation in eye disease Minisymposium
Wednesday, May 06, 2015 8:30 AM–10:15 AM
601/603 Minisymposium
Contributing Section(s): Cornea, Glaucoma, Retina
Non-Canonical Autophagy in Cellular and Organismal
Homeostasis
Douglas Green. St Jude Children’s Research Hospital, Memphis, TN.
Presentation Description: I will focus on the phenomenon of
LC3-Associated Phagocytosis (LAP), a form of non-canonical
autophagy which promotes the maturation of phagosomes via fusion
with lysosomes. LAP shares some, but not all, molecular features
with canonical autophagy. In addition, LAP involves molecular
interactions that are not involved in canonical autophagy, including
the stabilization of NADPH-oxidase-2 and of an atypical class III
PI3K complex by the protein, Rubicon, which otherwise acts as a
brake on canonical autophagy. LAP has a variety of functions in
innate immunity, homeostasis (especially in the response to dying
cells), and signaling. LAP also plays a major role in the vision cycle.
We will discuss the process and functions of LAP in the context of
macrophage biology.
Commercial Relationships: Douglas Green, None
Support: NIH Grant AI40646
Autophagy dysregulation and age-related macular degeneration
Yasuo Yanagi. Department of Ophthalmology, Graduate School of
Medicine and Faculty of Medicine, The University of Tokyo, Tokyo,
Japan.
Presentation Description: Elucidating the molecular mechanisms
underlying impaired retinal pigment epithelial (RPE) cell function
appears crucial both for improving our understanding of the
pathogenesis of retinal disease and for identifying novel targets
for prophylaxis and therapeutic intervention. In this presentation,
we show augmented autophagy in the retinal pigment epithelial
cell line ARPE-19 when cultured in the presence of the lipofuscin
pigment A2E. A2E alone does not induce RPE cell death, but cell
death was induced in the presence of A2E with the autophagy
inhibitor 3-methyladenine (3MA), with a concomitant increase
in the generation of mitochondrial reactive oxygen species. On
the other hand, the ATP production capacity of mitochondria was
decreased in the presence of A2E, and pharmacological inhibition of
autophagy had no additional effects. The altered mRNA expression
level of mitochondrial function markers was confirmed by realtime polymerase chain reaction, which showed that the antioxidant
enzymes SOD1 and SOD2 were not reduced in the presence of
A2E alone, but significantly suppressed with the addition of 3MA.
Furthermore, transmission electron micrography revealed autophagic
vacuole formation in the presence of A2E, and inhibition of
autophagy resulted in the accumulation of abnormal mitochondria
with loss of cristae. Spheroid culture of human RPE cells
demonstrated debris accumulation in the presence of A2E, and this
accumulation was accelerated in the presence of 3MA. These results
indicate that autophagy in RPE cells is a vital cytoprotective process
that prevents the accumulation of damaged cellular molecules. This
study provides new evidence that autophagy protects RPE cells
against lipofuscin A2E toxicity.
Commercial Relationships: Yasuo Yanagi, Bayer Healthcare (F),
Novartis Pharmaceuticals (F), Santen Pharmaceuticals (F)
Autophagy and Inflammation
Rajendra S. Apte. Washington Univ., St. Louis, MO.
Presentation Description: Macrophages are emerging as central
regulators of inflammation associated with diverse eye diseases.
Autophagy is a basic homeostatic process that regulates macrophage
activation and function. A review of the current data regarding the
role of autophagy and inflammation in eye disease will be presented.
Commercial Relationships: Rajendra S. Apte, None
Support: NIH Grant EY019287, RPB Career Development
Award, RPB Physician Scientist Award, RPB Unrestricted Grant to
Washington University
Autophagy in Neuroretinal Degeneration
David N. Zacks. Ophthalmology and Visual Sciences, University of
Michigan, Ann Arbor, MI.
Presentation Description: The role of autophagy in photoreceptor
and retinal pigment epithelium health and homeostasis will be
discussed.
Commercial Relationships: David N. Zacks, None
Support: NIH/NEI R01-EY-020823; Beckman Initiative for Macular
Research; Research to Prevent Blindness, Inc.; Foundation Fighting
Blindness
Preserving vision through healthy (self)-eating
Thomas A. Ferguson. Ophthalmology and Visual Sciences,
Washington University, St. Louis, MO.
Presentation Description: Autophagy is a crucial cellular process
responsible for the continuous clearance of cellular components,
damaged proteins, and mitochondria by delivering these cargos to
the lysosomes for degradation. The metabolic roles of autophagy
can be classified into two types, basal and induced. In nutrientrich conditions, autophagy is suppressed but still occurs at low
levels (basal autophagy); however, when cells are subjected to
starvation or other stresses, autophagy is activated immediately
(induced autophagy). Constitutive autophagy functions as a cellrepair mechanism that is important for long-lived post-mitotic cells;
while induced autophagy maintains the cells amino acid pool to
adapt to starvation and provide energy needs. It has been suggested
that decreased autophagy may be one factor in the development of
age-related diseases; however, evidence for this idea is currently
incomplete. We are examining the role of autophagy in the eye
by deleting essential autophagy genes in specific ocular cell types
and have documented several critical functions for this process
in preserving visual function. In the retinal pigment epithelial
(RPE) autophagy links two critical processes, phagocytosis of
photoreceptors outer segments and the visual cycle, to recover
retinoids for the production of the visual chromophore 11-cisRAL. This process is termed LC3-associated phagocytosis (LAP)
and involves the recruitment of autophagy proteins to the single
membrane phagosome to promote cargo degradation and vitamin
A recovery. In rod photoreceptors deletion of essential autophagy
genes revealed that the process is critical to the long term health
and survival of these neurons. Without autophagy rods degenerate
leading to loss of vision and it was determined that autophagy is also
imporant for maintaining appropriate levels of the phototransduction
proteins such transducin. Deletion of essential autophagy genes
in cone photoreceptors also led to degeneration and revealed the
importance of this process to maintaining cone cell function. In the
absence of autophagy cones have a number of defects including
accumulation of damaged mitochondria, increased cell death in bright
light, sensitivity to starvation stress, and an inability to control their
rates of transducin activation and inactivation. Together our studies
demonstrate that basal and induced autophagy regulate processes that
are critical to maintaining healthy vision.
Commercial Relationships: Thomas A. Ferguson, None
Support: EY002687
413 Bacterial pathogenesis and infection
Wednesday, May 06, 2015 8:30 AM–10:15 AM
Program #/Board # Range: 4043–4078/A0220–A0255
Physiology/Pharmacology, Retina
Program Number: 4043 Poster Board Number: A0220
Pseudomonas aeruginosa adaptation to the ocular surface:
transcriptional changes and virulence determinants
Matteo M. Metruccio1, Yvonne Wu1, David J. Evans1, 2, Suzanne M.
Fleiszig1, 3. 1School of Optometry, University of California Berkeley,
Berkeley, CA; 2College of Pharmacy, Touro University California,
Vallejo, CA; 3Graduate Groups in Vision Science, Microbiology, and
Infectious Disease & Immunity, University of California, Berkeley,
Berkeley, CA.
Purpose: Pseudomonas aeruginosa is a leading cause of contact
lens-related corneal infection. Using a lens-wearing rodent model,
we previously showed that bacteria could infect the cornea more
efficiently if first pre-exposed to the ocular surface. This is consistent
with the increased risk of infection with extended lens wear in
humans. Here, we studied bacterial adaptions to the ocular surface
that subsequently enable them to penetrate the corneal epithelium.
Methods: RNA-seq was used to compare the transcriptional profile
of P. aeruginosa strain PAO1 exposed to human tear fluid for 5 h at
37 °C to PBS controls. Tn-seq was used to identify bacterial genes
required for traversal of human corneal epithelial cell multilayers in
vitro. For the latter, a pooled transposon mutant library of PAO1 was
generated, and ~106 cfu of bacterial mutants incubated with Transwell
filter-grown human telomerase-immortalized corneal epithelial cells
for 4 h at 37 °C. Transposon insertion sites were deep sequenced for
the input and traversed populations. HiSeq 2000 (Illumina) was used
for sequencing and data analysis performed with Galaxy and IGB.
Results: RNA-sequencing showed many P. aeruginosa genes
were deregulated (up- or down-regulated) by exposure to tear fluid
(~180 genes ≥ 8 fold). They included; the phoP/Q two-component
regulatory system (down-regulated), oprH for antimicrobial
resistance (down-regulated), and algF for biofilm formation and
antiphagocytic activity (up-regulated). Two small non-coding RNAs,
rsmZ and phrS, associated with virulence factor regulation, response
to oxygen availability and quorum sensing were also down- and
up-regulated respectively. Tn-seq identified ~200 gene insertions
differentially represented in traversed populations as compared to
the input (cut-off ≥ 18 fold), showing roles in epithelial traversal.
Insertions mapped in or near 150 genes belonging to numerous
important virulence categories, including pcrV (type three secretion),
motC (motility and attachment) and mexF (multidrug efflux pump).
Conclusions: Use of an unbiased global genetic approach to study
P. aeruginosa interaction with ocular surface components in vitro
identified genes and genomic regions involved in bacterial adaptation
to the host environment and ocular pathogenicity.
Commercial Relationships: Matteo M. Metruccio, None; Yvonne
Wu, None; David J. Evans, None; Suzanne M. Fleiszig, Allergan
Inc. (C)
Support: NIH EY024060
Molecular mechanisms of host-pathogen interactions in
pseudomonas aeruginosa microbial keratitis
Ahmad Elsahn1, 2, Maria del Mar Cendra-Gascon1, Pawez Hossain1,
2
, Myron Christodoulides1. 1Infection, Inflammation & Immunity,
University of Southampton, Southampton, United Kingdom;
2
University Hospitals Southampton, Southampton, United Kingdom.
Purpose: To examine the molecular mechanisms of interactions
between P. aeruginosa bacteria and primary human corneal fibroblasts
(hCF) in an invitro model of microbial keratitis
Methods: Human CF were extracted from clinical samples, cultured
to confluence in vitro and incubated with live PAO1 wild type
bacteria as well as mutant strains deficient in type IV pilus (∆pilA),
flagella (∆fliM) or double mutants (∆pilA∆fliM) for 3h. Bacterial
association was quantified and compared across the strains. Confluent
hCF were also pre-treated with SRC kinase inhibitors genstein (GST)
and PP2 and actin microfilament inhibitor cytochalasin D (CD),
and bacterial internalization was compared across pre-treated cells
at 3h using a gentamicin protection assay. Wild type (WT) PA14 as
well as mutant strains deficient in type III secretion system (T3SS)
needle apparatus (∆popB) and flagella (∆flgK) were incubated with
confluent hCF for 9h, and bacterial cytotoxicity was assessed by a
lactate dehydrogenase (LDH) assay.
Results: Mutant PAO1strains ∆pilA, ∆fliM and ∆pilA∆fliM adhered
significantly less than WT to hCF (P<0.05). Bacterial internalization
in hCF pre-treated with CD, GST and PP2 was significantly less
than untreated cells (P<0.05). Mutant PA14 strains ∆popB and ∆flgK
caused significantly less cytotoxicity to hCF than WT PA14 (P<0.05).
Conclusions: PAO1 bacteria use type IV pilus and flagella to adhere
to hCF. Bacterial internalization to hCF is dependent on the actin
microfilament and SRC kinase systems. Bacteria use flagella to
adhere to hCF and T3SS to induce cytotoxicity.
Commercial Relationships: Ahmad Elsahn, None; Maria del
Mar Cendra-Gascon, None; Pawez Hossain, None; Myron
Christodoulides, None
Correlation of P. aeruginosa Type III Secretion Profiles Diversity
and Ocular Disease Syndromes
Jorge Maestre, Edith Perez, Eduardo C. Alfonso, Harry W. Flynn,
Darlene Miller. Ophthalmology, University of Miami, Miami, FL.
Purpose: Pseudomonas aeruginosa associated ocular infections
are among the most diverse and difficult to manage. Clinical
presentations and pathology range from purulent conjunctivitis and
ulcerative keratitis to invasive scleritis and persistent, destructive
biomaterial centered infections. Virulence, disease course and patient
outcomes are strain specific. We used a combination of molecular,
metabolic and in vitro resistance markers to determine and correlate
Pseudomonas aeruginosa Type lll effector protein (T3SS) profiles
with biochemical patterns, ocular disease presentations and in vitro
susceptibility.
Methods: Type III Profiles: A multiplex PCR was used to
characterize and compare the prevalence of exoS (invasive), exoT
(invasive), exoU (cytotoxic) and exoY (adenyl cyclase) (genotypes
for 155 Pseudomonas aeruginosa strains collected from ocular
sources (cornea-n=96, contact lens-n=20, conjunctiva-n=23, lacrimal
system-n=8, Intraocular fluids-n=8) from 2008 to 2014. Biochemicals
and vitro susceptibility profiles were determined using the Vitek 2
system.
Results: Exo Y and T effector proteins were detected in all 155
isolates, while exoS ( N=70) and exoU (N=63) were generally
mutually exclusive. Prevalence of the four identified genotypes
were exoS+U- (45.2%), exoS-U+ (40.6%), exoS+U+ (7%) and exoU-S(9.7%). ExoU (cytotoxic strain) was the predominant profile of
isolates recovered from cornea (46.9%) and contact lens cases (70%)
but was evident in all sources. ExoS genotypes were documents
in 50% or higher for isolates recovered from conjunctiva (56.5%),
intraocular fluids (50%) and lacrimal system (50%). Strains with
absence of T3SS effectors were seen most often in isolates associated
with conjunctivitis (34.8%).
No significant correlation of metabolic profiles (N=100) were
associated with these isolates. Strains with genotype exoS-U+ had
the higher MIC90 (1 ug/ml) for ciprofloxacin vs 0.5 ug/ml or less
for exoS+U- and exoU-S- . Ciprofloxacin resistance ranged from 1%
(cornea) to 6% (conjunctiva). MIC90 for moxifloxacin was 2 ug/
ml and ranged from 3% (cornea) to 8% (conjunctiva and lacrimal
system).
Conclusions: Ocular Pseudomonas aeruginosa strains are diverse
and associated with specific disease syndromes and antibiotic
profiles. Characterizing the T3SS profiles may serve as an important
adjunct in understanding the pathology and management of these
destructive and recalcitrant infections
Commercial Relationships: Jorge Maestre, None; Edith Perez,
None; Eduardo C. Alfonso, None; Harry W. Flynn, None; Darlene
Miller, None
Support: Core Grant Department Ophthalmology Bascon Palmer
Eye Institute
Photodynamic therapy to treat Methicillin-Resistant
Staphylococcus Aureus (MRSA) keratitis: An in vitro study
Heather A. Durkee1, Francisco Halili1, Mukesh Taneja2, Darlene
Miller3, 4, Alejandro Arboleda1, Cornelis J. Rowaan1, Mariela C.
Aguilar1, Guillermo Amescua4, Harry W. Flynn4, Jean-Marie A.
Parel1, 5. 1Ophthalmic Biophysics Center, Bascom Palmer Eye
Institute, University of Miami Miller School of Medicine, Miami, FL;
2
LV Prasad Eye Institute, Hyderabad, India; 3Ocular Microbiology
Laboratory, Bascom Palmer Eye Institute, University of Miami Miller
School of Medicine, Miami, FL; 4Anne Bates Leach Eye Hospital,
Bascom Palmer Eye Institute, University of Miami Miller School
of Medicine, Miami, FL; 5Brien Holden Vision Institute, UNSW,
Sydney, NSW, Australia.
Purpose: To assess the in vitro efficacy of rose bengal (RB) and
riboflavin (Ribo) mediated photodynamic therapy (PDT) for the
inhibition of a methicillin-resistant Staphylococcus aureus (MRSA)
strain.
Methods: A MRSA type II strain was isolated from the corneal
scraping of a patient with confirmed bacterial keratitis. Twentyfour hours prior to experimentation, a parent culture was plated on
nutrient agar. Next, a culture of MRSA was transferred into tryptic
soy broth and adjusted to a concentration of 1.5x108 colony forming
units per mL (cfu/mL). The MRSA suspension was diluted to a
concentration of 1.5x107cfu/mL with the appropriate solution and
1mL aliquots were inoculated in triplicate onto nutrient agar plates.
The eight groups were: (1) Control (high purity water) (2) UV-A
irradiation (3) 0.1% Ribo (4) 0.1% Ribo + UV-A (5) 0.1% RB (6)
0.1% RB + 518nm light (7) 0.03% RB (8) 0.03% RB + 518nm light.
All experiments were performed in minimal lighting conditions (4
lux) except for irradiation test plates. The UV-A and green lights
are custom built LED sources. The UV-A light, activate Ribo,
has a central wavelength of 375nm and an irradiance of 2.91mW/
cm2 over a 13.8cm2 surface. The 518nm light, activate RB, has a
central wavelength of 518nm and an irradiance of 2.2mW/cm2 over
a 28.3cm2 surface. Plates were either exposed to UV-A (Ribo) or
518nm (RB) irradiation for 20 minutes. All plates were immediately
placed upside down, wrapped in foil, and placed in an incubator at
30°C. Plates were photographed every 24 hours for 6 days.
Results: Riboflavin without irradiation did not inhibit MSRA growth;
however in Ribo with irradiation it did inhibit MRSA growth. 0.1%
RB with and without irradiation inhibited the growth of the MRSA.
0.03% RB inhibited MRSA growth with irradiation but did not
inhibit MRSA growth in the non-irradiated group. Inhibition in all
photosensitizer groups occurred as soon as 24 hours after irradiation.
Conclusions: Rose Bengal strips of 1.0% concentration are clinically
used to detect epithelial defects. Our study demonstrates MRSA
can be inhibited with 0.1% RB without irradiation. The RB will be
activated even when the patient is exposed to ambient light levels
in daily activities. PDT could be an excellent adjunct treatment for
MRSA keratitis as it provides a different mechanism of inhibition.
Results of 0.1% Ribo and 0.1% RB
when the bacteria were planktonic. However, the PMNs were
concomitantly sluggish and not successful in phagocytosis when the
bacteria developed into biofilms at later times of infection. The early
role of PMN NETs as basic host defence may perhaps be considered
for novel drug targets to prevent biofilm formation and to augment
bacteria killing
Commercial Relationships: Padmanabhan Saraswathi, None;
Roger Beuerman, None
Results of 0.03% Rose Bengal PDT at 72 hours
Commercial Relationships: Heather A. Durkee, None; Francisco
Halili, None; Mukesh Taneja, None; Darlene Miller, None;
Alejandro Arboleda, None; Cornelis J. Rowaan, None; Mariela C.
Aguilar, None; Guillermo Amescua, None; Harry W. Flynn, None;
Jean-Marie A. Parel, None
Support: Florida Lions Eye Bank, Drs KR Olsen and ME
Hildebrandt, NIH P30EY1481 (Center Grant), Research to Prevent
Blindness, Henri and Flore Lesieur Foundation (JMP). Technical
support was provided by: Karam Alawa, Victor Hernandez, and Nidhi
Relhan Batra.
Interaction of Polymorphonuclear neutrophils (PMNs) with
Pseudomonas aeruginosa in biofilms in corneal infections in the
mouse
Padmanabhan Saraswathi1, Roger Beuerman1, 2. 1SERI, Singapore
Eye Research Inst (SERI), Singapore, Singapore; 2Department of
Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore,
Singapore.
Purpose: Pseudomonas aeruginosa, is a common ocular pathogen
found in cornea infections, often associated with contact lens
wear. The survival of this bacteria as a “Biofilm” within a
extrapolysaccharide substances sheltered from innate immune
defence and antimicrobials. In this study, the interaction of
polymorphonuclear neutrophils (PMNs) with Pseudomonas in
planktonic and biofilm models was examined using a mouse model of
experimental keratitis infection
Methods: A suspension (108 CFU/ml) of Pseudomonas aeruginosa
(ATTC 9027) was used to infect the mouse cornea (C57BL/6).
The infection was monitored by slit lamp. Infected eyes were
enucleated at post infection (PI) day1 to describe the planktonic
status and PI day3 and 5 for potential biofilm status. The response
of polymorphonuclear neutrophils (PMNs) was characterised
using histological, ultra-structural imaging and quantifying
myeloperoxidase levels
Results: Histology showed the expected large numbers of PMNs in
the stroma at PI day1, with diverse morphologies. At later stages of
infection they were seen on the corneal surface, but more spherical
shape. Scanning Electron Microscopy (SEM) demonstrated the
release of Neutrophil Extracellular Traps (NETs) as long fibrils and
entangling many bacteria which were seen as free cells at the early
stage of infection. However, at PI day3 and 5 the PMNs were seen
to change their morphology and lacking pseudopodia appeared
immobile, surrounding the clusters of Pseudomonas within a biofilm
while other PMNs were on the corneal surface. Transmission Electron
Microscopy (TEM) further confirmed the active phagocytosis at PI
day1 and immotile PMNs settled beneath the layer of the biofilm at
later stages of infection. Enhanced myeloperoxidase activity from 4.1
units at day1 to 16.9 units at day5 PI indicated the increased PMN
activity in the course of the infection
Conclusions: The murine neutrophils were seen to be highly
activated during an infection and produce NETs as an early defence
Interactions of tear-film neutrophils with clinical bacteria
Maud Gorbet1, 2, Mark D. Willcox2. 1Systems Design Engineering,
University of Waterloo, Waterloo, ON, Canada; 2School of Optometry
and Vision Science, University of New South Wales, Sydney, NSW,
Australia.
Purpose: During sleep, in the closed-eye environment, a shift in tear
film composition leads to the recruitment of leukocytes to the ocular
surface. Neutrophils (also known as polymorphonuclear leukocytes;
PMN) are considered our first line of defense and play an essential
role in preventing infection. Following chemical stimulus, a lack of
upregulation of cell activation markers has previously been observed
on tear film neutrophils (TF-PMN). This pilot study was conducted
to investigate the response of TF-PMN to clinical bacteria and assess
whether overnight lens wear effected on their response to bacteria.
Methods: Acuvue Oasys lens wearers and non-lens wearers
were recruited to collect their cells upon awakening using an eye
wash “at-home collection kit”. Collected cells were counted and
resuspended in tubes containing either ATS-2% NHS (artificial tear
solution supplemented with 2% of normal human serum) or PBS10% Heat Inactivated (HI) FBS. Collected cells were incubated
with Pseudomona aeruginosa 6294 (an invasive clinical strain) and
P. aeruginosa 6206 (a cytotoxic clinical strain) at bacteria to PMN
ratios of 10:1 and 1:10. Following incubation, samples were serially
diluted in PBS, plated on agar and incubated overnight at 35oC. The
next day, bacteria colonies were counted. To further characterize the
response to bacteria, TF-PMN were analysed by flow cytometry for
receptor upregulation and oxidative burst.
Results: With a bacteria:TF-PMN ratio of 10:1 in ATS-2% NHS,
no reduction in bacteria growth was observed for 6206 and 6294.
Fewer 6294 CFU were counted when interactions with TF-PMN
occurred in PBS-HIFBS. Furthermore, regardless of incubation
medium, bacteria:TF-PMN interactions at a ratio of 1:10 resulted in
a significant increase in the number of 6206 cells (p<0.05). For both
6206 and 6294, interactions at the 1:10 ratio with TF-PMN collected
following overnight lens wear led to a significant increase in CFU
when compared to the 10:1 ratio (p<0.04). Flow cytometry results
confirmed the differential TF-PMN response to 6206 and 6294.
Conclusions: Our results suggest that at low bacteria to PMN ratio,
a condition that may closely mimic the closed-eye environment,
TF-PMN are unable to phagocytose clinical strains of Pseudomona
aeruginosa and may be releasing factors that promote bacterial
survival/growth. This may have an important impact during overnight
lens wear and may contribute to the higher risks of microbial
keratitis.
Commercial Relationships: Maud Gorbet, None; Mark D.
Willcox, None
Support: American Optometric Foundation VISTAKON® Research
Grant
Pseudomonas aeruginosa Ocular Strains Resistant to Ceftazidime
in Northeast of Mexico
Pedro M. González, Alejandro F. Ibarra-Lozano, Jorge E. Valdez,
Jaime Torres. Ophthalmology, Instituto Tecnológico de Monterrey,
San Pedro Garza García, Mexico.
Purpose: The purpose of this case series is to report Pseudomonas
aeruginosa resistance to ceftazidime in keratitis isolates in northeast
of Mexico. Concern exists around the growing antibiotic resistance
in different ocular pathogens worldwide. Ceftazidime is known to be
one of the antimicrobial agents used with a high sensitivity profile in
bacterial keratitis caused by Pseudomonas aeruginosa.
Methods: Patient records from January 2010 to November of 2014
were revised. Twenty eight patients were found with the diagnosis of
infectious keratitis. Eight cases were culture positive for Pseudomona
aeruginosa. variables studied included: gender, age, time of
presentation to consultation, visual acuity, comorbidities, antibiotic
sensitivity/resistance pattern and complications encountered.
Results: Eight culture positive P. aeruginosa cases were found
between 2010 to the end of 2014. There were 2 females and 6 males,
the age range was from 19 to 67 years old. Ceftazidime resistance
was reported in seven cases. Susceptibility was reported for
ciprofloxacin, meropenem, amikacin and gentamicin. Ciprofloxaxin
and meropenem were the ones with the lowest minimal inhibitory
concentration. Final visual acuity had a strong relation with the
initially reported. Two cases were associated to traumatism and one
to soft contact lenses use. Two cases had corneal perforation.
Conclusions: This study reviews a series of cases of P. aeruginosa
keratitis in northeast of Mexico; the microbiologic profile showed
87.5% resistance to ceftazidime. Sensitivity to meropenem,
ciprofloxacin, amikacin and gentamicin was of 100%. This data
should influence the management of this entity in the geographic
zone examined.
Commercial Relationships: Pedro M. González, None; Alejandro
F. Ibarra-Lozano, None; Jorge E. Valdez, None; Jaime Torres,
None
Corneal epithelial cells suppress vacuolar escape of P. aeruginosa
Abby Kroken, David J. Evans, Suzanne M. Fleiszig. School of
Optometry, University of California, Berkeley, Berkeley, CA.
Purpose: Previously we reported that invasive strains of P.
aeruginosa invade corneal epithelial cells wherein they replicate
and induce formation of plasma membrane blebs to which they
traffic. These events depend on the bacterial Type Three Secretion
System (T3SS), specifically the T3SS toxin ExoS. Other investigators
have studied ExoS using HeLa cells (not normally targeted by P.
aeruginosa) and PA103 (does not natively express ExoS), and have
not noted these phenomena. Here, we tested the hypothesis that the
impact of ExoS depends on bacterial strain and cell type.
Methods: Epithelial cell lines (HeLa or corneal) were infected with
P. aeruginosa strain PAO1 (natively expresses ExoS) or PA103
(engineered to express ExoS). Intracellular bacteria were quantified
using a gentamicin protection assay. A T3SS-driven GFP reporter was
used to monitor T3SS expression in individual bacteria. Images were
captured using time lapse wide-field or confocal microscopy.
Results: In both cell types, PAO1 and PA103 expressing ExoS
induced membrane blebbing, but only PAO1 invaded, replicated
intracellularly, or trafficked to blebs. In contrast to corneal cells,
HeLa cells even supported intracellular replication of PAO1
mutants lacking all known T3SS toxins (including ExoS). That
replication remained dependent on the T3SS, which was expressed
intracellularly, and occurred in the cytoplasm, without blebs, and in
cells possessing acidified vacuoles - all differentiating it from ExoSdependent replication in corneal cells. T3SS machinery mutants
remained in vacuoles in HeLa cells, showing that vacuolar escape is
mediated by a T3SS component or unknown effector, but not ExoS.
This contrasts with ExoS-dependent vacuolar escape in corneal cells.
Conclusions: ExoS has been mostly studied using PA103 and HeLa
cells. Our data show that neither accurately model how natively
encoded ExoS influences P. aeruginosa interactions with corneal
epithelial cells. This explains why its role in intracellular survival was
overlooked by investigators using those tools. Our data show that;
1) corneal epithelial cells have innate defenses against internalized
bacteria that are lacking in HeLa cells and which ExoS can overcome,
and 2) unknown T3SS factors allow intracellular replication in cells
lacking those innate defenses. Identifying mechanisms involved
could lead to novel strategies to combat infection.
Commercial Relationships: Abby Kroken, None; David J. Evans,
None; Suzanne M. Fleiszig, Allergan (C)
Support: NIH Grant AI079192
Pseudomonas aeruginosa induces autophagy in human corneal
epithelial cells
VIDYARANI MOHANKUMAR1, LakshmiPriya Jeganathan1, Lalitha
Prajna1, Chidambaranathan Gowri Priya2. 1Ocular Microbiology,
Aravind Medical Research Foundation, Madurai, India; 2Immunology
and Stem Cell Biology, Aravind Medical Research Foundation,
Madurai, India.
Purpose: Keratitis caused by Pseudomonas aeruginosa is a
serious ocular infection which may lead to corneal perforation if
the intracellular bacteria are not cleared completely. Autophagy, a
normal catabolic process, has been shown to play a major role in
the clearance of intracellular pathogens. We propose that autophagy
induced by P. aeruginosa in human corneal epithelial cells (HCET),
may have a role in the clearance of intracellular bacteria.
Methods: HCET cells transfected with LC3-GFP plasmid were
infected with three different ocular isolates of P. aeruginosa and
autophagy was monitored after 1h using a Leica TCS SP8 confocal
microscope. EBSS treated (amino acid starvation) HCET cells were
used as positive control for autophagy. Another set of HCET cells
were infected with P. aeruginosa to study the mRNA expression of
autophagy related protein, beclin1 by real time PCR. To study the
intracellular survival and replication of the bacteria, HCET cells were
infected with P. aeruginosa in the presence of EBSS or 3 –methyl
adenine (3mM in EBSS), an inhibitor of autophagosome formation.
After 3h, the extracellular bacteria were killed with gentamicin
and the cells were incubated for another three hours to allow for
intracellular bacterial replication. Diluted cell lysates were plated on
to MacConkey agar, and the colonies were counted after an overnight
incubation.
Results: The HCET cells upon infection with three different P.
aeruginosa isolates showed an increased LC3 punctation, which is
a classical marker for autophagosome formation. The total number
of LC3 positive cells and the relative number of LC3-puncta per cell
varied upon infection with the different isolates which may be due to
the difference in the intracellular bacterial load. Correspondingly, the
mRNA expression of beclin1 was higher in P. aeruginosa infected
cells compared to uninfected controls.The ocular isolates were able
to efficiently invade and replicate inside the epithelial cells and the
bacterial load was relatively higher when the cells were pretreated
with 3-methyl adenine.
Conclusions: Altogether, the results suggest that P. aeruginosa
induces autophagy in human corneal epithelial cells, which inturn may limit the intracellular bacterial load. Since the ocular P.
aeruginosa isolates can efficiently invade and replicate inside the
epithelial cells, autophagy may represent a host defensive mechanism
to curtail infection in P. aeruginosa keratitis.
Commercial Relationships: VIDYARANI MOHANKUMAR,
None; LakshmiPriya Jeganathan, None; Lalitha Prajna, None;
Chidambaranathan Gowri Priya, None
Role of IL-24 in Pseudomonas Aeruginosa Keratitis in a C57BL/6
Mouse Model
Bing Xu, Nan Gao, Fushin X. Yu. Anatomy & Cell Biology and
Ophthalmology, Wayne State University School of Medicine, Detroit,
MI.
Purpose: The cytokines in interleukin (IL)-10 family are known
by their anti-infection and anti-inflammatory activities. However,
the biology of IL-24, a member of IL-10 family cytokines, is
largely unknown, particularly in the ocular tissue. In this study, we
investigated the role of IL-24 in a mouse Pseudomonas Aeruginosa
(PA) keratitis model.
Methods: Epithelium-injured B6 mouse corneas were pretreated
with or without flagellin for 24h, followed by PA (ATCC 19660)
inoculation. Samples were collected at various time points post
infection and subjected to PCR and immunofluorescence. Mice were
subconjunctivally injected with either IL-24 siRNA to knock down
IL-24 expression or mouse IL-24 recombinant protein before the
inoculation of PA to explore the biological functions of IL-24.
Results: PA infection induced IL-24 transcription on corneal
epithelial cells; flagellin pretreatment not only alleviated the
infection, but also reduced the mRNA expression of IL-24, but not
IL-19 and IL-20, the other two members of IL-10 family that share
common receptors. STAT3, a signal mediator in IL-24 pathway,
was phosphorated in response to PA infection, and p-STAT3 was
mainly expressed in the infiltrated cells, indicating that immune cells
were recruited and activated by the infection. IL-24 downregulation
alleviated the severity of PA keratitis, and the application of IL-24
recombinant protein exacerbated PA infection on mouse cornea.
Conclusions: These data demonstrate that IL-24 exhibits a
detrimental effect on the host defense against PA infection, suggesting
that IL-24 pathway may be a new intervention site for treating
infectious keratitis.
Commercial Relationships: Bing Xu, None; Nan Gao, None;
Fushin X. Yu, None
Support: NIH/NEI EY017960
Thrombomodulin protects against bacterial keratitis and is not
angiogenic
Linda D. Hazlett, Sharon A. McClellan, Cui Li. Anatomy & Cell
Biology, Wayne State Univ Sch of Med, Detroit, MI.
Purpose: Thrombomodulin (TMB), a cell surface glycoprotein
composed of 5 domains, is expressed in a variety of cells. The
N-terminal lectin-like domain (TMD1) interacts with Lewis Y
antigen to inhibit angiogenesis and is responsible for TMB’s antiinflammatory properties; it also binds high mobility group box-1
(HMGB1), preventing its deleterious effects, while domains 2 and
3 (TMD23) induce neovascularization, with response regression
within 24 days. Despite data regarding activities of specific domains
of TMB, nothing has been reported regarding testing its protective
effects in experimental microbial keratitis, and whether it induces
corneal vascularity which is the purpose of this study.
Methods: C57BL/6 mice were injected (subconjunctivally and i.p.)
with recombinant (r)TMB and infected with P. aeruginosa. PBS
controls were similarly treated. Clinical score, photography with a slit
lamp, real time RT-PCR, MPO assay, and ELISA were used to assess
the disease response.
Results: Data show that treatment of C57BL/6 mice with rTMB
reduced clinical disease scores, corneal opacity and the neutrophil
infiltrate. mRNA levels for IL-1β, MIP-2, TLR4, and RAGE were
decreased, while anti-inflammatory molecules, including SIGIRR
and ST2 levels were increased when compared with controls. ELISA
confirmed the PCR data showing significant reduction of both IL-1β
and MIP-2 protein level (3 and 5 days post infection) after rTMB
treatment. VEGF, VEGF-R1 and VEGF-R2 were no different, or
reduced after TMB (5 days p.i.).
Conclusions: TMB is protective in keratitis, reducing disease
severity, and with no angiogenic effect .
Commercial Relationships: Linda D. Hazlett, None; Sharon A.
McClellan, None; Cui Li, None
Support: NIH Grants EY016058 and P30EY04068
Conjunctival chemosis as a specific feature of Pseudomonas
aeruginosa corneal ulcers
Kaleena B. Michael. Tennents institute of ophthalmology, Glasgow,
United Kingdom.
Purpose: Corneal ulcer is a common ocular problem often
complicated by delayed treatment from late diagnosis. We looked
for presence of conjunctival chemosis in 44 consecutive cases of
confirmed infective corneal ulcers.
Methods: Retrospective examination of early ocular photographs of
44 consecutive cases of infective corneal ulcers.
Results: Conjunctival chemosis was observed in 13 out of 44 cases.
12 of these were culture positive for pseudomonas aeruginosa, and 1
for colliform bacilli. Statistical analysis with Fishers Exact test was
significant p>0.000001, Odds Ratio 112 (95% CI: 10.6 - 1190).
Conclusions: Our findings suggest a strong association between
conjunctival chemosis in pseudomonas aeruginosa corneal ulcers.
This ocular feature could potentially help predict the presence of of
pseudomonas aeruginosa in new corneal ulcers. This would enable
individuals to receive the treatment of choice at an earlier stage,
before microbiological confirmation.
Commercial Relationships: Kaleena B. Michael, None
Microbiota and P. aeruginosa Genotype adhesion difference
on Worn Cosmetic Contact Lenses (CL) with Comparison of
Disinfectant Sensitivity
Elizabeth Shen1, Fung Rong Hu2. 1Ophthalmology, Taipei Tzu
Chi Hospital, Taipei, Taiwan; 2Ophthalmology, National Taiwan
University Hospital, Taipei, Taiwan.
Purpose: To analyze attached microbes found on worn cosmetic
CL materials and compare difference in adhesion of two Type III
secretion genotypes of P. aeruginosa. Disinfection ability of four
disinfectants is compared between two genotypes.
Methods: Healthy volunteers with myopia < -6 D and astigmatism
< -1.0 D with signed informed consent were recruited in this
prospective randomized study. Subjects wore nelfilcon (Alcon
Freshlook Colorblends gray), hilafilcon (Bausch & Lomb Naturelle
black),or etafilcon (Johnson & Johnson Acuvue 2 Define Accent style
black) daily disposable CL for at least 8 hours/day for 2 weeks. The
lenses are collected aseptically. Half of the lens is used to identify
attached microbes. The other half is incubated for 2 hrs with108cfu/
ml of cytotoxic PA103 or invasive PAK strains. Viable cell culturing
was done to determine number of bacteria attached. P. aeruginosa
incubated lenses were soaked with various disinfectants (Renu,
Aosept, Puremoist, and Replenish) at 25%, 50%, 75%, and 100% of
the suggested disinfection time. The number of bacteria remaining on
CL is counted and compared.
Results: The most commonly identified microbes attached to nonsymptomatic volunteers were coagulase negative Staphylococcus,
P. aeruginosa, and Streptococci. Adhesion of the cytotoxic strain
PA103 was found to be greatest for hilfilcon lenses (34.8x104cfu/
ml) > etafilcon (33.1 x104cfu/ml) > nelfilcon (15.5x104cfu/ml)
(P<0.05; ANOVA). Similar trend is noted for adhesion of the invasive
strain PAK: hilfilcon (30.9x104 cfu/ml)>etafilcon(37.5x104 cfu/ml)
>nelfilcon(10.1x104 cfu/ml) (P<0.05; ANOVA). At 100% suggested
time, all tested disinfectants were able to kill all bacteria. However,
at less than 75% of suggested disinfection time, significant number of
viable cytotoxic P. aeruginosa remained (P=0.03, t-test).
Conclusions: With increasing popularity of cosmetic CL, clinicians
are should be aware that different cosmetic materials attract different
microbial adhesions. P. aeruginosa, a commonly identified pathogen
in CL-associated microbial keratitis, is frequently found attached
to CL of asymptomatic individuals. Cytotoxic strains are more
resistant to MPS solution especially Renu. Proper lens care with
strict adherence to suggested disinfection time is strongly advised to
prevent sight threatening infections related to cosmetic lens wear.
Commercial Relationships: Elizabeth Shen, None; Fung Rong
Hu, None
Support: NSC-102-2628-B-002-051-MY3
Effect of the interaction Fusarium solani-Staphylococcus aureus
over limbocorneal fibroblasts
Antonio Bautista-Hernandez1, 2, Beatriz Buentello-Volante3, Victor
M. Bautista1, José Luis Gómez Olivares2. 1Microbiology and
Ocular Proteomics, Inst of Ophthalmology “Conde de Valenciana”,
Mexico City, Mexico; 2Department of Health Sciences, Autonomous
Metropolitan University., Mexico City, Mexico; 3Genetics, Inst of
Ophthalmology “Conde de Valenciana”, Mexico City, Mexico.
Purpose: To study the effect of the interaction Fusarium solaniStaphylococcus aureus over limbocorneal fibroblasts.
Methods: The limborcorneal fibroblasts were obtained from
limbocorneal tissue and grown in media DMEM-F12 supplemented
with serum fetal bovine 10%, were evaluated expression of vimentin
and cytokeratin. F. solani and S. aureus were isolated from human
corneal ulcers. The microorganisms were identified by classical
microbiology. The biofilm formation was evaluated in media
DMEM-F12 by the Christensen method and fluorescence microscopy.
Fibroblasts were exposed to S. aureus and/or F. solani to evaluate the
expression of CD34 and production of INFγ.
Results: According to classical microbiology studies, bacterial strain
corresponded to S. aureus and fungal strain to F. solani. Fluorescence
microscopy showed a formed biofilm by the interactions between
S. aureus and F. solani, that generated a extracellular matrix. The
limbocorneal fibroblasts had characteristic morphology, vimentin
positive and cytokeratin negative. The expression of CD34 decreased
and increased content of INFγ with stimuli of S. aureus, F. solani and
S. aureus-F. solani.
Conclusions: Microorganism S. aureus and F. solani showed ability
to form biofilm. The limbocorneal fibroblasts were vimentin positive
and cytokeratin negative. Expression of CD34 was decreased and
increased INFγ production with stimuli of S. aureus, F. solani and S.
aureus-F. solani.
Commercial Relationships: Antonio Bautista-Hernandez, None;
Beatriz Buentello-Volante, None; Victor M. Bautista, None; José
Luis Gómez Olivares, None
Support: This work was supported by “Conde de Valenciana”
Foundation
Involvement of endoplasmic reticulum (ER) stress in the
pathogenesis of bacterial endophthalmitis
Ajay Kumar1, Pawan Kumar Singh1, Ashok Kumar1, 2.
1
Ophthalmology, Wayne State University School of Medicine,
Detroit, MI; 2Anatomy and Cell Biology, Wayne State University
School of Medicine, Detroit, MI.
Purpose: Endoplasmic Reticulum (ER), through the unfolded protein
response (UPR), regulates various cellular functions, including
inflammation. Here, we have investigated the role of UPR signaling,
specifically IRE1α in regulating intraocular inflammation in an
experimental model of Staphylococcus aureus (SA) endophthalmitis
Methods: Endophthalmitis was induced by intravitreal injection of S.
aureus in WT (C57BL/6), TLR2-/-, and MyD88-/- mice. After 24 hours
of infection, retinal tissue was collected; RNA was extracted, and
RT-PCR was performed to assess ER stress marker genes (XBP-1s,
IRE1α, PD1, CHOP, WSF, BiP, and ERDj4) using specific primers.
To determine the role of IRE1α, inhibition studies were performed
using IRE1α specific inhibitor, 4μ8C, given prior to S. aureus
challenge. Secretion of inflammatory mediators and the activation of
TLR-downstream signaling pathways (JNK1/2, MAPK, and NF-kB)
were assessed using ELISAs and western blot analyses respectively.
Mechanistic studies were performed using cultured BV2 microglial
cells.
Results: S. aureus did not induce the classical UPR response as
evidenced by upregulation of ER stress markers (PD1, CHOP,
WSF, BiP, and ERDj4) in the infected retina. However, S. aureus
caused the splicing of XBP1 in both the mouse retina and the BV2
microglia. Concomitant with increased XBP-1s levels, the level of
IRE1α was also increased in infected cells/tissue. The IRE1 inhibitor
(4μ8C) reduced the levels of both XBP-1s and IRE1α in WT mice
and microglia, but not in TLR2-/- and MyD88-/- mice. Similarly, the
expression and secretion of inflammatory cytokines were attenuated
in 4μ8C-treated WT mice and microglia cells, while no reduction
was observed in TLR2-/- and MyD88-/- mice. Furthermore, S. aureusinduced the activation of ERK1/2, p38 MAPK, and NF-kB signaling
was down regulated by the IRE1α inhibition.
Conclusions: Taken together, our study, for the first time, provides
evidence of IRE1α-mediated UPR stress signaling in generating
retinal innate responses in bacterial endophthalmitis. Furthermore,
these findings suggest a potential cross-talk between TLR and UPRsignaling in orchestrating inflammatory responses in the retina.
Commercial Relationships: Ajay Kumar, None; Pawan Kumar
Singh, None; Ashok Kumar, None
Bacterial Impediment of Corneal Cell Migration
Kimberly Brothers, Nicholas A. Stella, Kristin M. Hunt, Regis
P. Kowalski, Jes Klarlund, Robert M. Shanks. Ophthalmology,
University of Pittsburgh, Pittsburgh, PA.
Purpose: The loss of corneal epithelium in patients with microbial
keratitis is well known but the underlying mechanisms are poorly
understood. This study set out to determine the impact of bacterial
secreted factors on ocular wound healing.
Methods: Bacterial secretomes from ocular keratitis isolates were
prepared from overnight cultures and filtered to remove bacteria.
Using a plate based cell migration assay, secretomes were added
to stratified human corneal limbal epithelial (HCLE) cells and
incubated. Calcein AM viability stained cell layers were imaged
by confocal microscopy. Porcine corneal organ culture was used
to assess epithelial wound healing phenotypes ex vivo. Transposon
mutagenesis of the Serratia marcescens genome was conducted
to identify bacterial genes responsible for corneal cell migration
inhibition. LPS was purified by the hot phenol method. The waaG
lipopolysaccharide (LPS) gene was cloned using yeast homologous
recombination into vector pMQ131.
Results: Corneal cells treated with bacterial secretomes from
4/5 Pseudomonas aeruginosa, 26/27 S. marcescens, and 2/14
Staphylococcus aureus isolates showed dose dependent inhibition
of HCLE migration. No inhibition was seen with secretomes
from 4 other ocular pathogens. Using an ex vivo porcine corneal
wound model, we have recapitulated S. marcescens inhibition of
wound healing. A transposon mutation in the S. marcescens LPS
biosynthetic locus that prevented bacterial inhibition of epithelial
cell migration was identified and complemented with the wild-type
gene on a plasmid. Supporting the importance of LPS in the cell
migration phenotype, depletion of LPS with polymyxin B agarose
inactivated the inhibitory ability of the bacterial secretomes. Purified
S. marcescens LPS, but not E. coli LPS was able to inhibit corneal
cell migration.
Conclusions: Together these data support that multiple ocular
pathogens secrete factors able to inhibit corneal epithelial wound
healing. Genetic and biochemical data using S. marcescens as a
model organism indicate that S. marcescens LPS is sufficient to
prevent corneal epithelial cell migration and wound healing. This
study presents a novel host-pathogen interaction with implications
for corneal ulcers and other medical problems where bacteria impact
wound healing, such as chronic wounds and provides evidence that
LPS may be a key factor in the inhibitory mechanism.
Commercial Relationships: Kimberly Brothers, None; Nicholas
A. Stella, None; Kristin M. Hunt, None; Regis P. Kowalski, None;
Jes Klarlund, None; Robert M. Shanks, None
Support: NIH 2T32 EY017271-06A1 NIH EY08098 NIH AI085570
Toxicity and Virulence of Viridans Group Streptococci Isolated
from Endophthalmitis
Mary E. Marquart, Hannah R. Rice. Microbiology, Univ of
Mississippi Med Ctr, Jackson, MS.
Purpose: Endophthalmitis due to viridans group streptococci (VGS)
has been shown to result in poor visual prognosis despite antibiotic
therapy. We hypothesized that VGS possess toxins that are involved
in ocular pathogenesis. The purpose of this study was to identify the
virulence characteristics of endophthalmitis strains of VGS.
Methods: Concentrated extracellular milieu from 20 endophthalmitis
strains of VGS, and concentrated culture media (controls), were
subjected to 3 assays: lysis of sheep erythrocytes, retinal pigmented
epithelial (RPE) cell toxicity, and Western blot for detection of
pneumolysin (the major toxin of S. pneumoniae). Each strain was
assigned a profile based on assay results. Four strains with different
profiles were chosen for intravitreous injection of 100 colonyforming units (CFU) of each strain into the left eyes of each of 3
rabbits, followed by clinical examination and bacterial recovery from
the vitreous. The concentrated extracellular milieu from each of the 4
strains was also tested for protease activity by gelatin zymography.
Results: Eight of 20 strains (40%) lysed sheep erythrocytes and 15
(75%) were cytotoxic at a level of ≥2 on a scale from 0 (no activity)
to 4 (full activity). Pneumolysin was detected in 6 (30%). Two of
the 4 strains tested in vivo caused anterior chamber inflammation in
addition to vitreous haze, with severity increasing with time up to 72
hours after infection. The most severe eyes, which were infected with
strain E664, had clinical scores with a mean of 25.33 ± 2.08 (scale
of 0 to 32) and bacterial recovery with a mean of 6.95 ± 0.53 log10
CFU/mL at 72 hours. Interestingly, E664 was not hemolytic nor did it
produce pneumolysin, but was highly toxic to RPE cells. The secondmost virulent strain in vivo, 144065, was negative for all of the in
vitro assays. The least virulent strain in vivo, E618, was positive for
all of the in vitro assays. Zymography of the 4 strains tested in vivo
showed protease activity in E664 and 144065, but not in the other 2
strains.
Conclusions: VGS from endophthalmitis are diverse in toxin activity,
and toxin activity cannot necessarily predict virulence in the rabbit
eye. Strains with protease activity induce more damage in the rabbit
eye than those without detectable protease. Determination of whether
protease activity contributes to pathogenesis will aid in the first steps
of characterizing the virulence of VGS in the eye.
Commercial Relationships: Mary E. Marquart, None; Hannah R.
Rice, None
Measuring Severe Inflammatory Trachoma (TI) when prevalence
is low provides indirect data on infection with C. trachomatis in
endemic communities
Andrea I. Zambrano2, Beatriz E. Munoz1, Laura Dize2, Harran
Mkocha4, Charlotte Gaydos3, Thomas Quinn2, 3, Sheila K. West1.
1
Ophthalmology, Wilmer Eye Inst Johns Hopkins Univ, New York,
NY; 2Infectious Disease, International Chlamydia Laboratory, Johns
Hopkins School of Medicine, Baltimore, MD; 3National Institute of
Allergy and Infectious Disease, Bethesda, MD; 4Kongwa Trachoma
Project, Kongwa, United Republic of Tanzania.
Purpose: Inflammatory trachoma (TI) is not measured when
assessing the impact of trachoma programs because it is felt to
indicate non-trachoma disease; only follicular trachoma (TF) is
measured. We tested the supposition that TI was not associated
with infection when disease prevalence is low, and does not add to
understanding the effect of programs.
Methods: In each of 52 communities in Kongwa Tanzania,
100 children ages 1-9 years were randomly selected for survey
at baseline, 6, and 12 months. In 17 communities, Mass Drug
Administration (MDA) was stopped at baseline because infection
rates were 1% or less; 35 had MDA. Both eyelids were graded for
evidence of TF and TI and a swab for detection of infection was
taken. The swabs were tested using Aptima (GenProbe/Hologic)
for presence of C. trachomatis. Overall prevalence rate of active
trachoma in communities with and without MDA at each time point
were compared, and the proportion of active trachoma cases that
were TI alone was estimated. Proportion of active trachoma cases
that were TI alone were compared among the treated and not treated
communities. All comparisons were analyzed using Fisher’s exact
test.
Results: Overall prevalence of active trachoma at baseline was 6%
(318 cases); 15% were TI alone. The prevalence of infection in TF
cases was 36% and 37% in TI alone. At 6 months, the prevalence of
active trachoma was 5.5% in communities where MDA was stopped;
18% of the cases were TI alone, for whom the rate of infection
was 41.2%. In treated communities, prevalence of active trachoma
was 3.9% and 10% of cases were TI alone for whom the rate of
infection was 30.8%. At 12 months, prevalence of active trachoma
in communities with MDA stopped was 5.8% and 17% of cases
were TI alone; 59% had infection with C. trachomatis. In treated
communities, prevalence rate was 5.1% with 22% of cases being TI
alone for whom the rate of infection was 37.5%. Infection in the TI
cases in communities where MDA was stopped was significantly
greater than in treated communities (p=0.02).
Conclusions: Despite low prevalence, the clinical sign of TI was
better correlated with infection than TF, and particularly so when the
survey was a year or more after MDA. TI should be measured as part
of impact surveys and particularly at surveys several years after the
last MDA.
Commercial Relationships: Andrea I. Zambrano, None; Beatriz
E. Munoz, None; Laura Dize, None; Harran Mkocha, None;
Charlotte Gaydos, None; Thomas Quinn, None; Sheila K. West,
None
Support: National Eye Institute EY022584
Evaluation of effectiveness of real-time PCR for bacterial
keratitis diagnosis
Daisuke Shimizu, Dai Miyazaki, Keiko Yakura, Tomoko Haruki,
Yoshitsugu Inoue. Ophthalmology and Visual Science, Tottori
University Faculty of Medicine, Yonago city, Japan.
Purpose: To determine the effectiveness of measurement of bacterial
DNA amount by real-time PCR for the diagnosis of bacterial
keratitis, we characterized the sensitivity and specificity profile and
determined cut-off value for diagnosis by using Receiver Operating
Characteristic (ROC) analysis.
Methods: Consecutive case series (241 eyes of 241 cases), suspected
of infectious keratitis and measured for the amount of bacterial
DNA in corneal tissue samples, were retrospectively analyzed. The
measurement of bacterial DNA amount (16S rDNA by real-time
PCR), smear examination by Gram and
Fungiflora staining, and culture testing were evaluated for diagnostic
efficacy by ROC analysis.
Results: One hundred and nine eyes were diagnosed as definitive
bacterial keratitis. Eighty four eyes were diagnosed as keratitis
of other causes. In the definitive bacterial keratitis eyes, average
bacterial DNA copy number was 1.6x106, culture positive rate was
54%, and the smear positive rate was 45%. In the non bacterial
keratitis eyes, they were 6.1x103, 0.0%, and 7.0%, respectively. Area
under the curve (AUC) was calculated to show diagnostic efficacy
based on ROC analysis of these testing. The AUC for definitive
bacterial keratitis was 0.73, 0.65, and 0.60 for smear testing,
bacterial DNA copy measurement, and culture testing, respectively.
To determine the most useful combination of each testing, AUC of
combined outcome was calculated using propensity scoring analysis.
Combination of smear testing and bacterial DNA amount indicated
highly efficacious diagnostic value (AUC: 0.80), and cut off value of
bacterial DNA copy for diagnosis of bacterial keratitis was 1.0X103.
Conclusions: Combined testing of bacterial DNA quantification and
smear testing was highly efficacious to definitively diagnose bacterial
keratitis at initial visit.
Commercial Relationships: Daisuke Shimizu, None; Dai
Miyazaki, None; Keiko Yakura, None; Tomoko Haruki, None;
Yoshitsugu Inoue, Alcon Japan Inc. (F)
Evaluation of real-time PCR for the diagnosis of intra-ocular
tuberculosis
Soumyava Basu, Manas R. Barik, Praveen K. Balne, Soveeta S.
Rath, Mamatha Reddy, Savitri Sharma. LV Prasad Eye Institute,
Bhubaneswar, India.
Purpose: Definitive diagnosis of intraocular tuberculosis has
remained challenging despite recent advances in molecular
diagnostic techniques. Here we report the development of a realtime polymerase chain reaction (PCR) assay for detection of
Mycobacterium tuberculosis complex in aqueous and vitreous
samples from eyes with intraocular tuberculosis.
Methods: Aqueous or vitreous humor samples were collected
from patients with clinically suspected ocular tuberculosis (based
on previously published diagnostic criteria; Gupta et al, Surv
Ophthalmol’ 2007) and non-uveitis eyes undergoing vitrectomy or
cataract surgeries (controls). mpb64 gene of M. tuberculosis genome
and human RPPH1 (RNase P RNA component H1) were amplified
from the extracted DNA and detected real-time by customized
FAM-labeled probes. The ratio of copy numbers of mpb64 and
RPPH1, obtained from each test and control sample was used to
generate Receiver Operating Characteristic (ROC) curves. The
optimum cut-off value of real-time PCR was identified from the
experimental data that had the highest Youden index (Youden index =
sensitivity+specificity-1).
Results: M. tuberculosis complex genome was detected in 33 of 47
test samples (70.2%) and 2 of 18 healthy controls (11.2%) based
on optimum cutoff value of copy number ratios (0.025) obtained
from ROC curve having highest Youden index number, 0.727. At
this cutoff value the sensitivity was 81.0% and specificity 91.7%.
The copy number ratios varied widely in different clinical samples,
with the highest median value seen in intermediate uveitis sub-group
(0.387±0.664). The numbers were not sufficient to compare aqueous
and vitreous samples.
Conclusions: We could develop a highly specific and sensitive
PCR assay for detection of M. tuberculosis complex in aqueous
and vitreous samples. There was wide variation in copy numbers in
different disease sub-types that need to be analyzed in larger studies.
Commercial Relationships: Soumyava Basu, None; Manas R.
Barik, None; Praveen K. Balne, None; Soveeta S. Rath, None;
Mamatha Reddy, None; Savitri Sharma, None
Models of microbial keratitis in ex vivo rabbit and human
corneas
Abigail Pinnock1, Nagaveni Shivshetty2, Sanhita Roy2, Stephen
Rimmer1, Ian Douglas1, Sheila MacNeil1, Prashant Garg2. 1University
of Sheffield, Sheffield, United Kingdom; 2LV Prasad Eye Institute,
Hyderabad, India.
Purpose: To study microbial keratitis, cultured cells and in
vivo animal models are commonly used but, these are either not
representative of the in vivo situation or involve the use of many
animals. Recently, there is increased use of ex vivo corneal models
to replace or reduce animal use but there is no direct comparison of
bacterial and fungal keratitis in these models. Accordingly, we aimed
to establish reproducible models of bacterial and fungal infections in
rabbit and human ex vivo corneal models to aid studies of keratitis.
Methods: Wild brown rabbit and donated human corneas were
maintained in corneal organ culture as previously described
(Deshpande et al., Biomater. 2013). Corneas were wounded
with a scalpel, and exposed to, or injected intrastromally with,
108Staphylococcus aureus, Pseudomonas aeruginosa or Candida
albicans for 24 or 48h at 37°C. Corneas were lysed, the resulting
suspension serially diluted and spotted onto agar plates for colony
enumeration. Corneal tissue was histologically processed and
sections were Gram-stained. Corneas not exposed to microbes were
used as controls.
Results: After 24h, using the scalpel wounding method, S.aureus,
P.aeruginosa and C.albicans were recovered at 6.4±1.5x105,
5.5±0.8x106 and 2.2±0.8.1x104 CFU/rabbit cornea respectively
and 3.8±0.8x106, 4.4±0.6x108 and 1.9±0.3x105 CFU/human cornea
respectively. No difference in the CFU/cornea after 48h was observed
compared with 24h. The injection method yielded a 10-fold increase
(p<0.05) in detectable organisms for P.aeruginosa but no difference
for S.aureus or C.albicans, compared with the scalpel method.
Histology of the scalpel-wounded and injection models indicated
extensive infiltration of P.aeruginosa, throughout the entire cornea,
with less infiltration observed for S.aureus and C.albicans.
Conclusions: Bacterial and fungal infections were initiated in ex
vivo corneal models after 24 and 48h, with both scalpel wounding
and injection methods suitable for inducing infection. Differences
between the CFU/cornea for rabbit and human corneas may be due to
the expression of different antimicrobial peptides or surface receptors
influencing colonisation of the tissue. These simple and reproducible
ex vivo models will be useful as an alternative to monolayer cells and
in vivo models for investigating microbial keratitis.
Commercial Relationships: Abigail Pinnock, None; Nagaveni
Shivshetty, None; Sanhita Roy, None; Stephen Rimmer, None; Ian
Douglas, None; Sheila MacNeil, None; Prashant Garg, None
Support: Wellcome-DBT grant 0998800/B/12/Z
The use of pediatric blood culture bottles in the diagnosis of acute
postoperative endophthalmitis
Tatiana Tanaka1, Joao N. Almeida2, Thais S. Di Gioia2, Flavia
Rossi2, Juliana M. Kato3, Bruno F. Ferreira1, Aline D. Ruppert1,
Yoshitaka Nakashima1, Sergio L. Pimentel1, Joyce H. Yamamoto1.
1
Ophthalmology, University of São Paulo, São Paulo, Brazil;
2
Microbiology, University of São Paulo, São Paulo, Brazil; 3Faculty
of Medicine, University of São Paulo, São Paulo, Brazil.
Purpose: Sample culture is an essential laboratory procedure
necessary to confirm the microbiological etiology and to prompt and
appropriate treatment of endophthalmitis. Techniques for culturing
vitreous samples vary. The traditional method for culturing the
undiluted vitreous uses solid media plates and tioglicolate. Direct
inoculation of blood culture bottles may be alternative.1 Pediatric
blood culture bottles may be suitable for samples of small amount.2
The present study evaluated the culture yield in the diagnosis of
endophthalmitis using for inoculation conventional methodology
(CM) or pediatric blood culture bottle (PBCB).
Methods: This retrospective study included cases of clinically
suspected acute postoperative endophthalmitis treated between
January 2010 and December 2013 at Department of Ophthalmology,
University of São Paulo, SP, Brazil. Undiluted vitreous were
cultivated in CM from January 2010 to December 2011 and in PBCB
from January 2012 to December 2013. The isolated agents and
culture yield were analysed for each methodology. The study was
approved by the Institutional Ethics Committee.
Results: Fourty two cases were included during this 4-year period.
These cases were associated with phacoemulsification (n=20, 47.6%),
trabeculectomy (n=9, 21.4%), extracapsular cataract extration (n=6,
14.3%), pars plana vitrectomy (n=4, 9.5%), phacoemulsification/
trabeculectomy (n=2, 4,8%) and intravitreal bevacizumab injection
(n=1, 2.4%). The most prevalent agents were Staphylococcus
epidermidis (n=5, 23.8%), Streptococcus viridans (n=3, 14.3%),
coagulase-negative Staphylococus (n=2, 9.5%) and Enterococcus
faecalis (n=2, 9.5%). The culture yield of the 23 eyes cultured in CM
was 36.4 % and of the 20 eyes cultured in PBCB was 65% (Table).
Conclusions: In spite of this non-comparative and retrospective
study, pediatric blood culture bottle yielded substantially high
positivity and seems a good alternative to CM if access to
microbiological facilities is suboptimal. PBCB had some advantages
over conventional methodology: easy inoculation, reduce the risk
of contamination with transport and possibility of storage at room
temperature.
1
Joondeph BC et a. A new culture method for infectious
endophthalmitis. Arch Ophthlamol 1989;107:1334-7; 2 Heggers JP et
al. The efficacy of pediatric bllod culture sets in the determinations of
burn bacteremia. J Burn Care Rehabil 1990; 11:419-22
Table
Commercial Relationships: Tatiana Tanaka, None; Joao N.
Almeida, None; Thais S. Di Gioia, None; Flavia Rossi, None;
Juliana M. Kato, None; Bruno F. Ferreira, None; Aline D.
Ruppert, None; Yoshitaka Nakashima, None; Sergio L. Pimentel,
None; Joyce H. Yamamoto, None
Gene expression analysis of severe bacterial, fungal and
acanthamoeba keratitis: role of immune and inflammatory
biomarkers of disease
Jaya D. Chidambaram1, 2, Namperumalsamy Venkatesh Prajna2,
3
, Palepu Srikanthi2, Manisha Shah3, Lalitha Prajna3, Elakkiya
Shanmugam3, Julien Bauer4, Martin Holland1, Matthew J. Burton1,
5 1
. Clinical Research Dept, London School of Hygiene & Tropical
Medicine, London, United Kingdom; 2Cornea Department,
Aravind Eye Hospital, Madurai, India; 3Aravind Medical Research
Foundation, Madurai, India; 4Pathology Dept, University of
Cambridge, Cambridge, United Kingdom; 5London School of
Hygiene & Tropical Medicine, International Centre for Eye Health,
London, United Kingdom.
Purpose: Microbial keratitis (MK) is a leading cause of blindness
worldwide. Excessive host inflammatory responses are thought to
be the cause of tissue damage in MK. In this study, we investigated
the expression of 45 genes involved in immune and inflammatory
pathways, and extracellular matrix (ECM) modulation in corneal
cells taken from late stage human bacterial (BK), fungal (FK) and
acanthamoeba keratitis (AK), as compared to normal cadaver corneal
tissue.
Methods: Corneal swab samples from 239 patients presenting to
Aravind Eye Hospital Cornea Clinic in India from Feb 2012 to
Feb 2013 with MK (>=3mm diameter stromal infiltrate). Final
outcome was recorded at 21 days post-enrolment (i.e. “nonhealing”=perforation or descemetocoele or intervention, e.g.
corneal glue, transplant or intrastromal antifungal). Cadaver
corneal tissue (n=13) was collected from Aravind Eye Bank. Total
RNA was extracted from swabs/tissue (Qiagen, Netherlands), and
RTqPCR performed with custom Taqman Low Density Arrays (Life
Technologies, USA). Genes were normalized to HPRT1. Pairwise
comparisons between BK, FK or AK versus Controls, BK versus FK
and FK healed versus FK non-healed were performed and statistical
significance of differential expression assessed with Wilcoxon rank
sum test in Stata 12.1 (Bonferroni adjusted p-values).
Results: 218 patients were microbiologically positive for fungus
(n=185, mainly Fusarium sp. or Aspergillus flavus), bacteria (n=20,
mainly Streptococcus pneumoniae) and Acanthamoeba sp. (n=13).
Differential expression (FC >2 or <2, p<0.0005) was found in 15, 9
and 3 genes in FK, BK and AK versus Controls, with no significant
difference between BK and FK expression. Upregulated genes
included ECM modifiers MMP9 (BK), MMP10 (FK, BK) and
COL5A1 (BK, FK, AK); cytokines IL8 (BK, FK), IL18 (FK), and
neutrophil antimicrobial effectors S100A9 and PI3 (all 3). In FK, PI3
was upregulated in non-healing ulcers (FC 3.6, p=0.002).
Conclusions: Several MMPs and immune effectors are significantly
upregulated in BK, FK and AK. PI3, expressed in healing epithelia
and known to be an immune modulator, correlates with poor
prognosis in other diseases (e.g. cutaneous graft-vs-host disease).
Further research is needed into PI3 as a possible prognostic
biomarker for FK.
Commercial Relationships: Jaya D. Chidambaram, None;
Namperumalsamy Venkatesh Prajna, None; Palepu Srikanthi,
None; Manisha Shah, None; Lalitha Prajna, None; Elakkiya
Shanmugam, None; Julien Bauer, None; Martin Holland, None;
Matthew J. Burton, None
Support: Wellcome Trust PhD Research Fellowship Grant no.
097437/Z/11/Z
Microbiome of contact lens cases following corneal infiltrative
events
Ajay Kumar Vijay1, Jacqueline Tan1, Lily Ho1, Anahit Penesyan2,
Ian Paulsen2, Mark D. Willcox1. 1School of Optometry & Vision
Science, University of New South Wales, Sydney, NSW, Australia;
2
Department of Chemistry and Biomolecular Sciences, Macquarie
University, Sydney, NSW, Australia.
Purpose: Contact lens cases become contaminated with bacteria
during use and this can lead to corneal infiltration or infection. Many
bacterial species remain non-culturable with standard laboratory
techniques. We performed culturing and next-generation DNA
sequencing to identify both culturable and non-culturable organisms
that reside on contact lens cases of patients during corneal infiltrative
events.
Methods: Contact lens cases were collected from patients with
corneal infiltrative events and both wells were individually swabbed.
Swabs from the right wells were cultured to isolate and identify
viable microbes using standard culture techniques. Microbial DNA
was extracted from the swabs of the left wells, and 16S rRNA
amplicon sequencing performed using PCR primers 515/806
targeting the V4 variable region of 16S rRNA gene.
Results: Six lens cases were collected from five subjects with corneal
infiltrative events: microbial Keratitis (n=1), contact lens peripheral
ulcer (n=1) and infiltrative keratitis (n=3). All six lens cases were
culture positive for Gram-negative bacteria; no Gram-positive
bacteria, fungi or Acanthamoeba were grown. Several bacterial
strains were cultured from 5/6 of the lens case wells; S. marcescens
(n=5), A. xylosoxidans (n=2), S. maltophilia (n=1), A. faecalis
(n=1) and P. fluorescens (n=1). 16S rRNA sequencing of DNA from
lens cases identified multiple microbial species (median = 30, max
= 79, min = 21) including members of Proteobacteria (95.9%),
Bacteroidetes (2.2%), Actinobacteria (1.8%), Firmicutes (0.1%),
Cyanobacteria (0.01%), Candidate division OP11 and Deinococcus
thermos (combined 0.01%).
Conclusions: The results of this study confirm that a large number
of microbes remain non-culturable and DNA analysis of lens cases
can provide valuable information that may help understand the
pathogenesis of contact lens related microbial keratitis and corneal
infiltrative events.
Commercial Relationships: Ajay Kumar Vijay, Bausch+Lomb (F);
Jacqueline Tan, None; Lily Ho, None; Anahit Penesyan, None; Ian
Paulsen, None; Mark D. Willcox, Bausch+Lomb (F)
Human Ocular Surface Microbiome Composition Revealed By
Next-Generation Sequencing
Thuy Doan1, Lakshmi Akileswaran1, Dallin Andersen1, Narae
Ko1, Angira Shrestha1, Cecilia S. Lee1, Aaron Lee2, Russell Van
Gelder1. 1Ophthalmology, University of Washington, Seattle, WA;
2
Ophthalmology, University of British Columbia, Vancouver, BC,
Canada.
Purpose: Human mucosal surfaces are thought to be colonized
by a diverse community of microorganisms that help shape the
immune system and when altered may lead to infections or cause
inflammation in the host. Conventional culture techniques have failed
to identify the composition and to characterize the diversity of these
communities because a majority of these microbes are unculturable.
In this study, we sought to characterize the ocular surface bacterial
community in healthy subjects by using 16S rRNA gene deep
sequencing on the Illumina platform.
Methods: Conjunctiva samples of the upper and lower fornices of
both eyes were collected using forensic DNA recovery swabs from
35 healthy volunteers. Along with appropriate negative control
samples, the conjunctiva samples underwent DNA extraction, library
preparation, and 16S rRNA gene deep sequencing on the Illumina
platform. Quantitative PCR for Torque Teno Virus (TTV) was also
performed. Data were analyzed in R.
Results: Sequencing resolution to the genus and species levels were
obtained for 140 conjunctiva samples from 35 healthy volunteers.
Propionibacterium acnes, Arthrospira fusiformis, Corynebacterium
tuberculostearicum, Enterobacter hormaechei, and Chryseobacterium
indologenes were the most abundant species across all samples.
Principal component analyses showed that the genera responsible
for the majority of the variance across all conjunctiva samples were
Corynebacterium, Propionibacterium, and Staphylococcus. TTV was
detected in 63% of the patients (17/27). Subgroup analyses revealed
that the TTV load was statistically higher in men compared to women
(0.012 TTV copy/epithelial cell ± 0.0028 TTV copy/epithelial cell
vs. 0.001 TTV copy/epithelial cell ± 0.0006 TTV copy/epithelial cell,
mean ± SEM, p = 0.0078).
Conclusions: The ocular surface microbiome bacterial composition
in healthy volunteers is diverse. The variability across samples
is largely determined by Corynebacterium, Propionibacterium,
and Staphylococcus. Low TTV levels are found in the majority
of the samples and TTV viral load is dependent on gender.
Future experiments using unbiased next-generation sequencing
to characterize the bacterial, fungal, and viral composition of the
ocular surface will further our understanding of ocular infectious and
inflammatory diseases.
Commercial Relationships: Thuy Doan, None; Lakshmi
Akileswaran, None; Dallin Andersen, None; Narae Ko, None;
Angira Shrestha, None; Cecilia S. Lee, None; Aaron Lee, None;
Russell Van Gelder, None
Support: EY022038, P30EY001730, Unrestricted Research
Departmental Grant From Research To Prevent Blindness
Mucosal microbiome in Sjögren Syndrome
Stephen C. Pflugfelder1, Cintia S. De Paiva1, Dan B. Jones2, Quianta
Moore1, Shani Corbiere5, Joseph Petrosino3, Diane Smith3, Michael
E. Stern4, 1, Nadim Ajami3. 1Ophthal-Ocular Surf Ctr, Baylor College
of Medicine, Houston, TX; 2Ophthalmology, Baylor College of
Medicine, Houston, TX; 3Alkek Center for Metagenomics and
Microbiome Research, Baylor College of Medicine, Houston, TX;
4
Biological Sciences, Allergan, Irvine, CA; 5Sjogren Syndrome
Support Group, Houston, TX.
Purpose: To compare the ocular, oral and fecal microbiome in
patients with Sjögren syndrome (SS) with control subjects
Methods: Conjunctival, tongue and fecal samples were obtained
from 10 patients with primary SS meeting revised ACR criteria and
controls (normal eyes and fecal samples, and tongue samples from
patients with rosacea). Severity of oral and ocular surface disease was
graded. Conjunctival goblet cell density was counted in impression
cytology. 16s ribosomal DNA gene sequencing was performed
by 454 (conjunctival) and MiSeq sequencing and sequences were
mapped to microbial databases. Relative abundance of phyla and
genera and alpha and beta diversity of observed OTUs between
groups were compared.
Results: A low abundance ocular surface microbiome consisting
of core phyla Actinobacteria,, Bacteroidetes, Proteobacteria and
Firmicutes was identified. There were no differences in alpha or beta
diversity between normal and dry eyes; however, there was greater
abundance of Firmicutes in the SS group. Compared to control, alpha
diversity was greater in the tongue and reduced in the stool in the
SS group. Between group differences in relative abundance were
observed with greater Streptococcus and Hemophilus and reduced
Neisseria and Fusobacterium genera in the SS tongue and greater
abundance of Blautia, Escherichia/Shigella, and Streptococcus and
reduced Akkermansia, Subdoligranulum, Faecalibacterium and
Prevotella in the SS stool. Distinct clustering of OTUs was seen in SS
stool and subjects with most severe clinical severity scores had the
least diversity of observed OTUs in the stool.
Conclusions: Minimal differences in abundance and diversity were
found in the SS ocular microbiome. In contrast, SS stool showed
a less diverse microbiome that contained a greater number of
inflammatogenic and lower number of homeostatic flora.
Commercial Relationships: Stephen C. Pflugfelder, None; Cintia
S. De Paiva, None; Dan B. Jones, None; Quianta Moore, None;
Shani Corbiere, None; Joseph Petrosino, None; Diane Smith,
None; Michael E. Stern, None; Nadim Ajami, None
Support: Sjögren Syndrome Metagenomics Research Fund, NIH
Grant EY11915 (SCP), Research to Prevent Blindness, Oshman
Foundation, William Stamps Farish Fund, Hamill Foundation
Metaproteomic analysis in infected ocular surface
Diana Gabriela Ponce-Angulo1, 2, Antonio Bautista-Hernandez1, 3,
Gerardo Aparicio-Ozores2, Victor M. Bautista1. 11. Research Unit/
Microbiology and Ocular Proteomics, Institute of Ophthalmology
“Fundación de Asistencia Privada Conde de Valenciana”, Mexico
City, Mexico; 22. Laboratory of Medical Bacteriology, Department of
Microbiology, National School of Biological Sciences- IPN, Mexico
City, Mexico; 33. Doctoral Program in Biological Sciences and
health, Autonomous Metropolitan University (UAM), Mexico city,
Mexico.
Purpose: To realize a comparative metaproteomic analysis of the
ocular surface in patients with ocular infection.
Methods: The samples were obtained from patients with healthy
and infected ocular surface. The human tears samples were taken
following the internal protocols in both groups. Steril saline solution
was applied over the ocular surface and collected by capillar
tubes. Microorganisms were identified by automated microbiology
methods. Chocolate agar, blood agar, Sabouraud agar were seeded
with samples from both groups, samples also were added to Brain
Heart Infusion. The bacterial identification was determined by Vitek
2 Compact System. The fungi were identificated by morphologycal
caracteristics. The tears samples of each patient were analysed by 2D
electrophoresis and differential protein expression between groups
was performed using Dymensión 2 Software.
Results: Microbiological analysis showed the presence of
Staphylococus aureus, S. epidermidis, S. lentus, Kokuraria rosea,
Streptococcus thoraltensis, Rothia mucilaginosa, Granulicatella
adiacens and Candida guillermondi in healthy group; S. capitis, S.
epidermidis and C. albicans were isolated and identified in infected
group, three patients not shown microbiology growth. Proteomic
analysis showed
In the results of healthy group, there were differential growt of
Staphylococus aureus, S. epidermidis, S. lentus, Kokuraria rosea,
Streptococcus thoraltensis, Rothia mucilaginosa, Granulicatella
adiacens and Candida guillermondi. In the infection group, the
findings in the isolated microorganism were S. capitis, S. epidermidis
and C. albicans and in three of all them had not microbiology grown.
At the end of the analysis of differential expression in 2D gels
without infection group compared with the control group the
following results: in the first sample were obtained 5 differentially
expressed spots, in the second sample 4 spots, en the third sample
2 spots, in the fourth sample 3 spots, in the fifth sample 2 spots and
finally in the sixth sample 2 spots. The metaproteomic analysis shows
the relevant expression of diferential proteins. The diferential proteins
will be identificated by mass spectrometry.
Conclusions: The bacterial species were obtained in both groups
were Staphylococcus epidermidis and Staphylococcus capitis.
The analysis have difencial protein expression in helthy group in
compararising with infection group.
Commercial Relationships: Diana Gabriela Ponce-Angulo, None;
Antonio Bautista-Hernandez, None; Gerardo Aparicio-Ozores,
None; Victor M. Bautista, None
Support: This work was supported by “Conde de Valenciana”
Foundation
Microbiologic spectrum of post-injection endophthalmitis by
indication for intravitreal anti-VEGF therapy
Charles Calvo1, Nadim Rayess1, Ehsan Rahimy1, Chirag Shah2,
Jeremy D. Wolfe3, Eric Chen4, Francis DeCroos5, Sunir J. Garg1,
Jason Hsu1. 1Retina Service, Wills Eye Hospital, Philadelphia,
PA; 2Ophthalmic Consultants of Boston, Boston, MA; 3Associated
Retinal Consultants at William Beaumont Hospital, Royal Oak, MI;
4
Retina Consultants of Houston, Houston, TX; 5Southeastern Retina
Associates, Chattanooga, TN.
Purpose: To describe the microbiologic spectrum of endophthalmitis
following intravitreal anti-VEGF injections for treatment of
neovascular age-related macular degeneration (AMD), diabetic eye
disease, and retinal vein occlusion (RVO).
Methods: A multicenter, retrospective, consecutive case series.
Results: Between January 1, 2011 and September 30, 2013, a total
of 503,890 intravitreal anti-VEGF injections were performed at the
five participating clinical sites. Presumed infectious endophthalmitis
occurred in 159 of 416,133 injections performed for neovascular
AMD (1/2617), 16 of 40,982 for diabetic eye disease (1/2561), and
8 of 46,775 for RVO (1/5846). For patients with neovascular AMD,
61 of the 159 cases (38%) of endophthalmitis were culture positive,
corresponding to a culture positive rate of 1/6822. The cultured
organisms included 13 cases of coagulase-negative Staphylococcus,
12 of S. epidermidis, 5 of S. pneumonia, 5 of S. mitis, 5 of
methicillin-sensitive S. aureus, 4 of Staphylococcus lugdunesis, 4
of Enterococcus fecalis, 3 of S. viridans, 3 of non-differentiated
gram-positive cocci, and 1 case of each of the following organisms:
Staphylococcus auricularis, Staphylococcus homininis, Streptococcus
sanguis, alpha-hemolytic Streptococcus, Candida parapsicolosis,
Propionibacterium, and Lactobacillus. For patients treated for
diabetic eye disease, 8 of the 16 cases (50%) were culture positive,
providing a culture positive rate of 1/5123. Four eyes grew
coagulase-negative Staphylococcus, 2 had S. epidermidis, and there
was 1 case of S. pneumonia and 1 case of H. influenzae. For patients
with RVO, 4 of the 8 cases (50%) were culture positive, resulting in
an overall culture positive rate of 1/11,694. There was 1 case of S.
pneumoniae, methicillin-sensitive S. aureus, S. mitis, and coagulasenegative Staphylococcus.
Conclusions: Gram-positive coagulase-negative Staphylococcus
was the most common bacteria isolated in cases of post-injection
endophthalmitis. A greater proportion of coagulase-negative
Staphylococcus was found in endophthalmitis following injections
for diabetic eye disease (75%) compared to AMD (51%) and RVO
(25%). Oral flora pathogens accounted for 22% of the cases.
Commercial Relationships: Charles Calvo, None; Nadim Rayess,
None; Ehsan Rahimy, None; Chirag Shah, None; Jeremy D.
Wolfe, None; Eric Chen, None; Francis DeCroos, None; Sunir J.
Garg, None; Jason Hsu, None
Microbiologic Analysis of Dacryocystitis at LAC + USC Medical
Center
Mica Bergman1, 2, Jesse berry1, 2. 1USC Eye Institute, Los Angeles,
CA; 2Ophthalmology, LAC + USC Medical Center, Los Angeles, CA.
Purpose: To investigate the microbiologic profile of dacryocystitis at
a major county hospital in Southern California.
Methods: IRB approved retrospective review of
dacryocystorhinostomy (DCR) operations performed from 1/1/2000
– 11/3/2014. Patients with a diagnosis of dacryocystitis in whom at
least one culture was performed during the course of their care were
included in this study.
Results: Sixteen cases of dacryocystitis were identified in fifteen
patients. Of these, eleven underwent culture one time, four underwent
culture two times, and one underwent culture three times, yielding
a total of 22 cases. Of the 22 cases, 19 cases had a positive yield,
12 cases were monomicrobial, 6 cases were bimicrobial, and 1 case
was trimicrobial. In lacrimal sacs undergoing culture two or more
times, the same organism was found in multiple specimens two out
of five times. In total, there were 14 isolates of gram-positive cocci,
10 isolates of gram-negative bacilli, 1 isolate of gram-negative
diplococci, and 2 fungal isolates. The most commonly identified
bacteria was klebsiella pneumoniae (4 isolates), followed by
staphylococcus aureus and streptococcus viridans (3 isolates each).
Bacteria thought to be nonpathogenic (eg staphylococcus epidermidis
and diphtheroids) were excluded.
Conclusions: This series demonstrates that there is a wide range
of pathogens implicated in dacryocystitis in our county hospital in
Southern California. The most commonly isolated bacteria were
klebsiella pneumoniae, staphylococcus aureus, and streptococcus
viridans. One third of patients had polymicrobial culture results.
Given that gram-negative and gram-positive pathogens were found
in similar numbers, it is advisable to treat initially with a broadspectrum antibiotic.
Commercial Relationships: Mica Bergman, None; Jesse berry,
None
Support: An Unrestricted grant from Research to Prevent Blindness,
New York, NY 10022
15 Years of Microbial Keratitis at an Urban University Practice
in Saint Louis: The Isolates
Hugo Y. Hsu1, 2, Sean Edelstein3. 1Doheny Eye Institute, Los Angeles,
CA; 2Ophthalmology, David Geffen School of Medicine at UCLA,
Los Angeles, CA; 3Ophthalmology, Saint Louis University School of
Medicine, Saint Louis, MO.
Purpose:
The spectrum of micro-organisms causing infectious keratitis varies
between geographic areas and through time. We wish to identify
the isolated micro-organisms from infectious keratitis cases and
to observe any trends in the spectrum of causative organisms
over a 15-year period at Saint Louis University’s Department of
Ophthalmology.
Methods:
We searched the database of the microbiology department and the
diagnosis database of the ophthalmology department at Saint Louis
University to identify cases of microbial keratitis from 1999-2013.
Records of culture-positive cases were reviewed retrospectively.
Non-contaminant isolates were tabulated into three 5-year periods
(1999-2003; 2004-2008; and 2009-2013) and compared.
Results:
229 non-contaminant isolates were identified: 45 from 1999-2003; 84
from 2004-2008; and 100 from 2009-2013. Overall, Gram-positive
organisms were the most commonly isolated (47%) followed by
Gram-negative organisms (34%). Fungi represented 18% of the
overall isolates. Separated into the three 5-year periods, Gram+
organisms represented 53%, 44%, and 46% of the total; Gramrepresented 38%, 27%, and 33% of the total; and fungal organisms
represented 9%, 20%, and 20% of the total. Pseudomonas was the
most commonly isolated organism overall (21%) as well as in each
of the 3 time periods. Streptococcus species were the next most
common isolates overall (15%) followed by coagulase-negative
staphylococcus and Staphylococcus aureus (14% each). The
percentage of Staphylococcus aureus isolates that were oxacillinresistant (ORSA) increased in each of the three 5-year periods from
22% to 44% to 69%.
Conclusions:
The number of non-contaminant isolates increased in each of the
three 5-year periods. Pseudomonas was the most common isolate
found in infectious keratitis at Saint Louis University over the
15-years period. The two biggest changes we observed were in the
number and percentage of fungal isolates as well as the increase
in the proportion of ORSA isolates over time. Fungal organisms
accounted for 9% of isolates at the start of the 15-year period but then
doubled to 20% during the mid 2000’s and continued through the
end of the 15-year period in 2013. The percentage of ORSA isolates
tripled over the 15 years period.
Commercial Relationships: Hugo Y. Hsu, None; Sean Edelstein,
None
Clinical and Microbiological Characteristics of Infectious
Keratitis in Mexico
Alejandro F. Ibarra-Lozano, Pedro M. Gonzalez, Jaime Torres, Jorge
E. Valdez. Ophthalmology Residence, Instituto Tecnológico y de
Estudios Superiores de Monterrey, San Pedro Garza Garcia, Mexico.
Purpose: Geographical differences play a major role to the clinical
and microbiological characteristics of infectious keratitis. The
purpose of this observational retrospective study is to present these
aspects of the infectious keratitis found in Mexico to be able to
establish empiric treatment guidelines.
Methods: Patient records from January 2010 to November of 2014
were revised. Cases in which corneal scrape cultures were made
were selected. Variables studied included were: gender, age, time of
presentation to medical attention, visual acuity, microbiologic agent
encountered, comorbidities, and complications.
Results: Twenty-eight cases of microbial keratitis with corneal scrape
culture were found. Male:Female ratio was 18:10 respectively and
age at presentation varied from 2 to 85 years. The most commonly
encountered microbiologic agent was Pseudomona aeruginosa, found
in 8 patients; 5 cases were positive to fungal agents, 2 for Fusarium
spp and 2 for Candida spp; S. pneumonie was found in two patients,
Acantamoeba spp in one patient, and Corynebacerium matruchotti
in another patient. Other isolated microorganisms included M.
catharralis, H. influenzae, A. xyloxidans, Aspergillus, and S.
marcescens. Twenty one percent of the cultures were negative.
Ocular trauma was associated to 32.1% of the cases, including
vegetal and metallic objects. Diabetes Mellitus was found as a
comorbidity in 32.1% of the cases and one case was HIV positive.
Only 3 contact lens users were identified in the total of cases. Time
from the beginning of symptoms to time of medical attention ranged
from 1 to 60 days. Patients who waited more time to get medical
attention were those with fungal and parasitic infections. Gramnegative bacteria dominated the microbiologic pattern being the
encountered organism in 36.3% of the positive cultures. Four patients
had corneal perforation, two of them associated to Pseudomona
aeruginosa.
Conclusions: This study revised the epidemiology of microbial
keratitis in northern Mexico. The microbiologic pattern encountered
varies importantly from that in other geographic zones, especially
in the unusually high Gram-negative predominance. There was a
tight relation between microbial keratitis with trauma and Diabetes
Mellitus. This study will aid the generation of new treatment
guidelines in the geographic zone studied.
Proteus mirabilis (n=12/30%), Serratia marcescens (n=8/20%), and
Klebsiella pneumoniae (n=7/17.5%). NonEnterobacteriaceae were the
predominant group -68% (n= 85/125). Top pathogens: Pseudomonas
aeruginosa (41/48%), Haemophilus influenzae (n=15/17.6%) and
Moraxella species (n=8/9.4%).
In period II (2000-2014), gram negative pathogen frequency
was 11.6% (n=121/1039), 75/62% were nonEnterobacteriaceae.
Top pathogens were: Pseudomonas aeruginosa (n=37/49.3%),
Haemophilus influenzae (15/20%), Moraxella osloensis (8/10.7%).
For Enterobacteriaceae, the rate was 38% (n= 46). Serratia
marcescens (n=16/34.8%), Enterobacter cloacae (9/19.6%), and
Klebsiella pneumoniae (5/10.9%).
Antimicrobial susceptibilities for Enterobacteriaceae from period
I were: amikacin-80%; ceftazidime-83% and ciprofloxacin-89%.
Non-Enterobacteriaceae: amikacin-100%; ceftazidime-90%; and
ciprofloxacin-98%. Susceptibilities for Enterobacteriaceae from
period II: amikacin, 95%; ceftazidime, 97%; and ciprofloxacin- 89%.
Non-Enterobacteriaceae: amikacin, 84%; ceftazidime, 91%; and
ciprofloxacin-98%.
Moxifloxacin susceptibility for the Enterobacteriaceae (N=12) was
83% versus 41% for the Non-Enterobacteriaceae (N=17).
The highest resistance and/or nonsusceptible trends for all pathogens
were observed for moxifloxacin-41% (n=12/29), followed by
amikacin (10%), ceftazidime (6%) and ciprofloxacin (4%).
Conclusions: Gram negative isolates from patients with
endophthalmitis have remained stable over the 25 year period.
Emerging resistance trends to commonly used antibiotics amikacin,
ceftazidime, ciprofloxacin and moxifloxacin have been identified.
Commercial Relationships: Benjamin D. Wilson, None; Darlene
Miller, BPEI (E); Harry W. Flynn, BPEI (E)
Support: RPB Unrestricted Award, NIH Core Grant P30EY014801
Commercial Relationships: Alejandro F. Ibarra-Lozano, None;
Pedro M. Gonzalez, None; Jaime Torres, None; Jorge E. Valdez,
None
Gram-negative Isolates from Patients with Endophthalmitis:
Incidence Rates and Antibiotic Susceptibilities
Benjamin D. Wilson, Darlene Miller, Harry W. Flynn. Bascom Palmer
Eye Institution, Miami, FL.
Purpose: To report incidence rates and antibiotic susceptibilities
among gram-negative isolates from patients with endophthalmitis.
Methods: Microbiology reports were reviewed to identify the
incidence rates of gram negative pathogens (Enterobacteriaceae vs
NonEnterobacteriaceae) recovered from endophthalmitis patients
during a 25 year period (1990-2014). A combination of disk diffusion,
Vitek 2 and Etests were used to susceptibility trends for amikacin,
ceftazidime, and ciprofloxacin. Etests were used to evaluate
moxifloxacin. Identifications were confirmed using conventional
methods and Vitek 2.
Results: In period I (1990-1999, period I) Gram negative pathogens
represent 11.4% (n=125/1095) of the cases. Organism spectrum
included: Enterobacteriaceae (32%, n=40/125). Top pathogens were
Detection of virulence factors with multiplex PCR Technique of
Conjunctival Coagulase Negative-Staphylococcus (CNS) from
patients undergoing cataract surgery
Veronica E. Castillo1, Yolanda Lopez2, Margarita Samudio2, Norma
Farina2, Sonia Abente2, Nilsa Gonzalez2, Florentina Laspina2,
Agustin Carron1, Diogenes Cibils1, Herminia Mino de Kaspar3.
1
Ophthalmology, Hospital de Clínicas, National University of
Asunción, Asuncion, Paraguay; 2Research Institute for Health
Sciences, National University of Asunción, Paraguay, Asuncion,
Paraguay; 3Ophthalmology, Ludwig-Maximilians-University,
Munich, Germany.
Purpose: We performed a prospective study to identify by multiplex
PCR (Polymerase Chain Reaction), genes encoding virulence factors
(ica, Atle and mecA) in CNS isolates from the ocular microbiota
of patients undergoing cataract surgery and to investigate possible
changes in the Coagulase Negative-Staphylococcus (CNS) profile
due to antibiotic prophylaxis.
Methods: After approval of the Institutional Review Board of our
institution had been obtained, patients undergoing cataract surgery
were recruited for this study at the Department of Ophthalmology,
National University of Asuncion, Paraguay. Patients (n= 162)
received in the eye to be operated moxifloxacin 0.5% eye drops, four
times the day before surgery and a last drop one hour before surgery
(T1). The other eye remained as control (T0). Conjunctival swabs
were taken from both eyes one hour after the last drop. Presence of
genes encoding biofilm formation (ica and atlE) and gene encoding
methicillin resistance (mecA) were detected by a multiplex PCR in
CNS isolates from both timepoints.
Results: Of the 162 patients, 87 eyes were positive for CNS in T0,
yielding 96 CNS isolates and in T1, 70 eyes were positive, yielding
77 isolates CNS. We used for this study 43 CNS isolates from T0 and
45 from T1. Of the total CNS isolates, 81,8% (72/88) had at least one
virulence factor gene (37/43 from T0 and 35/45 from T1 (p=0.314),
simultaneous detection of ica and Atle genes was more frequent in
T0 (n=25, 58.0%) than T1 (n=21, 46.7%), but the difference was not
significant (p=0.28).
Conclusions: A high frequency of genes encoding virulence factors
was observed in the coagulase-negative Staphylococcus isolates. The
use of moxifloxacin did not modify significantly the CNS virulence
factor profiles.
Commercial Relationships: Veronica E. Castillo, None; Yolanda
Lopez, None; Margarita Samudio, None; Norma Farina, None;
Sonia Abente, None; Nilsa Gonzalez, None; Florentina Laspina,
None; Agustin Carron, None; Diogenes Cibils, None; Herminia
Mino de Kaspar, None
Comparison of two different evaluation methods for biofilm
formation of S.epidermidis isolated from ocular surface
Berna Akova Budak1, Sertac Argun Kivanc1, Meral Yildiz1, Merih
Kivanc2, Gulay Gullulu3. 1Uludag University, Bursa, Turkey;
2
Anadolu University, Eskiehir, Turkey; 3Armedica Eye Center, Izmit,
Turkey.
Purpose: To compare of two different evaluation methods for
biofilm formation of Staphylococcus epidermidis isolated from ocular
surface.
Methods: S.epidermidis strains that were isolated from ocular surface
previously were studied. Microtiter plate method (MPA) and Congo
red agar (CRA) method were used for measuring biofilm formation.
Ica A, ica D, bap genes positivity and multi-antibiotic resistance were
determined. Accurracy of measuring biofilm production capacity of
MPA and CRA were compared.
Results: 8 of the strains produced strong biofilm according to MPA
method, and 4 of these strains also produced strong biofilm according
to CRA method. 8 strains produced strong biofilm according to CRA
method. 11 strains were biofilm negative with MPA however 7 (64
%) of these were positive with CRA. 15 strains were resistant to 3
or more antibiotics, 7 (46%) and 6 (40 %) of these strains produced
strong biofilm according to MPA and CRA methods respectively. 8
strains were resistant to 4 or more antibiotics, 6 (75 %) and 3 (37 %)
of these strains produced strong biofilm according to MPA and CRA
methods respectively.
Conclusions: Strains that produced strong biofilm with MPA had
more multi-antibiotic resistance than with CRA.These two methods
may not be consistent with each other.
Commercial Relationships: Berna Akova Budak, None; Sertac
Argun Kivanc, None; Meral Yildiz, None; Merih Kivanc, None;
Gulay Gullulu, None
Microbiology and Biofilm Growth on Clinically Infected Silicone
Implants within the Lacrimal System: A Thirty Year Review
Lilangi S. Ediriwickrema1, David Samimi2, 1, Brett Bielory2, Darlene
Miller2, Thomas V. Johnson2. 1Ophthalmology, USC Eye Institute,
Los Angeles, CA; 2Bascom Palmer Eye Institute, University of
Miami Miller School of Medicine, Miami, FL.
Purpose: To investigate the pathogens and biofilms responsible for
clinically significant infection of silicone stents implanted within the
lacrimal system.
Methods: Retrospective review of culture results for silicone
lacrimal stents removed early for clinically significant infection over
a thirty year period. As a control, routinely removed, clinically noninfected stents were prospectively sent for culture over a six month
period. Four clinically infected stents and six clinically non-infected
stents showing mucus within the lumen at removal were sent for
scanning electron microscopy to grade the presence of organisms,
matrix deposits, organisms within a matrix, and a significant biofilm
by a masked electron micrographer.
Results: Nineteen stents were included in the study; 100% of
clinically non-infected stents (n=9) and of those removed for
infection (n=10) were culture positive. None of the non-infected
stents were culture positive for mycobacteria compared with 90% of
infected specimens (p < 0.001). Of non-infected stents, 89% grew
gram-positive organisms compared with 50% of infected stents
(p=0.07). Sixty-seven percent of non-infected stents had gramnegative organisms versus 50% of infected stents (p=0.46). Electron
microscopy of the four infected stents revealed organisms consistent
with mycobacteria (size, shape) encased within a matrix. Of the six
non-infected stents examined, organisms were identified within the
lumens that were consistent with culture results but were without
clear biofilm formation. A masked electron micrographer was able
to identify and grade the presence of organisms, matrix deposits,
organisms within a matrix, and a significant biofilm all with statistical
significance.
Conclusions: In our study population, atypical mycobacteria
comprise the primary pathogen responsible for clinically significant
infection of silicone stents in the lacrimal system. Robust biofilm
production by this organism likely plays a role in pathogenesis.
Development of biofilm resistant implant material, targeted liposomal
antibiotic delivery, and physical or chemical disruption strategies are
potential methods towards reducing rates of infection.
Commercial Relationships: Lilangi S. Ediriwickrema, None;
David Samimi, None; Brett Bielory, None; Darlene Miller, None;
Thomas V. Johnson, None
Support: Research to Prevent Blindness, New York, NY 10022.
Antimicrobial Efficacy of Multipurpose Disinfecting Solutions
against Combined Inoculum of ISO Bacteria and Clinical Isolates
Marina Milenkovic1, James Cook2. 1Corneal R&D Microbiology,
Abbott Medical Optics, Santa Ana, CA; 2Corneal R&D, Abbott
Medical Optics, Santa Ana, CA.
Purpose: To compare antimicrobial efficacy of multipurpose
disinfecting solutions (MPS) against combined inoculum of ISO
bacteria and Gram-negative clinical isolates during 14 days storage.
Methods: The multipurpose disinfecting solutions studied were MPS-1: polyquaternium (PQ1)+alexidine dihydrochloride (ALX),
MPS-2: polyhexamethylene biguanide (PHMB)+poloxamer, MPS-3:
PQ1+myristamidopropyl dimethylamine (ALDOX)+nonanoylEDTA, MPS-4: PQ1+ALDOX+polyoxyethylene-polyoxybutylene
(EOBO-41), MPS-5: PQ1+ALDOX and MPS-6: PHMB+PQ1. Test
solutions were inoculated with combined inoculum of ISO bacteria
and Gram-negative clinical isolates in the test tube according
to ISO 14729. Test solution efficacy was evaluated at minimum
recommended manufacturer disinfection time of 6 hours, 24 hours,
7 days and 14 days. ISO bacteria tested were: Staphylococcus
aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027 and
Serratia marcescens ATCC 13880. Clinical isolates tested were:
Achromobacter sp., Serratia marcescens and Achromobacter
xylosoxidans.
Results: After 6 hours exposure, MPS-1, MPS-2 and MPS-6 showed
>2.4 log kill against combined inoculum of all ISO bacteria and
clinical isolates and did not exhibit reduced efficacy or regrowth
over 14 days. MPS-3 showed <1 log kill against combined inoculum
of all ISO bacteria and clinical isolates after 6 hours exposure, and
exhibited regrowth over 14 days. MPS-4 and MPS-5 exhibited <1
log kill against combined inoculum of all ISO bacteria and Serratia
marcescens and all ISO bacteria and Achromobacter xylosoxidans
clinical isolates after 6 hours exposure and showed reduced efficacy
over 7 days storage. MPS-4 and MPS-5 exhibited <1.5 log kill
against combined inoculum of all ISO bacteria and Achromobacter
sp. clinical isolate after 6 hours exposure and showed reduced
efficacy after 24 hours. Low numbers of ISO bacteria S. aureus and
S. marcescens were observed for MPS-3, MPS-4 and MPS-5 after
6 hours exposure when combined inoculum of these bacteria and
Achromobacter sp. was used.
Conclusions: MPS-1, MPS-2 and MPS-6 showed high ability
to reduce microbial load after 14 days storage, while MPS-4 and
MPS-5 exhibited reduced disinfection efficacy. MPS-3 exhibited low
disinfection efficacy at 6 hours and regrowth over 14 days.
Commercial Relationships: Marina Milenkovic, Abbott Meducal
Optics (E); James Cook, Abbott Medical Optics (E)
467 Bacterial and viral infection systems
Wednesday, May 06, 2015 3:45 PM–5:30 PM
702/704/706 Paper Session
MyD88 Adaptor Protein is Required for Adenovirus Keratitis
Mirja Ramke, Xiaohong Zhou, Ashish Chintakuntlawar, Jaya Rajaiya,
James Chodosh. Ophthalmology, Massachusetts Eye and Ear
Infirmary, Harvard Medical School, Boston, MA.
Purpose: The innate immune response is robust in the development
of epidemic keratoconjunctivitis (EKC). EKC is a hyperacute and
highly contagious ocular surface infection by human adenovirus
species D (HAdV-D) types 8, 37, 53, 54, 56, and 64. Toll-like
receptors (TLRs) are important contributors to innate immunity, and
recruit intracellular adaptor protein myeloid differentiation factor
88 (MyD88) to form active signaling complexes. Initiation of TLR
and MyD88 downstream signaling cascades leads to activation of
transcription factors and secretion of proinflammatory and antiviral
cytokines. The role of TLR-dependent pathways in adenovirus
keratitis is unknown.
Methods: Stromal keratitis was induced by intracorneal injection
of 105 infectious units of HAdV-D37 or dialysis buffer control in
C57Bl/6j (WT), TLR2-/-, TLR9-/-, MyD88-/-, and TLR2/9-/- mice.
Flow cytometry, ELISA, real-time RT-PCR, confocal microscopy,
and Western blot analysis were performed on the cornea 4 days post
infection (dpi). Cultured human corneal fibroblasts (HCF) at 2nd
passage were infected with HAdV-D37 or dialysis buffer control, and
treated with MyD88 inhibitory peptide prior to Western blot analysis
and real-time RT-PCR.
Results: Clinically evident keratitis developed in TLR2-/-, and
TLR9-/-mice within 1 dpi, whereas keratitis was not evident or was
significantly reduced in MyD88-/- and TLR2/9-/- mice, respectively.
Flow cytometric analysis showed reduction in CD45+ leukocytes
in infected MyD88-/- corneas as compared to WT. ELISA showed
significantly higher CXCL1, IL-6, CCL2, and CXCL2 expression in
infected WT corneas than in MyD88-/- or TLR2/9-/-. By confocal
microscopy using Cy3-labeled HAdV-D37, viral entry appeared
similar between WT and MyD88-/-, but E1A gene expression was
reduced in MyD88-/-corneas. Src kinase phosphorylation and
CXCL1 induction were also decreased in infected MyD88-/- corneas
as compared to WT. Pretreatment with MyD88 inhibitory peptide
in HCF in vitro reduced the phosphorylation of Src kinase and
decreased CXCL8 and CCL2 gene expression.
Conclusions: Cytokine expression in adenovirus keratitis is MyD88
and Src dependent, and relies on the synergistic activation of TLR2
and 9. The innate immune response is critical to the development of
stromal keratitis in EKC.
Commercial Relationships: Mirja Ramke, None; Xiaohong Zhou,
None; Ashish Chintakuntlawar, None; Jaya Rajaiya, None; James
Chodosh, None
Support: NIH grants EY013124, EY021558, and EY014104, and
Research to Prevent Blindness.
Interferon regulator factor 8 (IRF-8) limits ocular pathology
during HSV-1 infection by restricting the activation and
expansion of CD8 T cell
Lin Sun, Anthony St Leger, Chengrong Yu, Chang He, Rashid M.
Mahdi, Chi-Chao Chan, CHRALES E EGWUAGU. Immunology,
NEI, Bethesda, MD.
Purpose: Interferon Regulatory Factor 8 (IRF-8) plays an important
role in myeloid and B cell differentiation. Although expression of
IRF-8 is induced in T cells in response to antigen stimulation, its role
in T cell mediated regulation of immune responses or host immunity
is still unknown. In this study, we have used an ocular herpes simplex
virus 1 (HSV-1) infection model to investigate the function of IRF-8
in CD8 T cell mediated control of viral infection.
Methods: We generated mice with targeted deletion of IRF-8 in the
CD4 compartment (CD4-IRF-8KO) by breeding IRF-8fl/fl mouse
(kindly provided by Dr. Herbert Morse, NIH) with mice expressing
Cre recombinase under the CD4 promoter (CD4/Cre mice). WT
and CD4-IRF8KO mice were infected with HSV-1 by corneal
scarification and HSV-specific CD8 T cell responses were examined
and characterized using the virus-specific gB-Tetramer reagent, flow
cytometry and intracellular cytokine staining assays. Viral clearance
was determined by plaque assay and ocular pathology was assessed
by histological analysis.
Results: Compared to WT, the CD4-IRF8KO mice generated
robust immune responses characterized by increased numbers of
virus-specific CD8 T cells and markedly elevated levels of proinflammatory cytokines, resulting in excessive ocular inflammation.
CD4-IRF8KO CD8 T cells expressed high levels of chemokine
receptors and up-regulation of the chemotactic molecules facilitated
the infiltration of protective CD8 T cells into the trigeminal ganglia
(TG). In addition, viral clearance in the eyes of CD4-IRF8KO mice
was found to correlate with a dramatic increase of HSV-specific CD8
T cells in the TG.
Conclusions: The data presented here suggest that an important
function of IRF-8 in T cells may be to restrain lymphocyte activation,
proliferation and to regulate the expression of molecules that mediate
lymphocyte chemotaxis. Specifically, loss of IRF-8 led to exuberant
inflammatory response, resulting in exacerbated immune-mediated
ocular pathology in HSV-1 infected mice. Our data showing that
IRF-8 suppresses T cell expansion and effector functions suggest
that IRF-8 is a potential therapeutic target that can be exploited to
mitigate pathological autoimmunity.
Commercial Relationships: Lin Sun, None; Anthony St Leger,
None; Chengrong Yu, None; Chang He, None; Rashid M. Mahdi,
None; Chi-Chao Chan, None; CHRALES E EGWUAGU, None
Proteotyping as a tool to study the evolution of human
adenoviruses associated with epidemic keratoconjunctivitis
James Chodosh1, Gurdeep Singh1, Christopher Robinson1, Jeong
Yoon Lee1, Jaya Rajaiya1, Morris Jones2, David Dyer3, Donald Seto2.
1
Ophthalmology, Mass Eye & Ear Infirmary/HMS, Boston, MA;
2
George Mason University, Manassas, VA; 3University of Oklahoma
Health Sciences Center, Boston, OK.
Purpose: Human adenoviruses of species D (HAdV-D) cause
infections of mucosal epithelia, including the ocular surface.
HAdV-D types 8, 37, 53, 54, 56, and 64 are the known etiologic
agents of epidemic keratoconjunctivitis, a severe and highly
contagious ocular surface infection associated with prolonged
corneal inflammation. HAdV-D is also the largest and fastest growing
HAdV species, populated by 45 of 70 typed HAdVs in GenBank,
and 13 of the 18 HAdVs most recently typed. Because the viruses
within HAdV-D appear to evolve by multiple rounds of homologous
recombination, the degree of relatedness across numerous types
at specific genetic loci can be difficult to discern by traditional
bioinformatics tools. Proteotyping is a novel bioinformatics approach
that provides a unique overview of homologous recombination events
in genome evolution.
Methods: In proteotyping, maximum likelihood trees are used to
align amino acid sequences of hypervariable, frequently recombined
proteins. The clade-guided consensus sequence is determined, and
each amino acid is assigned a unique, arbitrary color. Consensus
residues are colored white, and gaps in the alignment are colored
black. A threshold of <10% sequence divergence is used to
distinguish unique proteotypes. Proteotyping analysis was applied to
open reading frames across 38 whole genome sequenced HAdV-Ds,
including those associated with epidemic keratoconjunctivitis.
Results: Among hypervariable regions, the hexon protein was found
to be the most variable (least recombined) with 28 proteotypes, while
the putative E3 CR1α protein had only 6 proteotypes for 38 viruses,
suggesting significant prior recombination. The E3 CR1β and CR1γ,
and fiber proteins had 14, 15, and 22 proteotypes, respectively. The
fiber proteotype most closely predicted corneal tropism. Highly
conserved genes such as DNA polymerase had only one proteotype.
HAdV-D proteotyping results correlated well with the results of
traditional recombination analyses, including Bootscan and SimPlot.
Conclusions: Proteotyping identifies unique amino acid
signatures across a virus species, providing both an overview
of recombination patterns as well as detailed information about
conserved and variant amino acid positions that phylogenetic trees
cannot offer. Proteotyping represents a valuable tool to discern
the evolution of viruses within HAdV-D associated with epidemic
keratoconjunctivitis.
Commercial Relationships: James Chodosh, None; Gurdeep
Singh, None; Christopher Robinson, None; Jeong Yoon Lee, None;
Jaya Rajaiya, None; Morris Jones, None; David Dyer, None;
Donald Seto, None
Support: NIH grants EY013124, EY021558, and P30EY014104, a
Senior Scientific Investigator Award (to JC) from Research to Prevent
Blindness, the Massachusetts Lions Eye Research Fund, and the Falk
Foundation
Identification and characterization of a novel microbial
virulence-associated transcription factor and its role in ocular
infection
Robert M. Shanks, Nicholas A. Stella, Kimberly M. Brothers,
Kristin M. Hunt, Xinu Liu, Eric G. Romanowski, Regis P. Kowalski.
Chemistry, University of Pittsburgh, Pittsburgh, PA.
Purpose: To identify bacterial regulators necessary for microbial
keratitis. Bacterial keratitis progresses rapidly and can cause vision
loss even with effective antibiotics. An unknown gene was isolated
in genetic screens. Its role in controlling virulence factor production,
cytotoxicity to human corneal cells, corneal wound healing, and
keratitis was determined.
Methods: Molecular genetic, biochemical, RNA-Seq, proteomic, and
metabolomic approaches were used to characterize an eepR mutant
in a keratitis isolate of the bacteria Serratia marcescens. HCLE cells
were used in cytotoxicity assays and cell migration assays. Pig eyes
were used for corneal organ culture, and NZW rabbits were used for
keratitis studies.
Results: Transposon mutations were isolated in an uncharacterized
gene in a screen for genes involved in secretion of protease and
hemolysin activity using S. marcescens. Targeted deletion of eepR
in clinical and laboratory strains and complementation analysis
confirmed the importance of EepR in transcription of metalloprotease
and hemolysin genes. EMSA analysis confirmed a direct binding
of EepR to the hemolysin gene promoter, but not the protease gene
promoter. Proteomic analysis supported the importance of EepR in
protease secretion. Mass-spec and metabolomic analysis confirmed
loss of hemolysin production in the eepR mutant. Bacteria lacking
the eepR gene lost cytotoxicity to HCLE cells. This defect could
be restored by expression of the metalloprotease gene in the eepR
mutant. The eepR mutant was unable to inhibit corneal cell migration
and wound healing. In a rabbit keratitis model, EepR was necessary
for proliferation of the bacteria with a significant reduction in CFU
recovered from corneas infected with the eepR mutant compared to
the parental strain.
Conclusions: These results indicate EepR is a novel bacterial
transcription factor required for expression of secreted protease and
hemolysin activity from ocular clinical bacterial isolates. The EepR
protein regulates cytotoxicity through protease regulation and hostpathogen interactions through a yet unknown mechanism. EepR is
required for bacterial proliferation in mammalian corneas. Further
studies are required to determine the mechanism behind EepR’s role
in proliferation and corneal wound healing inhibition.
Commercial Relationships: Robert M. Shanks, None; Nicholas
A. Stella, None; Kimberly M. Brothers, None; Kristin M. Hunt,
None; Xinu Liu, None; Eric G. Romanowski, None; Regis P.
Kowalski, None
Support: NIH Grant AI085570, NIH Grant EY08098, Research to
Prevent Blindness
Impact of microbiome on ocular immunity
Mihaela G. Gadjeva1, Abirami Kugadas1, Laura Ruiz2, Sharmila
Masli2. 1Medicine, Brigham and Womens Hospital, Boston, MA;
2
Department of Ophthalmology, Boston University, Boston, MA.
Purpose: How microbiome affects the ocular immunity is at its
early stage of accumulating experimental evidence. Here, we carried
out experiments to evaluate the repertoire of ocular commensals
in healthy mice, and autoimmune-prone mice. To determine the
significance of microbiome in promoting health, we compared the
susceptibility to infection of germ-free (GF) mice, conventional mice,
and GF mice reconstituted with murine or human microbiota.
Methods: To characterize the ocular commensals in healthy mice and
mice that develop Sjögren syndrome-like disease, conjunctival swabs
were collected from the conventionally bred wild type (C57BL6)
mice and thrombospondin-1 (Tsp-1 -/-) deficient mice and plated
on selective agar media. To evaluate the ocular immune status of
the different groups of mice, quantitative LC-MS/MS analysis of
ocular surface proteomes were carried out. To evaluate the impact of
microbiome on sensitivity to infection, mice were challenged with P.
aeruginosa 6294, bacterial presence in the cornea and the degree of
developed ocular pathology during keratitis were quantified.
Results: We found that the repertoire of the ocular microbiota in mice
was limited to Staphylococcus aureus, Staphylococcus coagulase
negative sp, and Streptococcus sp. The Tsp-1-/- mice showed
significant increase in Staphylococcus aureus, Staphylococcus
coagulase negative sp ocular commensals, suggestive of a defect
in the clearance mechanisms. Quantitative LC-MS/MS analysis
of ocular surface proteomes demonstrated that in the absence of
commensals, the tear-film components were altered. For example,
complement pathway components and iron-scavenging proteins
were significantly reduced in the GF animals. In agreement with this
finding, the GF mice were more sensitive to ocular P. aeruginosa–
induced keratitis than the conventional mice. This was exemplified
by increased bacterial presence and elevated corneal pathology. Upon
reconstitution of GF mice with either mouse or human microbiota,
the resistance to infection and the levels of ocular innate immune
mediators were recovered.
Conclusions: Our data suggest that tonic signals from local
commensal flora continuously induce increased synthesis of ocular
innate immune effectors to limit microbial presence at the ocular
surfaces. Furthermore, when the host fails to limit commensal
outgrowth, autoimmune disease states could occur.
Commercial Relationships: Mihaela G. Gadjeva, None; Abirami
Kugadas, None; Laura Ruiz, None; Sharmila Masli, None
Support: NIH/NEI R01 EY022054
Toxigenic Staphylococcus Aureus bacteria activate conjunctival
goblet cell NLRP3 inflammasomes using TLR 2, TLR1, and α
toxin
Darlene A. Dartt1, 2, Dayu Li1, 2, Robin R. Hodges1, 2, Michael
Gilmore3, 2, Meredith S. Gregory-Ksander1, 2. 1Schepens Eye Research
Institute/MEEI, Boston, MA; 2Ophthalmology, Harvard Medical
School, Boston, MA; 3Massachusetts Eye and Ear Infirmary, Boston,
MA.
Purpose: To determine the Toll-like receptor (TLR) and pore forming
signals used by toxigenic Staphylococcus aureus (S. aureus) bacteria
in conjunctival goblet cells to activate the NLRP3 inflammasome to
produce IL-1β.
Methods: Cultured human goblet cells were incubated for 72 hrs
with siRNA for NLRP3, TLR 1, 2, and 6; scrambled siRNA; or
vehicle. Cells were then stimulated with no additions or S. aureus
RN6390. NLRP3 activation was determined by measuring proIL1β expression by western blotting and mature IL-1β secretion by
ELISA. The amount of TLR-1 and -2 expression in cultured cells
was measured by Q-PCR. Cultured goblet cells were stimulated by
no additions, α toxin, toxigenic S. aureus RN6390, and S. aureus
ALC837 that only lacked α toxin. Caspase-1 activity was measured
by FLICA along with pro-IL-1 β expression and mature IL-1β
secretion.
Results: NLRP3 siRNA completely decreased NLRP3 expression,
pro-IL-1β expression, and mature IL-1β secretion stimulated by S.
aureus RN6390. Use of non-target siRNA had no effect. When cells
were stimulated with S. aureus RN6390, TLR2 siRNA completely
blocked pro-IL-1β expression and mature IL-1β secretion. Nontarget siRNA had no effect. Stimulation of pro-IL-1β expression
and mature IL-1β secretion by S. aureus RN6390 in the presence
of TLR1 siRNA was significantly, but not completely decreased. In
contrast in the presence of TLR6 siRNA, S. aureus RN6390 did not
significantly alter pro-IL-1β expression and mature IL-1β secretion.
Values were the same as in the presence of non-target siRNA. TLR1
was expressed at much lower levels than TLR2 in cultured goblet
cells. S. aureus RN6390, but not α toxin nor S. aureus ALC837,
stimulated pro-IL-1β expression and mature IL-1β secretion. The α
toxin was active as it stimulated caspase-1 activity to the same level
as S. aureus RN6390. Simultaneous addition of α toxin and S. aureus
ALC837 restored pro-IL-1β expression and mature IL-1β secretion to
the same level as produced by S. aureus RN6390.
Conclusions: We conclude that in conjunctival goblet cells toxigenic
S. aureus stimulates the NLRP3 inflammasome to secrete mature IL1β by activating TLR2, to a lesser extent TLR1, but not TLR 6 and
by the pore forming action of its α toxin.
Commercial Relationships: Darlene A. Dartt, None; Dayu Li,
None; Robin R. Hodges, None; Michael Gilmore, None; Meredith
S. Gregory-Ksander, None
Support: NIH grant EY017381
Pseudomonas aeruginosa isolates with lasR mutations were
associated with worse visual outcomes in the Steroids For
Corneal Ulcer Trial (SCUT)
Michael E. Zegans1, Kathryn Ray2, Wesley Hebert1, Amanda Naimie1,
John H. Hammond1, Lalitha Prajna3, Deborah Hogan1, Antonio
DiGiandomenico4, Thomas M. Lietman2, Muthiah Srinivasan3.
1
Microbiology and Immunology, Geisel School of Medicine at
Dartmouth, Lebanon, NH; 2Ophthalmology, F.I Proctor Foundation,
UCSF, San Francisco, CA; 3Ophthalmology, Aravind Eye Hospital,
Maduri, India; 4MedImmune, Gaithersburg, MD.
Purpose: Pseudomonas aeruginosa (PA) uses quorum sensing
to measure cell density and regulate production of virulence
factors. In PA, quorum sensing is mediated through the activity
of 3 hierarchically arranged signaling cascades, with the LasR
transcription factor positioned as the master regulator. While lasR up
regulates known virulence factors such as proteases, lasR mutants are
frequently isolated from infections, suggesting that lasR is selected
against in certain clinical contexts. We investigated 101 PA keratitis
isolates from the SCUT study for evidence of lasR mutations and the
effect of these mutations on visual outcomes. This is the first large
scale study of lasR among PA keratitis strains.
Methods: 101 PA SCUT isolates were screened for the lasR mutant
sheen colony phenotype by streaking them on a lysogeny broth agar
plate. Colonies with an iridescent sheen were considered possible
lasR mutants. The lasR gene including the promoter was sequenced
in all candidate isolates. Normal LasR function is associated with
production of proteases, surfactant and homoserine lactones (HSL).
Our candidate lasR mutants were assessed for these phenotypes. As
a control, all of the above assays were performed with randomly
selected SCUT PA isolates without the iridescent sheen phenotype.
Finally, we compared vision at presentation and at 3 months in
patients infected with las-intact vs. las- deficient Pseudomonas.
Results: 22 of 101 PA isolates from the SCUT study were noted
to have colony morphology with a sheen phenotype characteristic
of lasR mutations. Mutations in the lasR gene or promoter were
identified among the candidate lasR mutants, but not in the other
SCUT PA isolates. We verified the loss of LasR signaling by
demonstrating decreased protease activity (p<0.01), decreased
surfactant production (p<0.01), and a lack of production of HSL.
Finally, SCUT patients infected with strains with lasR mutations had
worse vision at presentation and at 3 months.
Conclusions: Mutations of lasR were common (22/101) among
PA keratitis isolates from the SCUT study and associated with
worse vision at presentation and at 3 months compared to patients
infected with las-intact strains. These data suggest that conventional
assumptions about the relationship between quorum sensing and
virulence may need to be re-evaluated.
Commercial Relationships: Michael E. Zegans, None; Kathryn
Ray, None; Wesley Hebert, None; Amanda Naimie, None; John H.
Hammond, None; Lalitha Prajna, None; Deborah Hogan, None;
Antonio DiGiandomenico, MedImmune (E); Thomas M. Lietman,
None; Muthiah Srinivasan, None
Support: NEI U10 EY015114
Clinical Trial: NCT00324168.
469 Corneal inflammation due to allergy, dry eye, or transplant
Wednesday, May 06, 2015 3:45 PM–5:30 PM
Program #/Board # Range: 4871–4895/A0120–A0144
Contributing Section(s): Cornea, Physiology/Pharmacology
Microbial Keratitis Outcome in Corneal Grafts
Arthur Okonkwo1, 2, Jeffry Hogg1, 2, Hamed Anwar2, We Fong Siah2,
Francisco C. Figueiredo1, 2. 1Newcatle University, Newcastle, United
Kingdom; 2Royal Victoria Infirmary, Newcastle, United Kingdom.
Purpose: Identify risk factors for microbial keratitis in corneal grafts
and subsequent outcomes.
Methods: A retrospective analysis of medical records of all patients
presenting with a microbial keratitis (excluding herpetic keratitis)
in corneal grafts to the Royal Victoria Infirmary, Newcastle upon
Tyne, United Kingdom, between January 2003 and July 2014.
Demographics, predisposing factors, clinical, microbiological
findings and final outcomes were analyzed.
Results: Fifty-eight episodes of microbial keratitis were identified
in 40 eyes of 40 patients (38 penetrating keratoplasty and 2 deep
anterior lamellar keratoplasty). Eleven (27.5%) patients presented
with multiple episodes of infection of which 8 of them had a failed
graft. Mean age of patients was 65.9 (± SD 19.6, range 25.0-89.6)
years. The median time interval between corneal graft transplantation
and the incidence of microbial keratitis was 49.5 months (range
2-158 months). In these eyes, the predisposing factors for a microbial
keratitis were failed corneal graft (61.4%), concurrent use of topical
glaucoma medication(s) (59.6%) or topical antibiotic (28.1%),
history of herpetic eye disease (30.4%), contact lens wear (29.8%),
co-existing ocular surface problem such as keratoconjunctivitis
sicca (17.5%) or atopic eye disease (8.8%). About 75% of the cases
had 2 or more predisposing factors. Corneal scrape culture showed
gram-positive organism in 38.3% of cases, gram-negative organism
in 23.4%, fungal species in 4.3% and no growth in 34.0%. Four eyes
developed failed corneal grafts following the microbial keratitis
while 2 eyes were complicated with a perforated ulcer (1 eye had
underlying Peter’s anomaly, failed graft and Molteno tube; 1 eye had
a large Staphylococcal ulcer in a failed graft). Best-corrected visual
acuity at final follow-up visit was 20/40 or better in 12 (20.7%)
cases, between 20/40 and 20/200 in 17 (29.3%) cases and worse than
20/200 in 29 (50%) cases. Twelve eyes (30.0%) had lost visual acuity
post-keratitis compared to their baseline vision.
Conclusions: Our analysis revealed that the majority of cases
presenting with microbial keratitis in a corneal graft have multiple
predisposing risk factors; failed graft is a major determinant. The
occurrence of microbial keratitis in corneal graft is associated with a
high ocular morbidity rate with poor visual outcome.
Commercial Relationships: Arthur Okonkwo, None; Jeffry Hogg,
None; Hamed Anwar, None; We Fong Siah, None; Francisco C.
Figueiredo, None
Mechanisms of V-domain Ig suppressor of T cell activation
(VISTA)-mediated acceptance of corneal allografts
Tomoyuki Kunishige1, Hiroko Taniguchi1, Tatsukuni Ohno2, Miyuki
Azuma2, Junko Hori1. 1Nippon Medical School, Tokyo, Japan; 2Tokyo
Medical and Dental University, Tokyo, Japan.
Purpose: V-domain Ig suppressor of T cell activation (VISTA) is a
novel and structurally distinct Ig superfamily inhibitory ligand. We
have previously demonstrated that survival of allografts treated with
anti-VISTA mAb was less than that of the control, and that VISTA
plays important role in induction of alloantigen-specific ACAID. (1)
To further investigate the mechanism of VISTA-mediated corneal
allograft survival, we examined destruction of corneal endothelial
cells (CECs) by allo-reactive T cells in vitro. (2) As the next, we
examined infiltrating T cells in the graft-bearing eyes from the
recipients treated with anti-VISTA or control IgG.
Methods: The corneas from C57BL/6 (B6) eyes pre-treated with
anti-VISTA mAb or control rat IgG were incubated with CD4+ T
cells for 6h. Dead CECs stained with propidium iodide were counted
and compared. (2) Normal corneas of C57BL/6 were transplanted
into normal eyes of BALB/c mice. Recipients were administrated
with 0.2 mg of anti-VISTA mAb or control rat IgG, three times a
week after grafting. For Immunohistochemical staining, graft-bearing
eyes were removed.
Results: No significant differences were observed between the
number of dead CECs treated with anti-VISTA monoclonal
antibodies (mAb) and that treated with control IgG after incubation
with allo-reactive T cells. It is indicated that VISTA does not have
protective effect in the cornea from the allo-specific killing by CD4
T cells. (2) Immunofluorescent staining of the graft-bearing eyes at
3 to 5 weeks after grafting revealed that the number of infiltrating
CD4+ T cells and CD8+ T cells at graft center and host-graft junction
were significantly higher in anti-VISTA treated recipients compared
to control.
Conclusions: Taken together of our present and previous data, it
is suggested that VISTA plays an role in the acceptance of corneal
allografts by inducing allo-specific ACAID which suppress T cell
infiltration into the cornea, although VISTA doesn’t have a local
protective effect in the interaction between CECs and CD4+ T cells.
Commercial Relationships: Tomoyuki Kunishige, None; Hiroko
Taniguchi, None; Tatsukuni Ohno, None; Miyuki Azuma, None;
Junko Hori, None
Support: Grant-in-Aid for Scientific Research (C) from the Japan
Society for the Promotion of Science.
Mesenchymal stem cell based therapy for restoring corneal graft
transparency
Vijayalakshmi Rajendran1, Rosie Fordyce1, Izabela Klaska1, John V.
Forrester1, 3, May Griffith2, Lucia Kuffova1. 1Immunity, Infection and
Inflammation, University of Aberdeen, Aberdeen, United Kingdom;
2
Integrative Regenerative Medicine Centre, Linköping University,
Linköping, Sweden; 3Immunology and Virology, University of
Western Australia, Crawley, WA, Australia.
Purpose: Mesenchymal stem cells (MSC) are being extensively
tested in the field of organ transplantation due to their potent antiinflammatory and tissue regenerative properties. In the case of
corneal transplantation, the additional anti-angiogenic properties
of MSC make them an attractive candidate for maintaining corneal
clarity. This project aims to investigate the effect of allogeneic vs
syngeneic MSC in preventing loss of and/or restoring corneal graft
clarity in mouse corneal allograft model
Methods: Recipient-derived (syngeneic-Balb/c) and donor-derived
(allogeneic-B6) bone marrow MSC were isolated, depleted of
CD45+ cells and characterized as lineage negative. The effect of
MSC on in vitro T cell proliferation was assessed by CFSE labelling.
MSC(1x106 cells) were administered in vivo either intravenously
(IV) or subconjuctivally (SC) 7 days before (1st dose) and on the
day (2nd dose) of corneal allotransplantation (B6 to Balb/c). Corneal
graft opacity was evaluated for 2 months post grafting. Calcein/
CFSE labelled MSC were administered IV to determine where
MSC trafficked after inoculation. In addition, in vivo induction of T
regulatory cells (Tregs) in secondary lymphoid organs was assessed
by flow cytometry
Results: Both allo and syn-MSC had strong inhibitory effects
on T cell proliferation in vitro. Only IV administered allo-MSC
significantly reduced corneal opacification (p<0.0001) compared to
syn-MSC and untreated (PBS) controls at 2 months post-transplant.
Calcein/CFSE labelled splenocytes were tracked to the spleen and
the eye-draining lymph nodes 16 hrs after inoculation by both IV
and SC routes. However, labelled MSC could not be detected when
administrated IV. Flow cytometric analysis at day 4 after IV showed
syn-MSC but not allo-MSC induce significantly higher frequency
of splenic Tregs (CD4+ Foxp3+) compared to PBS treated controls
(n=4/group)
Conclusions: Our data show that IV but not SC administration of
allo-MSC significantly improved corneal allograft survival. SynMSC were not effective. This data suggest that allo-MSC induce
immunological tolerance to corneal allograft. However the effect
did not correlate with induction of Tregs. In addition, the inability
to track MSC after IV inoculation suggests that they are rapidly
removed from the system presumably by phagocytic engulfment.
Engulfment of apoptotic allo-MSC may be a possible mechanism for
inducing allospecific tolerance in this model
Commercial Relationships: Vijayalakshmi Rajendran, None;
Rosie Fordyce, None; Izabela Klaska, None; John V. Forrester,
None; May Griffith, None; Lucia Kuffova, None
Second corneal allografts ablate T regulatory cells that are
induced by first corneal allografts and produce a permanent loss
of immune privilege in both eyes
Jerry Y. Niederkorn1, Kathryn J. Paunicka2, jessamee Mellon1, joseph
R. brown1. 1Ophthalmology, Univ Texas Southwestern Med Ctr,
Dallas, TX; 2Neurobiology, Stanford University, Stanford, CA.
Purpose: To determine the mechanism whereby an initial penetrating
keratoplasty (PK) abolishes immune privilege of subsequent corneal
allografts
Methods: Corneas from C57BL/6 donors were grafted orthotopically
to BALB/c mice. A 2.0 mm trephine was used to make shallow
circumferential incisions in left eyes prior to applying corneal
allografts to right eyes. T regulatory cell (Treg) activity was measured
in vivo using a local adoptive transfer (LAT) of DTH assay. Tregs
were also assessed by flow cytometry.
Results: C57BL/6 corneal allografts underwent rejection in 50% of
naïve BALB/c hosts. However, PK to one eye abolished immune
privilege and resulted in 100% graft rejection in the contralateral,
unmanipulated eye, even when the first corneal graft was syngeneic
and did not evoke an immune response. This effect could be
mimicked with either 360° or 180° corneal incisions, which induced
rejection of 90-100% corneal grafts transplanted to the opposite
eye, even 100 days after the initial corneal incision. PK induced
a 300-fold increase in substance (SP) production in both eyes.
Intravenous injection of 1.0 pg of SP abolished immune privilege of
corneal allografts (>90% rejection), which persisted for ≥100 days.
Placing shallow 360° corneal incisions in one eye also induced the
immune rejection of long-standing (>60 days) corneal allografts
in the opposite eye. The surgery-induced loss of immune privilege
coincided with a steep down-regulation in GITR expression on
CD4+CD25+ T regulatory cells (Tregs) but had no effect on Treg
expression of CTLA-1, Foxp3, or TGF-β on corneal allograftinduced Tregs.
Conclusions: First time C57BL/6 corneal transplants enjoy a 50%
acceptance rate in naïve BALB/c mice. However, circular corneal
incisions used in preparation for a second PK disable disable T regs
by down-regulating GITR expression. The ablation of T reg function
induced by second PK also abolishes the immune privilege of longstanding corneal allografts in the opposite eye and leads to immune
rejection of previously healthy corneal transplants.
Commercial Relationships: Jerry Y. Niederkorn, None; Kathryn
J. Paunicka, None; jessamee Mellon, None; joseph R. brown,
None
Support: NIH grants EY007641 and EY020799 and Research to
Prevent Blindness
The contribution of T cells to corneal angiogenic privilege in
transplantation
Antonio Di Zazzo, Brinda Subbarayal, Thomas H. Dohlman, Takenori
Inomata, Sunil Chauhan, Reza Dana. Ophthalmology, Schepens Eye
Research Institute, Boston, MA.
Purpose: To investigate the effect of T effector cells (Teffs) and/
or regulatory T cells (Tregs) from high-risk (HR) or low-risk (LR)
transplanted mice on vascular endothelial cell (VEC) proliferation.
Methods: Ipsilateral lymph nodes were collected from Balb/c
mice 14 days after low-risk and high-risk corneal transplantation.
CD4+CD25- effector T cells and CD4+CD25+ Tregs, sorted by
magnetic beads and stimulated with anti-CD3, were co-cultured in a
96-well plate with MILE SVEN 1 cells, a vascular endothelial cell
line, in DMEM/FBS10%. VEC proliferation was measured using the
BrdU incorporation assay 19 hours after coculturing.
Results: CD4+CD25- Teffs from HR and LR transplanted mice
significantly promoted VEC proliferation compared with positive
(VEGF-A 20ng/ml) and negative controls. VEC proliferation was
significantly higher when cocultured with Teffs from HR than Teffs
from LR and naïve mice (Teff HR 0.9 ± 0.03;Teff LR 0.7234 ± 0.1;
VEGF-A 0.6 ± 0.03; Negative Control: 0.5± 0.02 p<0.0001). After
culturing Teffs and VECs with CD4+CD25+ Tregs from transplanted
mice, VEC proliferation was significantly decreased compared with
VECs cultured with Teffs alone (Teff 0.9 ± 0.03; Teff+Treg 0.6 ±
0.06; p<0.0005). Tregs alone had no effect on VEC proliferation.
Conclusions: Our data show that CD4+CD25- effector T cells
isolated from HR and LR transplanted mice directly induce
VEC proliferation. Further, Tregs inhibitTeff cell-mediated VEC
proliferation. Our results suggest that a balance between proaniogenic Teffs and anti-angiogeneic Tregs is essential to sustain
angiogenic privilege in HR corneal
Commercial Relationships: Antonio Di Zazzo, None; Brinda
Subbarayal, None; Thomas H. Dohlman, None; Takenori
Inomata, None; Sunil Chauhan, None; Reza Dana, None
Support: NIH EY 012963
Interleukin-12 and Interleukin-23-mediated IFN-γ Secretion by T
Helper 17 Cells Exacerbates Dry Eye Disease
Yihe Chen, Sunil Chauhan, Jing Hua, Masahiro Omoto, Reza Dana.
Schepens Eye Research Ins /MEEI, Boston, MA.
Purpose: Previously, we found that IL-17 and IFN-γ double-positive
T helper (Th17/1) cells can induce dry eye disease (DED). In this
study, we aimed to determine the cytokines that promote conversion
of Th17 cells to Th17/1 cells.
Methods: Dry eye disease (DED) was induced in wild-type (WT)
C57BL/6 mice using the controlled environmental chamber (CEC)
for 14 days. The draining lymph nodes (DLN) of DED mice were
harvested, and Th17 (IL-17+IFN-γ-) cells were sorted using cytokine
secretion assay kits. Subsequently, sorted Th17 cells were injected
intravenously into naïve Rag-/- mice (no B or T cells), which were
then placed in the CEC for 5 days. These Rag-/- mice were injected
with anti-IL12p40, anti-IL12, anti-IL23R antibodies, or isotype IgG
one day before and one day after the transfer of Th17 cells. Disease
severity was evaluated using corneal fluorescein staining (CFS). T
cell cytokine secretion was analyzed using real-time PCR and flow
cytometry.
Results: On day 2 and day 5 after the transfer of Th17 cells, mice
treated with anti-IL12p40, anti-IL12, or anti-IL23R antibodies
showed significantly milder disease severity than those treated with
isotype IgG (CFS scores on day 2: 2.5±1.0, 3.5±0.5, 1.3±0.8, vs.
6.3±0.8, p < 0.05; day 5: 3.5±0.3, 4.0±1.0, 2.5±0.9, vs. 7.5±1.0, p
< 0.05). The disease severity among the different antibody-treated
groups was similar. On day 5 after transfer, 36% of the purified Th17
cells converted to Th17/1 cells in the DLNs of mice treated with
isotype IgG. In contrast, only 12%, 23%, and 20% of the transferred
Th17 cells became double positive (IL-17/IFN-γ) in mice treated
with anti-IL12p40, anti-IL12, or anti-IL23R antibodies, respectively.
The mRNA expression of IFN-γ was significantly reduced on the
conjunctivae in all three antibody treated groups compared to isotype
IgG treated mice (mRNA copy numbers per 106 GAPDH copies:
22.1±2.3, 26.4±0.2, 20.0±4.3, vs. 40.0±4.6 in anti-IL12p40, antiIL12, anti-IL23R, vs. isotype IgG treatments).
Conclusions: These data demonstrated that blockade of IL-12 or
IL-23 ameliorates DED by preventing the conversion of Th17 to
double-positive Th17/1 cells and diminishing IFN-γ production in the
conjunctiva.
Commercial Relationships: Yihe Chen, None; Sunil Chauhan,
None; Jing Hua, None; Masahiro Omoto, None; Reza Dana, None
Proinflammatory Th17 cells are more effective than Th1 cells in
providing B cell help in dry eye disease.
Brinda Subbarayal, Antonio Di Zazzo, Susanne Eiglmeier,
SANGMOK LEE, Sunil Chauhan, Reza Dana. Opthalmology,
Schepens Eye Research Institute, Quincy, MA.
Purpose: T cell help is known to be fundamental for the induction
and subsequent organization of germinal centers (GCs), resulting
in the generation of memory B cells and long-lived plasma cells.
However, it is not known whether Th17 cells can act as effective
helper T cells in experimental dry eye disease (DED). The aim of this
study was to determine whether Th17 cells function as helper cells
for B cells in DED.
Methods: Dry eye was induced by exposing female C57BL/6 mice
to desiccating stress (subcutaneous injection of scopolamine [0.5
mg/ 0.1 mL] 3 times a day, humidity < 15%, and airflow rate of
15L/min) for 3 weeks. To determine the kinetics of GC formation
and isotype switched B cells, cervical lymph nodes were stained
for GCs (B220+GL7+FAS+) and isotype switched (B220+IgDIgM-) B cells on day 0, 7, 14, 21, and 28 using flow cytometry. On
day 21, formation of GCs was analyzed by immunhistochemistcal
staining of lymph node sections with anti-ki67-FITC and anti-IgMAPC antibodies. T cell-B cell coculture assays were performed to
determine T cell help on B cells. The cells were activated with 1μg/
ml of anti-CD3 and 1μg/ml of anti-IgM for 36hours before analyzing
B cell proliferation using flow cytometry.
Results: Germinal center B cell formation in the lymph nodes
of DED mice peaked on day 21, whereas no GC formation was
observed in naïve mice. T effector (Teff) cells from DED mice
significantly enhanced B cell proliferation in a contact dependent
manner. To determine whether Th1
(IFN-γ+) or Th17 (IL-17+) cells provide major B cell help, IFN-γ or
IL-17 depleted Teff (CD4+CD25-) cells were cocultured with B cells.
Although both subsets effectively reduced B cell proliferation in
vitro, Th17 cells were more effective than Th1 cells in helping B cell
proliferation in DED mice.
Conclusions: Th17 cells are more effective than Th1 cells in
inducing B cell proliferation in DED.
Commercial Relationships: Brinda Subbarayal, None; Antonio Di
Zazzo, None; Susanne Eiglmeier, None; SANGMOK LEE, None;
Sunil Chauhan, None; Reza Dana, None
Support: NIH Grant R01-EY-20889
Caspase-8 Mediated Activation of NLRP3 and NLRC4
inflammasomes in Experimental Dry Eye Mouse Model and
Human Corneal Epithelial Cells Exposed to Hyperosmolarity
Jin Li1, 2, Wei Chi1, 3, Xia Hua1, 4, Fang Bian1, Xiaoyong Yuan1, 4,
Ruzhi Deng1, 2, Zongduan Zhang1, 2, Cintia S. De Paiva1, Stephen
C. Pflugfelder1, De-Quan Li1. 1Ophthalmology, Baylor College
of Medicine, Houston, TX; 2Ophthalmology, Wenzhou Medical
University, Wenzhou, China; 3Ophthalmology, Zhongshan
Ophthalmic Center, Sun Yan-Sen University, Guangzhou, China;
4
Ophthalmology, Tianjin Eye Hospital, Tianjin Medical University,
Tianjin, China.
Purpose: To explore the underlying mechanisms of innate immunity
in response to desiccating stress using an experimental dry eye
mouse model and human corneal epithelial cells (HCECs) exposed to
hyperosmolarity, an in vitro model mimicking dry eye environment.
Methods: The experimental dry eye was induce by desiccating stress
in C57BL/6 mice exposed to an air draft in <40% ambient humidity
with subcutaneous injection of scopolamine hydrobromide for 5
days. Primary HCECs were established from donor limbal explants.
The cultures in iso-osmolar medium (312 mOsM) were switched
to hyperosmotic media (400, 450 and 500 mOsM), with or without
prior incubation of NLRP3 inhibitor (glybenclamide), caspase-1
inhibitor (z-YVAD fmk), or caspase-8 inhibitor (z-IETD fmk).
Reverse transcription and quantitative real time PCR (RT-qPCR),
immunofluorescent staining, Western blotting, caspase activity assays
and ELISA were performed to evaluate the concerned molecules.
Results: NLRP3 and NLRC4 inflammasomes were found to be upregulated with increased production and activity of ASC, caspase-1,
caspase-8, IL-1β and IL-18 in corneal and conjunctival epithelia
of the dry eye mice, as well as in HCECs exposed to hyperosmotic
media, as evaluated by RT-qPCR, immunofluorescent staining,
Western blotting, ELISA and caspase activity assays. Glybenclamide
inhibited NLRP3 activation, and also blocked the activation of
caspase-1. Both inhibitors to NLRP3 or caspase-1 suppressed
maturation of IL-1β and IL-18. Interestingly, blockage of caspase-8
by z-IETD fmk down-regulated the NLRP3 and NLRC4, and further
suppressed the caspase-1 dependent activation of IL-1β and IL-18.
Conclusions: These findings uncovered a novel mechanism and
signaling pathway of innate immunity by ocular surface mucosal
epithelium in response to desiccating environment stress, which
activates NLRP3 and NLRC4 inflammasomes through caspase-8
mediated pathway to promote the casapase-1 dependent activation of
IL-1β and IL-18 during dry eye development. These results provide
new insight in the innate immunity that contributes to pathogenesis of
dry eye.
Commercial Relationships: Jin Li, None; Wei Chi, None; Xia
Hua, None; Fang Bian, None; Xiaoyong Yuan, None; Ruzhi Deng,
None; Zongduan Zhang, None; Cintia S. De Paiva, None; Stephen
C. Pflugfelder, None; De-Quan Li, None
Support: NIH NEI Grants EY023598 (DQL) and EY11915 (SCP),
Allergan Inc., Research to Prevent Blindness, Oshman Foundation,
William Stamps Farish Fund.
A model of allergic conjunctivitis in transgenic mice with
humanized FcεRIα
Kaspar W. Schürch1, Andreas Ebneter1, Chantal Dysli1, Alexander
Eggel2, Martin Zinkernagel1. 1Ophthalmology Department, Bern
University Hospital, Bern, Switzerland; 2Immunology Department,
Bern University Hospital, Bern, Switzerland.
Purpose: Allergy has an increasing prevalence and severity
especially in industrialized countries. The spectrum of allergic
symptoms ranges from allergic rhinitis to dermatitis to allergic
conjunctivitis. Allergic conjunctivitis is an immunoglobulin E (IgE)mediated type I allergic reaction. Because IgE is the central molecule
responsible for allergic reactions, neutralizing IgE or inhibiting the
IgE synthesis represents a rational approach for the treatment. We
present a transgenic mouse model for allergic conjunctivitis using
mice expressing a humanized high-affinity IgE receptor (B.6CgFce1a(tm1Knt)Tg(FcERIa)1Bhk/J).
Methods: Tg(FcεRIα) transgenic mice and controls were injected
with subconjunctival NIP specific human-IgE (JW8, 100 nM in
PBS) with subsequent intravenous allergen challenge with NIP
(NIP25-BSA, 1.5 mg/ml in Saline). Allergic reaction was quantified
in vivo using spectral domain anterior segment OCT (Heidelberg
Engineering) and ex vivo using immunohistochemistry for
monoclonal rabbit anti-mouse mast cell tryptase, polyclonal rabbit
anti-human IgE and FcεRIα and HE staining.
Results: The transgenic FcεRIα group showed a statistically
significant (p<0.05) swelling of the conjunctiva using anterior
segment OCT compared to the control group. The IgE binding was
confirmed by immunohistochemistry which was co-localized to
tryptase positive and FcεRIα positive mast cells.
Conclusions: We present the preliminary results of a novel mouse
model for allergic conjunctivitis using a transgenic mouse with a
humanized FcεRIα receptor and quantify the allergic reactions using
anterior segment OCT in vivo. This model will be helpful to dissect
immunological pathways in allergic conjunctivitis and may be useful
to develop novel therapeutic options for this common disease.
Commercial Relationships: Kaspar W. Schürch, None; Andreas
Ebneter, Bayer (R); Chantal Dysli, None; Alexander Eggel, None;
Martin Zinkernagel, Heidelberg Engineering (F)
The roles of epithelial cell derived type 2 initiating cytokines
in experimental allergic conjunctivitisThe roles of epithelial
cell derived type 2 initiating cytokines in experimental allergic
conjunctivitis
Yosuke Asada1, 2, Susumu Nakae2, Kanji Hori1, Jobu Sugita1,
Nobuyuki Ebihara3, Akira Murakami1, Akira Matsuda1.
1
Ophthalmology, Juntendo University School of Medicine, Tokyo,
Japan; 2Laboratory of Systems Biology, Center for Experimental
Medicine and Systems Biology, The Institute of Medical Science, The
University of Tokyo, Tokyo, Japan; 3Department of Ophthalmology,
Juntendo Urayasu Hospital, Chiba, Japan.
Purpose: To clarify the possible involvements of type 2 initiating
cytokines, interleukin (IL)-25, IL-33, thymic stromal lymphopoietin
(TSLP) for the pathophysiology of allergic conjunctivitis using
ragweed (RW)-induced experimental allergic conjunctivitis (EAC).
Methods: IL-25-/-, IL-33-/-, TSLP receptor (TSLPR) -/- and congenic
BALB/C wild type mice were sensitized with ragweed (RW) in alum,
and then challenged 4 times using RW eye drops. Clinical score
was evaluated at 20 minutes after the last eye drop challenge. The
conjunctival tissue was collected for histological analyses and for
cytokine expression analysis using real time PCR at 24 hours after
the last eye drop challenge.
Results: Significant reduction for the clinical scores and the numbers
of infiltrating eosinophils was observed in the RW-induced EAC
models using IL-33-/- mice. There was no significant difference
of the numbers of infiltrating eosinophils of the RW-EAC models
among IL-25-/-, TSLPR-/- and the wild-type mice. The mean numbers
of eosinophil per one slide were 17.2 ±1.4 in IL-33-/- mice, 72.0 ±
4.9 in IL-25-/- mice, 79.4 ± 15.3 in TSLPR-/- mice, and 79.3 ± 5.3
in the wild type mice. Il4 mRNA, il13 mRNA and ccl5 mRNA
expression was significantly higher in the conjunctivae of RW-EAC
models using wild type mice compared to those of IL-33-/- mice.
Immunoshistochemical staining using mouse basophil marker mcp8
showed significant reduction of the basophil numbers in the RW-EAC
models using IL-33-/- mice (1.25 ± 1.8 per one slides) compared to
those of the wild type mice (7.9 ± 5.3).
Conclusions: IL-33 plays essential roles for eosinophil infiltration
and the expression of Th2-type cytokines in RW-EAC models.
Significant reduction of basophils, which is known as an abundant
source of IL-4, may also contribute to the attenuated inflammatory
responses in IL-33-/- mice.
Commercial Relationships: Yosuke Asada, None; Susumu Nakae,
None; Kanji Hori, None; Jobu Sugita, None; Nobuyuki Ebihara,
None; Akira Murakami, None; Akira Matsuda, None
The Role of Group 2 Innate lymphoid Cells (ILC2s) in Mouse
Models of Papain-Induced Conjunctivitis
Jobu Sugita2, 3, Akira Matsuda2, Yosuke Asada2, 3, Nobuyuki Ebihara1,
Akira Murakami2, Susumu Nakae3. 1Department of Ophthalmology,
Juntendo Urayasu Hospital, Chiba, Japan; 2Depatrment of
Ophthalmology, Juntendo University School of Medicine, Tokyo,
Japan; 3Laboratory of Systems Biology, Center for Experimental
Medicine and Systems Biology, The Institute of Medical Science,
The University of Tokyo, Tokyo, Japan.
Purpose: Group 2 innate lymphoid cells (ILC2s) produce large
amounts of Th2 cytokines in response to IL-33 and induce eosinophil
infiltration. We previously identified ILC2s in mouse lacrimal glands
and in conjunctivae in response to papain induced inflammation. To
clarify the role of ILC2s in papain-induced conjunctivitis, ILC2s
were depleted in mice models.
Methods: To make papain-induced conjunctivitis model mice,
papain-soaked soft contact lens (2mm diameter) was inserted into the
conjunctival sacs of C57/BL6 (B6)-Rag2-/- mice. To deplete ILC2s,
200 μg of anti-CD25 antibody was injected into the Rag2-/- mice two
days before contact lens insertion. We also used B6-Rag2/common
γ chain (cγ)-/-, which lack acquired immune systems and ILC2s. 4
days after contact lens installation, the eyes were subjected to flow
cytometry analysis or histological analysis. To identify mouse ILC2s
by a flow cytometer, anti-mouse lineage mix antibodies, ST2, CD25,
CD127 and CD90.1 antibodies were used to mouse conjunctival
tissues from papain-induced conjunctivitis model.
Results: Anti-CD25 antibody injection in Rag2-/- mice accomplished
93% depletion of ILC2s in the conjunctival tissue compared to PBS
injection. The numbers of infiltrated eosinophil in papain-induced
conjunctivitis models diminished by the anti-CD25 antibody
treatment (48 ± 8 versus 14 ± 6.4, mean number of eosinophil ± SD
per slides, n = 8, P< 0.05 by Mann-Whitney’s U test). ILC2 deficient
Rag2/cγ-/- mice also showed diminished numbers of eosinophil
compared to Rag2-/- mice (53 ± 20 versus 8 ± 4, n = 8, P< 0.05)
Conclusions: ILC2s depletion by anti-CD25 antibody could diminish
eosinophil infiltration in papain-induced conjunctivitis model.
Commercial Relationships: Jobu Sugita, None; Akira Matsuda,
None; Yosuke Asada, None; Nobuyuki Ebihara, None; Akira
Murakami, None; Susumu Nakae, None
Differential role of immunoregulatory cells in mediating allergic
conjunctivitis in mouse model of severe ocular allergy.
Amirah Mohd Zaki1, Sarah B. Dale1, 2, Malihe Eskandarpour1,
Daniel Saban2, 3, Virginia L. Calder1. 1ORBIT, UCL Institute of
Opthalmology, London, United Kingdom; 2Ophthalmology, Duke
University, School of Medicine, Durham, NC; 3Immunology, Duke
University, School of Medicine, Durham, NC.
Purpose: The pathology and mechanisms underlying allergic
conjunctivitis are still poorly understood. A few studies have
investigated cytokines involvement during allergic conjunctivitis.
Severe mouse model that represent the pathology of ocular allergy in
human is needed in order to study more extensively the mechanisms
underlying allergic conjunctivitis. Here, we have used a mouse model
of severe ocular allergy to investigate different cells and cytokines
involvement in ocular allergy.
Methods: C57Bl/6 mice were immunized via intraperitoneal
injection with ovalbumin in aluminium hydroxide and pertussis
toxin and left for 21 days. Immunized mice were then challenged
once daily for 7 days with topical ovalbumin (OVA) and were
sacrificed a few hours after the last topical OVA administration.
Samples were collected from each mouse (of both ocular allergy
induced versus normal C57Bl/6) for the whole eye for H&E staining
and immunofluoresence staining of different cell types. Cells were
explanted from conjunctiva for FACS analysis of different cell types
that are present in the conjunctiva. Lymph nodes were also collected
for single cell analysis on the presence of different T cell subsets by
intracellular cytokine staining.
Results: There is increased in the number of infiltrating cells in the
conjunctiva after 7 days challenged with OVA as compared to normal
conjunctiva. Double immunfluorescnce staining has confirmed that
some of these infiltrating cells consist of mast cells, Th2 and Th9
cells. Further evaluation on the different cells and cytokines that are
present in the conjunctiva in severe ocular allergy by intracellular
staining has found that the number of cells that secretes IL-9 and IL10 has increased to almost double during disease but these cytokines
did not mainly secreted by Th9 cells as there are no difference in the
level of IL-9+IL-10+ cells. This is different in the lymph nodes where
level of Th9 cells increased five-folds during disease suggesting
that these cells is present in the systemic but did not migrate to the
conjunctiva and contribute to the disease severity.
Conclusions: Different cell types are involved in the pathogenesis of
allergic conjunctivitis. IL-9 and IL-10 might contribute to the disease
severity in the eye and other cells, but not Th9 cells as the source of
IL-9 during allergic conjunctivitis.
Commercial Relationships: Amirah Mohd Zaki, None; Sarah B.
Dale, None; Malihe Eskandarpour, None; Daniel Saban, None;
Virginia L. Calder, None
Development of a RNAi therapeutic for the treatment of allergic
conjunctivitis
Ana Isabel Jimenez1, Tamara Martinez1, Victoria Gonzalez2, Carmen
Martinez-Garcia3, Covadonga Paneda1. 1R&D, Sylentis, Madrid,
Spain; 2Preclinical, Sylentis, Madrid, Spain; 3Universidad de
Valladolid, Valladolid, Spain.
Purpose: We have shown that Orai1, the pore forming subunit
of the CRAC calcium channel, is upregulated in ovoalbumininduced allergic conjunctivitis in mice. Down-regulation of Orai1
has been shown to reduce sneezing and nasal rubbing in animal
models of allergic rhinitis. We have designed a battery of siRNA to
silence Orai1 and selected the most efficacious product for further
development for the treatment of allergic conjunctivitis.
Methods: The efficacy of different siRNAs to down-regulate Orai as
well as their effect on cell survival was assessed in human and murine
cell lines. In vivo efficacy of the lead compound and chemically
stabilized versions was studied in a mouse model of ovoalbumininduced allergic conjunctivitis.
Results: SYL116011 was shown to be the most potent of the
sequences studied, causing a maximal reduction of Orai1 mRNA
levels of 80% in human cells and 70% in mouse cells. This reduction
was observed in absence of alteration of cell viability. Prophylactic
SYL116011 treatment of a mouse model of ovoalbumin-induced
ocular allergy caused a dose dependent-reduction of allergy related
clinical signs (chemosis and tearing) and a reduction in the infiltration
of mast cells and eosinophils in the conjunctiva; these observations
were equivalent to those observed in response to patanol and to
some extent better than those observed in response to levocabastine.
The reduction in clinical signs and cell infiltration correlated with a
reduction in the expression levels of allergy biomarkers in the mouse
eye (TLSP and CD137).
Conclusions: We have identified a compound that efficiently downregulates Orai1. Prophylactic treatment of allergic mice with this
compound causes a reduction in allergy related endpoints.
Commercial Relationships: Ana Isabel Jimenez, Sylentis (E);
Tamara Martinez, Sylentis (E); Victoria Gonzalez, Sylentis (E);
Carmen Martinez-Garcia, Sylentis (F); Covadonga Paneda,
Sylentis (E)
Support: INDREYE Innpacto Grant IPT-2012-0438-010000,
SURFEYE Retos Grant RTC-2014-2375-1
Clinical features associated with steroid-induced ocular
hypertensive response in eyes with refractory allergic ocular
diseases with proliferative lesions or corneal involvement
Dai Miyazaki1, Atsuki Fukushima9, Yuichi Ohashi2, Eiichi Uchio3,
Naoki Kumagai4, Jun Shoji5, Etsuko Takamura6, Yayoi Nakagawa8,
Kenichi Namba7, Hiroshi Fujishima10. 1Ophthalmology, Tottori
University, Yonago, Japan; 2Ehime University School of Medicine,
Toon, Japan; 3Fukuoka University, School of Medicine, Fukuoka,
Japan; 4Kumagai Eye Clinic, Yamaguchi, Japan; 5Nihon University
School of Medicine, Tokyo, Japan; 6Tokyo Women’s Medical
University School of Medicine, Tokyo, Japan; 7Hokkaido University
Graduate School of Medicine, Sapporo, Japan; 8Nakagawa Eye
Clinic, Osaka, Japan; 9Kochi Medical School, Kochi, Japan;
10
Opthalmology, Tsurumi University Dental Hospital, Kanagawa,
Japan.
Purpose: To identify clinical factors significantly associated with
steroid-induced ocular hypertension in refractory allergic ocular
diseases with proliferative lesion or corneal involvement.
Methods: The 1436 refractory allergic ocular disease patients
enrolled for topical tacrolimus treatment trial were retrospectively
analyzed. Of the 1436 eyes treated with topical tacrolimus, 410
patients necessitated with topical steroids in conjunction during the 3
months follow up, were analyzed.
The baseline intraocular pressures (IOP) were measured, and the
clinical signs were rated on a 4 grade scale. The change in the IOP
(ΔIOP) during the follow up were calculated by subtracting the base
line from the maximum IOP. Patients responded with ≥ third quartile
of IOP change was defined as steroid-induced hypertensive.
Results: The mean ΔIOP was 0.72 ± 0.17 mmHg (median, 0
mmHg; third quartile, 3 mmHg). The incidence of giant papillae
was significantly higher for patients who had steroid-induced
hypertension (P=0.0007). The IOP elevation was inversely correlated
with age (ρ=-0.142, P<0.005), and the incidence of IOP elevation
was significantly higher in patients without eczema (P<0.05).
Logistic analysis on the ocular hypertensive responders showed that
eyes with giant papillae had a significant high OR of 3.43 for the
maximum score after age adjustments (95% confidence interval, 1.577.49; P=0.002). The area under the receiver operating characteristic
curve for prediction of ocular hypertensive response was 0.60, 0.54,
and 0.56 for giant papilla, younger age (≤median), and eyes without
eczema, respectively.
Conclusions: Refractory allergic eyes with giant papillae, without
eczema, and younger age were significantly associated with positive
ocular hypertensive response. These findings may help to predict eyes
that will have steroid-induced hypertension.
Commercial Relationships: Dai Miyazaki, None; Atsuki
Fukushima, None; Yuichi Ohashi, None; Eiichi Uchio, None;
Naoki Kumagai, None; Jun Shoji, None; Etsuko Takamura,
None; Yayoi Nakagawa, None; Kenichi Namba, None; Hiroshi
Fujishima, None
Experience with the monoclonal anti IgE antibody Omalizumab
in severe refractory vernal keratoconjunctivitis in children
Serge Doan1, Flore Amat2, Eric E. Gabison1, isabelle cochereau1,
Jocelyne Just2. 1Ophthalmology, Bichat Hospital & A de Rothschild
Foundation, Paris, France; 2Allergology, Hopital Trousseau, Paris,
France.
Purpose: Vernal keratoconjunctivis (VKC) is a severe form
of pediatric ocular allergy, characterized by acute and chronic
corneoconjunctival inflammation that may lead to visual sequelae.
Although topical immunosuppressive drugs such as cyclosporine are
usually effective, some severe forms may be refractory and require
prolonged steroid therapy. Omalizumab is a monoclonal anti IgE
antibody, administered systematically and authorized for severe
asthma. We report our clinical experience with omalizumab in severe
VKC children.
Methods: We retrospectively reviewed the files of 4 boys treated
with omalizumab because of severe VKC, defined as persistent
corneal inflammation despite continuous topical 2% cyclosporine and
steroid eye drops.
Results: Four boys, aged 7 to 13 years old, were treated. All children
had asthma and 1 had severe lid eczema. Two patients had required
supratarsal steroid injections. Omalizumab was administered every 2
weeks by subcutaneous injections, at doses varying from 450 to 600
mg per injection. Three patients out of 4 responded to the treatment,
with a decrease in frequency and in duration of the inflammatory
flares, and also a decreased need for topical steroid. However,
the response was incomplete and they still had inflammatory
corneoconjunctival flares despite continuous topical cyclosporine. On
the other hand, asthma and lid eczema were completely controlled
in these 3 patients. The fourth child did not respond to o and needed
oral steroids for his VKC and his asthma. Noticeably, this patient
did not have detectable sensitization to any allergen, contrary to the
other cases. The treatment was stopped in this refractory case, but is
still ongoing in all other cases, with a median duration of 16 months
(range, 6 to 26 months).
Conclusions: Omalizumab is an interesting treatment in severe
refractory forms of VKC, but its efficacy is incomplete in these very
severe cases.
Commercial Relationships: Serge Doan, None; Flore Amat, None;
Eric E. Gabison, None; isabelle cochereau, None; Jocelyne Just,
None
Evaluation of a Novel PI3-kinase Inhibitor in a Murine Model of
Allergic Conjunctivitis: Treatment of Chronic Inflammation in
Ocular Surface Disease
Andy Whitlock1, Zhenhua Huang2, Yanli Wang2, Laura Belen1,
Claire M. Gelfman1. 1Pre-Clinical, Ora, Andover, MA; 2KBP
BIOSCIENCES CO., LTD, Jinan, China.
Purpose: Inflammation is a key player in the development of
ocular surface diseases including Dry Eye and Chronic Allergic
Conjunctivitis. Pharmacological intervention with PI3-Kinase (PI3K)
inhibitors has been shown to alleviate systemic inflammation and
thus represents an attractive therapeutic target for ocular disease.
The purpose of this study was to test the anti-inflammatory potential
of a novel PI3K inhibitor, KBP-7306, in a model of ocular surface
inflammation.
Methods: The anti-inflammatory effect of 0.06% KBP-7306 was
examined in a chronic Short Ragweed (SRW) Allergic Conjunctivitis
model. Female Balb/C mice were sensitized with a subcutaneous
injection of SRW, and then challenged 18 days later with topical
SRW to determine responsiveness to the allergen sensitization and
topical opththalmic challenge. Following the challenge, sensitized
mice were randomized into treatment groups (n=8) and given test
compounds four times daily on days 19-20 and three times daily on
days 21-24. SRW challenges were repeated twice daily on days 21-24
for a total of 8 challenges; their responses to allergen were evaluated
after challenges 1, 4, 6 and 8. Cyclosporine 0.05% (Restasis®)
was used as a clinical comparator and Prednisolone acetate 1.0%
was used as a positive control. KBP-7306 vehicle was used as the
negative control. Following challenges hyperemia was evaluated
using a Micron III imaging system. The allergic response was graded
on a 0-4 scale using a modification of Ora’s clinical grading scale.
Significance was determined using a Two-way ANOVA analysis and
Bonferroni post-test comparing all treatment groups to the vehicle
group.
Results: Challenged mice treated with vehicle maintained a high
level of allergen-induced hyperemia throughout the study, but
decreased by .5 points on the final day (day 24). 0.06% KBP-7306
significantly decreased hyperemia on days 22 and 23 (P < 0.01 and P
< 0.05 respectively). Treatment with both Prednisolone and Restasis
decreased hyperemia throughout the entire challenge period. This
effect was significant for both Prednisolone and Restasis on day 22 (P
< 0.001 and P < 0.05 respectively) and for Prednisolone on day 23 (P
< 0.01).
Conclusions: The data from this study confirms that the PI3K
inhibitor KBP-7306 is an efficacious anti-inflammatory drug in this
model of ocular surface inflammation.
Commercial Relationships: Andy Whitlock, Ora, Inc (E);
Zhenhua Huang, KBP BIOSCIENCES CO., LTD (E); Yanli Wang,
KBP BIOSCIENCES CO., LTD (E); Laura Belen, Ora, Inc (E);
Claire M. Gelfman, Ora, Inc (E)
Air pollution-related pediatric conjunctivitis: a prospective
observational study
Edoardo Villani, Matteo Sacchi, Vittoria Termine, Francesco Pichi,
Paolo Nucci. DISCCO. Eye Clinic., University of Milan. San
Giuseppe Hospital., Milan, Italy.
Purpose: To investigate, in pediatric patients, the rate and clinical
features of unspecific conjunctivitis of unknown origin (UCUO) and
to evaluate their relationship with air pollution.
Methods: Over the period January 2013 - December 2013, we
consecutively screened all the patients referred for symptomatic
ocular surface inflammation to the Unit of Pediatric Ophthalmology,
University Eye Clinic of San Giuseppe Hospital in Milan. Inclusion
criteria for this study were age<18, diagnosis of conjunctivitis,
residence in the region Lombardia. Exclusion criteria were previous
ocular surgery, ocular diseases (other than ocular surface diseases),
keratitis (other than punctate keratopathy), contact lens wearing,
systemic conditions or therapies with well-known effect on the ocular
surface, topical treatments in the 3 months before screening. All
included patients underwent a full eye exam. UCUO was defined as
conjunctivitis of unknown etiology, not clearly due to infection or
allergy. Based on addresses of residence and sites of 73 automatic air
pollution monitoring stations (location and mean annual particulate
matter, PM10 and PM2.5, concentrations were provided by the
Regional Environmental Protection Agency – ARPA Lombardia),
each patient was paired with a value of exposure to PM. Relationship
between UCUO and PM exposure was investigated.
Results: 132 of 251screened children were included in this study
(72 females and 60 males, mean age 5.9±3.4, range 1-12). Figure 1
reports diagnosis’ rate in included children. UCUO was diagnosed in
48 (36%) patients. The most common symptoms and signs in UCUO
children were foreign body sensation (37/48), photophobia (28/48),
conjunctival hyperemia (45/48), and limbal corneal vascularization
(22/48). PM10 exposure value was significantly higher in UCUO
(33.5±5.4mg/m3) compared to each other group (25.2±5.9,
28.9±6.9, 27.5±5.7, respectively; P<0.001, ANOVA). UCUO/total
conjunctivitis ratio was significantly higher in residents in areas with
more than 75 (Q3) days/year exceeding 50mg/m3 (EU legal limit)
compared to areas with less than 45 (Q1) exceedances/year: 24/39:
61% vs 8/35: 23%; P>0.001, Chi-square test.
Conclusions: Our data suggest a relationship between UCUO and air
pollution. This form of conjunctivitis is not rare in pediatric patients
and may be the most frequent in most polluted areas.
Figure 1. Diagnosis’ rate in included patients
Commercial Relationships: Edoardo Villani, None; Matteo
Sacchi, None; Vittoria Termine, None; Francesco Pichi, None;
Paolo Nucci, None
Soluble hyaluronan receptor CD44 levels secreted by human
conjunctival cells change in inflammatory conditions
Laura Garcia-Posadas1, 2, Laura Soriano-Romani1, 2, Antonio LopezGarcia1, 2, Margarita Calonge1, 2, Yolanda Diebold1, 2. 1IOBA (Institute
of Applied Ophthalmobiology), University of Valladolid, Valladolid,
Spain; 2CIBER-BBN (Biomedical Research Networking Center in
Bioengineering, Biomaterials, and Nanomedicine), Valladolid, Spain.
Purpose: Our group has reported the presence of soluble CD44
(sCD44) in human tears and measured sCD44 levels in corneal
and conjunctival epithelial cell lines under in vitro inflammatory
conditions. We also reported that those cell lines are not a good model
to perform hyaluronan-related studies. For that reason our purpose
is to determine if cultured human conjunctival primary cells release
sCD44 when exposed to different in vitro simulated inflammatory
conditions.
Methods: Human primary fibroblasts and epithelial cells were grown
from conjunctival tissue obtained from cadaveric donors (n=7) and
cultured in supplemented DMEM/F12 medium. This study was
in strict accordance with the tenets of the Declaration of Helsinki
and Spanish Regulations concerning the use of human tissue for
biomedical research. When confluence was reached cells were treated
for 24 h with inflammatory cytokines: TNF-α (25 ng/ml), IFN-y (10
ng/ml), and IL-13 (20 ng/ml). Then, supernatants were collected and
levels of sCD44 and IL-6 were measured by ELISA. In addition,
proliferation was studied with Alamar Blue™ reagent in cytokineexposed cells. Data (mean ± SEM) were analyzed by the Student t
test.
Results: Conjunctival fibroblasts basally released sCD44 (0.32
± 0.041 ng/ml); when exposed to TNF-α or IL-13, a significant
increase to 0.46 ± 0.038 ng/ml (p=0.022) and 0.59 ± 0.084 ng/ml
(p=0.012), respectively, was observed. Similar results were obtained
for IL-6 production. Untreated fibroblasts secreted 274.98 ± 63.80
pg/ml IL-6, and TNF-α and IL-13 increased secreted IL-6 levels to
7920.33 ± 2530.18 (p=0.006) and 1386.92 ± 444.39 (p=0.023) pg/
ml, respectively. In epithelial cells, a tendency to decrease IL-6 levels
was observed with IFN-y treatment, from 21.19 ± 1.41 ng/ml to 18.19
± 1.18 ng/ml (p=0.061). Fibroblast proliferation rate was reduced
(29% decrease, p=0.007) by IL-13 exposure whereas no changes
were observed in epithelial cells.
Conclusions: We demonstrated that basal release of sCD44 occurred
in both conjunctival fibroblasts and epithelial cells. Increased sCD44
levels measured under simulated inflammatory conditions would
suggest an involvement of sCD44 in conjunctival inflammation.
These results are consistent with our previous studies with
hyaluronan receptors, where we reported an increase in CD44 and
RHAMM levels in patients with immune-atopic diseases.
Commercial Relationships: Laura Garcia-Posadas, None; Laura
Soriano-Romani, None; Antonio Lopez-Garcia, None; Margarita
Calonge, None; Yolanda Diebold, None
Support: FEDER-CICYT Grant MAT2013-47501-C02-1-12 and FPI
Scholarship Program BES-2011-046381 (Ministry of Economy and
Competitiveness, Spain).
The clinical characteristics of conjunctivitis related with Asian
dust
Ryota Ko1, Masahiko Hayashi2, Hideyuki Hayashi1, Eiichi Uchio1.
1
Ophahtlmology, Fukuoka University, Fukuoka, Japan; 2The
department of science, Fukuoka University, Fukuoka, Japan.
Purpose: Asian dust is originated from Northern China and spreads
over China and some part of Japan by the wind. It has been known
as the major air pollutant exacerbating allergic diseases. We reported
the increased number of acute conjunctivitis in Fukuoka area during
the season of Asian dust and the presence of asian dust particles on
the ocular surface of the patients. To clarify the nature of asian dustrelataed conjunctivitis, clinical findings had been further analysed in
this study.
Methods: Forty five patients [men : women = 30 : 15, 49.6 ±22.7
years old (mean ± S.D.) ] with newly diagnosed acute conjunctivitis
from March 12 to May 24, 2013 in Fukuoka area were selected as
candidates. The degree of inflammatory reaction, itchy sensation,
hyperemia, eye discharge, foreign body sensation were clinically
recorded and scored. Asian dust particle on the ocular surface of
candidate was studied as the method we reported. Clinical findings of
Asian dust particle-positive patients were analysed.
Results: Forty four out of forty fives patients (97.8%) were Asian
dust positive. The presence of the percentage of the Asian dust
particle varied from 7 to 56 % (average= 26%). Only clinical finding
scores of hyperemia and itchy sensation had significant difference in
the subgroup where Asian dust /whole particulate ratio was higher
than the average of 26%. (t-test : P<0.01, respectively).
Conclusions: Clinical findings of the Asian dust –related
conjunctivitis suggested allergic reaction. Further studies should be
conducted.
Commercial Relationships: Ryota Ko, Kobayashi pharmaceutical
Co, Ltd. (I); Masahiko Hayashi, Kobayashi pharmaceutical Co,
Ltd (F); Hideyuki Hayashi, Kobayashi pharmaceutical Co, Ltd (I);
Eiichi Uchio, Kobayashi pharmaceutical Co, Ltd (I)
SAFETY EVALUATION OF OLOPATADINE
HYDROCHLORIDE OPHTHALMIC SOLUTION 0.77% IN
PEDIATRIC SUBJECTS WITH ASYMPTOMATIC EYES
Edward J. Meier1, Abhijit Narvekar2, Kyong Bae2, Kenneth N. Sall3,
David Wirta4. 1Apex Eye, Mason, OH; 2Alcon Research Ltd, Fort
Worth, TX; 3Sall Eye Research Medical Center, Artesia, CA; 4Eye
Research Foundation, Newport Beach, CA.
Purpose: Purpose. The objective of this study subgroup analysis was
to evaluate the safety profile of olopatadine hydrochloride ophthalmic
solution, 0.77% (Olopatadine) in pediatric subjects between ages 2 to
17 years old after 6 weeks of treatment.
Methods: Methods. This was a multicenter, randomized, doublemasked, vehicle controlled, parallel group study in subjects 2 years of
age or older with asymptomatic eyes (NCT01698814). Subjects were
randomized to receive either olopatadine 0.77% or vehicle solution
dosed in each eye once a day for 6 weeks. Eligible subjects had to be
asymptomatic for ocular allergies and other eye conditions. Safety
parameters (best-corrected visual acuity, ocular signs, intraocular
pressure, dilated fundus examination and vital signs) and adverse
events were assessed at Day 0 and weeks 1, 3 and 6; for which
descriptive statistics were performed.
Results: Results. A total of 75 pediatric subjects were included in
this study. Baseline and demographic characteristics were similar
between Olopatadine (n=51) and Vehicle (n=24) groups, with most
of the subjects completing the study (94.1% on Olopatadine & 87.5%
on Vehicle). No clinically relevant differences were observed in
any of the safety parameters between both treatment groups. Of the
subjects who experienced treatment-emergent adverse events (AEs)
(16 subjects [31.4%] and 6 subjects [25.0%] in the Olopatadine
and Vehicle groups, respectively), 8 in the Olopatadine and 2 in the
Vehicle group experienced treatment-related AEs. Ocular adverse
events (MedDRA SOC Eye disorders, related and nonrelated)
observed included abnormal sensation in the eye (4% vs 0%), pruritis
(2% vs 0%), irritation (0% vs 4%), foreign body sensation (2% vs
0%) and chalazion (0% vs 4%), in the Olopatadine and Vehicle
groups, respectively. No deaths or serious AEs were reported in this
study.
Conclusions: Conclusions. Olopatadine 0.77% was well-tolerated,
with no safety issues or meaningful trends identified in a pediatric
population aged 2 to 17 years.
Commercial Relationships: Edward J. Meier, Alcon (F); Abhijit
Narvekar, Alcon (E); Kyong Bae, Alcon (E); Kenneth N. Sall,
Alcon (F); David Wirta, Alcon (F)
POOLED ANALYSIS OF 2 CONFIRMATORY TRIALS
EVALUATING THE EFFICACY AND SAFETY OF
OLOPATADINE HYDROCHLORIDE OPHTHALMIC
SOLUTION 0.77% USING A CONJUNCTIVAL ALLERGEN
CHALLENGE MODEL
Eugene B. McLaurin1, Abhijit Narvekar2, Adeniyi Adewale2, Paul
J. Gomes3, Gail Torkildsen4. 1Total Eye Care, P.A., Savannah, GA;
2
Alcon Research Ltd, Fort Worth, TX; 3Ora, Inc., Andover, MA;
4
Andover Eye Associates, Andover, MA.
Purpose: Two phase 3 studies with similar designs were conducted to
evaluate the efficacy and safety of olopatadine 0.77% (HDO) for the
treatment of allergic conjunctivitis (AC). The objective of this pooled
analysis was to evaluate the integrated efficacy and safety findings
from a larger dataset.
Methods: Data were pooled from 2 similarly designed, randomized,
multicenter, double-masked, active (olopatadine 0.1% & 0.2%)
and vehicle controlled, parallel-group studies which used the
Conjunctival Allergen Challenge (CAC) model. The study designs
were identical except that one of the studies had an additional 16
hour duration efficacy evaluation visit and the other had an additional
active comparator, olopatadine 0.1%. Eligible patients in both studies
were ≥18 years with a history of AC. The pooled analysis employed
a mixed model repeated measures ANOVA statistical method. The
primary hypotheses were that HDO is superior to Vehicle (at onset of
action & 24 hour duration CACs) and olopatadine 0.2% (at 24 hour
duration CAC) with respect to ocular itching (OI) score evaluated at
3 time points (3, 5 & 7 minutes post-CAC).
Results: Data from a total of 448 patients were included in the
analysis. Baseline and demographic characteristics were similar
between HDO (n=164), olopatadine 0.2% (n=167) and Vehicle
(n=117) groups. HDO was superior to Vehicle (p<0.0001) for OI
at all 3 post-CAC time points at onset & 24 hours duration (avg.
treatment differences [over the 3 post-CAC time points] were -1.44
and -1.30 at onset and 24 hours duration, respectively) & also
superior to olopatadine 0.2% (p=0.0009) for OI after 24 hours at all
3 post-CAC time points (avg. treatment difference was -0.31). The
pooled analysis of safety parameters showed no discernable trends or
clinically relevant differences across the treatment groups; a review
of adverse events did not reveal any safety issues for HDO. No deaths
or treatment-emergent serious AEs were reported in these studies.
Conclusions: This analysis strengthens conclusions drawn from 2
studies that olopatadine 0.77% was superior to vehicle at onset and
after 24 hours, as well as superior to olopatadine 0.2% at 24 hours
after dosing with respect to reduction of OI associated with AC.
Olopatadine 0.77% had a safety profile comparable to olopatadine
0.2%.
Commercial Relationships: Eugene B. McLaurin, Alcon (R),
Allergen (R); Abhijit Narvekar, Alcon (E); Adeniyi Adewale, Alcon
(E); Paul J. Gomes, Alcon (C), Ora, Inc. (E); Gail Torkildsen, Alcon
(F), Alcon (R), Allergan (F), Ora, Inc (C)
Support: This research was supported by Alcon Research, Ltd., Fort
Worth, TX.
Clinical Trial: NCT01743027 & NCT01479374
Anti-Inflammatory Effects of Alcaftadine 0.25% Ophthalmic
Solution as Measured by In-Vivo Confocal Microscopy
Paul J. Gomes, Keith J. Lane, Endri Angjeli, Yesha Raval, Emily
Schoemmell, Donna L. Welch. ORA, Andover, MA.
Purpose: Mediators released from the mast cell initiate the
signs and symptoms of allergic conjunctivitis, and can cause the
disease to progress to allergic inflammation. The antihistamine,
alcaftadine, indicated for prevention of itching associated with
allergic conjunctivitis, has been shown preclinically to have antiinflammatory effects. The purpose of this study was to characterize
the anti-inflammatory effects (via blood vessel confocal microscopy)
and anti-histaminic effects of alcaftadine in human subjects.
Methods: This was a single-center, double-masked, randomized,
placebo-controlled, conjunctival allergen challenge (CAC) study
comprised of 3 visits (N=16). Main entry criteria were: a positive
history of allergic conjunctivitis; a positive allergic CAC reaction;
and a bilateral conjunctival inflammation score (CIS: leukocyte
migration and adherence) of ≥ 2 (0-4 scale) at 60-90 mins post-CAC
at Visit 1. At Visits 2 and 3, subjects received one drop of alcaftadine
in one eye and placebo in the contralateral eye. Visit 2 measured the
prophylactic effects of alcaftadine instilled 15 min prior to CAC.
Visit 3 measured the treatment effects of alcaftadine administered 20
min post-CAC. The primary endpoint was CIS of confocal images of
conjunctival blood vessels 60 mins post-CAC, scored by a masked
grader. Secondary endpoints included ocular itching, eyelid swelling,
redness and chemosis.
Results: At Visit 2, the CIS in alcaftadine-treated eyes (2.34 ± 0.98;
mean, SD) was lower than placebo eyes (2.94 ± 1.01): p=0.07.
Itching scores were > 1 unit (0-4 scale) lower in the alcaftadine–
treated eyes (p<0.0008). At Visit 3, itching scores remained > 1 unit
lower in alcaftadine-treated eyes even after the second challenge, 24
hours post-instillation of study drug at Visit 1 (p<0.0005). At Visit 3,
alcaftadine-treated eyes had a lower CIS (2.84 ± 0.87) than placebo
eyes (3.16 ± 0.87), but the differences were less than when treatment
was given prior to challenge (p=0.17).
Conclusions: Alcaftadine had a long-lasting (>24 hrs) effect on
itching, and inhibited the inflammation of an acute allergic reaction.
Prevention was more effective than treatment after allergy had
ensued. Consistent coverage will prevent the allergic response from
occurring, and the progression to a more complex and difficult to
treat allergic inflammation.
Commercial Relationships: Paul J. Gomes, Ora, Inc. (E); Keith J.
Lane, ORA (E); Endri Angjeli, ORA, Inc. (E); Yesha Raval, ORA
(E); Emily Schoemmell, ORA (E); Donna L. Welch, ORA (E)
Topical administration of beta-1,3-D-glucan to modulate immune
responses and suppress allergic conjunctivitis in a Murine Model
Ji Young Kwon1, HeeJung Ju1, Hyun Soo Lee1, 2, Choun-Ki Joo1, 2.
1
Catholic Institute for VIsual Science, Catholic University, Seoul,
Korea (the Republic of); 2Seoul St. Mary`s Hospital, Seoul, Korea
(the Republic of).
Purpose: Recently studies performed on beta-1,3-D-glucan, a
cell wall component of a variety of fungi, yeasts, and bacteria,
demonstrated that it affects the balance of Th1/Th2 immune response.
We therefore determined whether topical application of beta-1,3-Dglucan modulates ocular allergy in murine model.
Methods: 7-8 week-old BALB/c mice were sensitized via
intraperitoneal injection once with Ovalbumin (OVA) and aluminum
hydroxide. Mice were rested for 2 weeks and then challenged by
instillation of OVA eyedrops once daily for 13 days. beta-1,3-Dglucan (BG) eyedrop was administered 5 min after OVA challenge
once daily. Clinical signs were measured and we evaluated the
infiltration of eosinophils and mast cells into conjunctiva with flow
cytometry, and serum levels of OVA specific IgE production and Th2
cytokines in vitro stimulation of T cell in drainage lymph nodes (LN)
Results: BG treated mice showed less allergic conjunctivitis,
indicated by clinical signs and decreased production of serum OVA
specific IgE. In addition, BG treatment led to decreased infiltrations
of CD45 positive immune cells, eosinophil, and mast cells into
conjunctiva, compare to only vehicle treated (control) mice. BG
administration suppressed Th2 cytokine production in vitro T cells
assay through the induction of IL-10 producing CD4 T cells in
drainage LNs
Conclusions: Taken together, these results suggest that beta-1,3-Dglucan is capable of inducing IL-10–producing CD4+ T cells and
suppressing the Th2 response in drainage LNs and conjunctival
eosinophil infiltration, as the therapeutic potential of topical beta-1,3D-glucan for allergic conjunctivitis.
Commercial Relationships: Ji Young Kwon, None; HeeJung Ju,
None; Hyun Soo Lee, None; Choun-Ki Joo, None
Support: This study was supported by Research Fund of Seoul
St.Mary’s Hospital, The Catholic University of Korea
Evaluating Sustained Release Dexamethasone for the Treatment
of Chronic Allergic Conjunctivitis Using a Modified Conjunctival
Allergen Challenge (Ora-CAC®) Model
Michael Bassett, Deepa Mulani, Charles D. Blizzard, Arthur Driscoll,
Amar Sawhney. R&D, Ocular Therapeutix, Bedford, MA.
Purpose: To evaluate the efficacy and safety of Sustained Release
Dexamethasone (OTX-DP) as a one-time administered sustained
release drug (dexamethasone) depot when placed in the canaliculus
of the eyelid for the treatment of the signs and symptoms of chronic
allergic conjunctivitis.
Methods: The OTX-DP is a punctum plug containing dexamethasone
within a biodegradable PEG hydrogel matrix. The OTX-DP is
designed to release dexamethasone into the tear fluid in a tapered
profile. This was evaluated in a Phase 2 human study, wherein
subjects underwent a series of allergen challenges using a modified
Ora-CAC model to induce the inflammatory component of chronic
allergic conjunctivitis. After showing a reproducible allergen
response for itching and redness, and meeting eligibility criteria,
subjects were randomized (1:1) at Day 1 to receive OTX-DP or
Placebo Vehicle Punctum Plug (PV), bilaterally. The primary efficacy
measures (ocular itching and conjunctival redness) were assessed
at 14 days post-insertion, with scoring based on zero to 4 scales,
with zero being symptom free, and 4 representing the most extreme.
Subjects were followed further at 4 weeks and 6 weeks post-insertion
for assessing continued therapeutic effect for allergic conjunctivitis.
Results: 35 and 33 subjects were randomized to OTX-DP and PV,
respectively. At 14 days post-insertion, a single OTX-DP dose was
statistically superior to PV in ocular itching and conjunctival redness
at all-time points (3, 5, and 7 minutes for itching and 7, 15, and 20
minutes for redness) post-CAC, as shown in Table 1. The differences
(p-values) observed for itching and redness, were respectively:
-0.78 (0.0031), -0.98 (0.0002), -0.88(0.0007), and -0.51 (0.0100),
-0.70 (0.0006), -0.67 (0.0008). [JP1] The treatment difference was
> 0.5 units for itching and redness at 14, 28, and 42 days, showing
consistent efficacy on itching and redness, as shown in Figure 1. No
serious treatment-related adverse events were noted in either group.
Conclusions: Overall, OTX-DP treatment was shown to be well
tolerated. Treatment effects were evident for at least 42 days. The
data supports the continued development of this drug product in the
Ora-CAC® model.
Commercial Relationships: Michael Bassett, Ocular Therapeutix
(E); Deepa Mulani, Ocular Therapeutix (E); Charles D. Blizzard,
Ocular Therapeutix (E); Arthur Driscoll, Ocular Therapeutix (E);
Amar Sawhney, Ocular Therapeutix (E)
Correlations between allergen-specific IgE serum levels in
patients with ocular allergy
Julia Polido2, 1, Thiago Cabral2, 1, Maria de Fátima Fernandes2,
Paula Perini2, Denise Freitas1, Maria Emília Araújo2, 1, Pedro
Serracarbassa2. 1OPHTHALMOLOGY, UNIFESP, Vitória, Brazil;
2
OPHTHALMOLOGY, HSPE/IAMSPE, São Paulo, Brazil.
Purpose: To evaluate the ocular allergies of patients seen at the
Hospital do Servidor Publico Estadual de Sao Paulo (HSPE) and
correlations with serum allergen specific immunoglobulin E levels.
Methods: We performed a longitudinal study of patients with ocular
allergy who were treated at a Cornea and Immunology and Allergy
Department. Patients underwent an ophthalmologic examination to
identify their main presenting signs and symptoms. The eye allergy
types were divided into four groups. We conducted the following
laboratory tests: blood count, serum total IgE and specific IgE.
Results: We examined 61 patients, 16 (26.2%) with clinical
diagnosis of seasonal allergic conjunctivitis, 23 (37.7%) patients
with perennial allergic conjunctivitis, 19 (31.1%) patients with vernal
keratoconjunctivitis and 3 (4.9%) with atopic keratoconjunctivitis.
The Dermatophagoides pteronyssinus (dp) antigen was positive in
93.9% of patients, followed by mixed dust mites (D. pteronyssinus,
D. Farina, Blattella Germanica, house dust) in 93.8% of patients and
Blomia tropicalis in 78.8% of patients.
Conclusions: Perennial allergic conjunctivitis was the most prevalent
disorder and showed higher positivity in class V/VI for the specific
antigens mixed dust mites (HX2), D. pteronyssinus. and D. farinea.
Commercial Relationships: Julia Polido, None; Thiago Cabral,
None; Maria de Fátima Fernandes, None; Paula Perini, None;
Denise Freitas, None; Maria Emília Araújo, None; Pedro
Serracarbassa, None
518 Clinical studies in ocular infection and immunity
Thursday, May 07, 2015 8:30 AM–10:15 AM
Contributing Section(s): Anatomy/Pathology, Clinical/
Epidemiologic Research, Eye Movements/Strabismus/Amblyopia/
Neuro-Ophthalmology
A Case-Control Study of Herpes Zoster Ophthalmicus: Bronx
Epidemiology of HIV Eye Studies (BEHIVES)
Marianna Atiya1, David Poulsen1, Ethan K. Sobol1, Grace Honik2,
Jose Diaz1, Jonathan Powell2, David C. Gritz2, 1. 1Albert Einstein
College of Medicine, Bronx, NY; 2Ophthalmology and Visual
Sciences, Montefiore Medical Center, Bronx, NY.
Purpose: Herpes zoster ophthalmicus (HZO) has an estimated
incidence of 1.5 to 6.4 cases per 100,000 persons per year in the
general population. The purpose of this retrospective case-control
study is to examine the risk of developing HZO among patients who
are positive for human immunodeficiency virus (HIV), and among
patients with history of atopic disease.
Methods: This study utilized hospital-based controls for the study
period from May 1, 2006 to May 31, 2014. Inclusion criteria involved
Bronx residents diagnosed with new-onset HZO during the eight-year
study period at Montefiore Medical Center. Hospital-based controls
were drawn from unique outpatients visits at Montefiore Medical
Center during the study period. Controls were chosen randomly in
a 4:1 ratio and were time-matched to HZO cases. Medical records
were reviewed to confirm inclusion criteria and the data were used
to calculate odds ratios for developing HZO in HIV-positive patients
and in patients with history of atopic disease.
Results: 170 patients were diagnosed with new-onset HZO during
the study period. Compared to hospital-based controls (n=680), HIV
infection was shown to increase the odds of HZO by 6.63 (95% CI
2.96-14.8, p<0.001), and history of atopic disease was shown to
increase the odds of HZO by 2.55 (95% CI 1.50-4.37, p=0.001).
Conclusions: Infection with HIV and history of atopic disease are
significant risk factors for having a new-onset case of HZO. This is a
particularly important association in the Bronx, where the prevalence
of HIV infection is over 3.6 times greater than in the United States
overall.
Commercial Relationships: Marianna Atiya, None; David
Poulsen, None; Ethan K. Sobol, None; Grace Honik, None; Jose
Diaz, None; Jonathan Powell, None; David C. Gritz, None
Incidence of Herpes Simplex Eye Disease: Results from the
Pacific Ocular Inflammation (POI) Study
Durga S. Borkar1, 2, Vivien M. Tham3, John V. Parker4, Aileen
Uchida4, Aleli C. Vinoya4, Nisha Acharya1. 1F I Proctor
Foundation, Univ of California, San Francisco, San Francisco, CA;
2
Massachusetts Eye and Ear Infirmary, Boston, MA; 3Pacific Vision
Institute of Hawaii, Honolulu, HI; 4Kaiser Permanente Hawaii,
Honolulu, HI.
Purpose: To provide a population-based estimate of the incidence of
herpes simplex eye disease with comparisons across racial, gender,
and age groups.
Methods: The electronic medical record of Kaiser Permanente
Hawaii between January 1, 2006 and December 31, 2007, was
searched for International Classification of Diseases, 9th Edition
(ICD9) codes corresponding to herpes simplex eye disease. Chart
review was performed to confirm a diagnosis of herpes simplex eye
disease and to collect information on specific ocular manifestations.
Incidence rates were calculated per 100,000 person-years for the
entire population, as well as for age-, gender-, and race-specific
subgroups using a dynamic population model.
Results: In the Kaiser Hawaii population of 217,061 people,
ninety-four cases of herpes simplex eye disease were identified.
The overall incidence was 21.7 per 100,000 person-years (95%
confidence interval (CI): 17.5-26.5). For people 65 years of age and
over, the incidence rate was 37.4 per 100,000 person-years (95%
CI: 22.8-57.7), approximately twice the remainder of the population
(p=0.01). The most common manifestation of herpes simplex eye
disease was keratitis, followed by dermatitis and conjunctivitis. The
incidence of herpes simplex eye disease for Asians was 15.2 per
100,000 person-years (95% CI: 10.3- 22.0), which was significantly
lower than the rate for non-Asians (p=0.02). Prior and current use
of immunosuppressant medications were found to be risk factors for
herpes simplex eye disease.
Conclusions: These results provide a population-based estimate
of herpes simplex eye disease in the Hawaiian population and
demonstrate differences across age and racial subgroups. Various
genetic and environmental factors may explain these differences.
Commercial Relationships: Durga S. Borkar, None; Vivien M.
Tham, None; John V. Parker, None; Aileen Uchida, None; Aleli C.
Vinoya, None; Nisha Acharya, None
Support: Nisha Acharya, MD, MS: NEI U10 EY021125-01, UCSF
Research Evaluation and Allocation Committee Award; UCSF
Department of Ophthalmology: NEI EY02162, Research to Prevent
Blindness unrestricted grant
Herpes zoster ophthalmicus and associations with ocular
complications: Bronx Epidemiology of HIV Eye Studies
(BEHIVES)
Jonathan Powell, Marianna Atiya, David Poulsen, Ethan K. Sobol,
Jose Diaz, David C. Gritz. Ophthalmology, Albert Einstein College
of Medicine/Montefiore Medical Center, Bronx, NY.
Purpose: Herpes zoster ophthalmicus (HZO) has potentially serious
ocular complications. There is little data published on the strength
of association of risk factors for developing ocular complications.
We explored several risk factors potentially associated with
the development of iritis, keratitis, blepharoconjunctivitis, and
postherpetic neuralgia (PHN) for individuals with HZO.
Methods: A comparative, retrospective investigation among HZO
patients in the Bronx was performed. Cases of HZO from June 1,
2006 through May 31, 2014 were identified using Montefiore’s
Clinical Looking Glass software (CLG), which compiles medical
information for all individuals within the hospital outpatient system.
The inclusion criteria consisted of a clinical diagnosis of HZO and
Bronx residency. Relevant demographic and clinical information
was extracted from the CLG database and confirmed through chart
review. Potential associations evaluated included age, gender,
HIV status, diagnosis of diabetes and atopy, and presence of the
Hutchinson’s sign. Firth logistic regression modeling was used to
determine any significant associations between HIV status and other
potential risk factors for each outcome of interest. Odds ratios (OR)
and 95% confidence intervals (95% CI) were calculated to estimate
relative risk.
Results: The study group included 155 patients with HZO. While
controlling for confounders, HIV status was significantly associated
with the development of iritis (p=0.0275) and keratitis (p = 0.0013).
The adjusted OR estimates for iritis and keratitis manifestations
among HIV cases were 3.163 (95% CI: 1.146 to 8.648) and 5.230
(95% CI: 1.963 to 14.389) respectively. Also, HIV infection
had an estimated OR of 4.067 (95% CI: 1.469 to 12.910) for the
development of at least one of the four ocular complications while
adjusting for Hutchinson’s sign and age.
Conclusions: The presence of HIV was correlated with an increased
risk of developing various ocular complications for individuals with
HZO. To our knowledge, this study represents the first time the
strength of association has been measured between HIV status and
the onset of iritis, keratitis, blepharoconjunctivitis, and PHN among
HZO cases.
Commercial Relationships: Jonathan Powell, None; Marianna
Atiya, None; David Poulsen, None; Ethan K. Sobol, None; Jose
Diaz, None; David C. Gritz, None
Herpes simplex virus disease of the anterior segment in children
Juan Carlos Serna-Ojeda, Arturo J. Ramirez-Miranda, Alejandro
Navas, Aida Jimenez-Corona, Enrique O. Graue. Ophthalmology,
Institute of Ophthalmology “Conde de Valenciana””, Mexico City,
Mexico.
Purpose: To analyze the clinical presentation, characteristics,
treatment with topic and oral acyclovir, recurrences and final
outcomes and complications of patients aged 17 or younger with
herpes simplex virus disease of the anterior segment.
Methods: An observational and retrospective study was performed
with review of the medical records of all the children with
diagnosis of herpes simplex infection of the anterior segment at an
ophthalmologic reference center in Mexico City, from 2002 to 2012.
Patients were diagnosed based in history and examination, and in
specific cases with viral culture and polymerase chain reaction test.
Patients were treated with topical or oral acyclovir according to the
clinical presentation. Recurrent disease was analyzed with KaplanMeier curves. Main outcome measures included: final visual acuity,
bilaterality, recurrent disease and surgical procedures performed.
Results: One hundred and three patients were included with a median
age at presentation of 9 years, from which 6 had a bilateral and
simultaneous disease. The median follow up time was 18 months
(range 18 days - 12 years). The most common clinical manifestation
was epithelial dendritic keratitis in 42 eyes (38.5%) with 15 patients
presenting multiple forms of herpes simplex virus disease. Recurrent
disease was evident in 42 (38.5%) of the eyes. Eight patients
underwent a penetrating keratoplasty at a median age of 15 years.
The median final visual acuity in the group of patients was 20/40.
Conclusions: In this paper, one of the largest series of pediatric
population with herpes simplex virus of the anterior segment, we
conclude that these patients have a high rate of epithelial dendritic
manifestation and recurrent disease.
Commercial Relationships: Juan Carlos Serna-Ojeda, None;
Arturo J. Ramirez-Miranda, None; Alejandro Navas, None; Aida
Jimenez-Corona, None; Enrique O. Graue, None
Epidemiology of Uveitis at a Tertiary Eye Center in the MidAtlantic United States.
Susan Osmanzada, Diba Osmanzada, Asima Bajwa, James Patrie,
xin wenjun, Ashvini Reddy. Ophthalmology, University of Virginia,
Charlottesville, VA.
Purpose: To report demographic, etiologic, and clinical features of
patients with uveitis seen at a tertiary care center in central Virginia.
Methods: Retrospective review of uveitis cases seen at the
University of Virginia from 1984-2014. Descriptive statistics, and,
where applicable, Fisher’s exact test and exact logistic regression
analysis were used to report demographics, anatomical/etiological
classification of uveitis, clinical features, and management.
Results: 491 patients (644 eyes) were included. Mean age was 47
years (±21) at presentation. 278 (56.6%) patients were female, 153
(31.2%) patients had bilateral disease. 60.5% were Caucasian, 28.3%
were African American. Mean duration of follow-up was 5 years
(±6.7). The most frequent anatomic type was undifferentiated anterior
uveitis (n=126, 25.7%), followed by undifferentiated panuveitis
(n=21, 4.3%). The most common etiology was post-traumatic (n=60,
12.2%) followed by post-procedural (n=49, 10.0%) and herpetic
anterior uveitis (n=39, 7.9%). Herpetic disease was more common
among Caucasians (n=32, 10.8%) than African Americans (n=2,
1.5%) (gender-adjusted odds ratio (OR): 7.69, 95% CI [2.12, 50.00]),
and sarcoidosis was more common among African Americans (n=23,
17.2%) than Caucasians (n=9, 3.0%) (gender-adjusted OR: 6.54, 95%
CI [2.98, 15.29]). Herpetic anterior uveitis was more common among
females (n=30, 10.8%) than males (n=9, 4.2%) (race-adjusted OR:
3.03, 95% CI [1.32, 7.71]). Mean logMAR acuity was 0.54 and 0.52
at initial and final visits, respectively (P=0.002). 388 (79%) and 133
(27.3%) patients received local and systemic steroids, respectively.
52 (10.6%) patients received an antimetabolite.116 (23.7%) patients
were managed with topical glaucoma medication. 43 (8.8%), 129
(26.4%), and 46 (9.4%) patients underwent glaucoma surgery,
cataract surgery, and vitrectomy, respectively.
Conclusions: Undifferentiated anterior, traumatic, post-procedural,
herpetic disease, HLA-B27 disease, and sarcoidosis were most
common causes of uveitis. Sarcoidosis was more commonly seen
in African American males, herpetic anterior uveitis was more
frequently seen in Caucasian females. Mean visual acuity improved
significantly for the cohort from initial to final visit with majority
receiving local or systemic corticosteroids.
Table 1: Specific uveitis diagnoses
Table 2: Multivariate analysis for race and gender
Commercial Relationships: Susan Osmanzada, None; Diba
Osmanzada, None; Asima Bajwa, None; James Patrie, None; xin
wenjun, None; Ashvini Reddy, None
Validation of ICD-9 Codes Used in the Pacific Ocular
Inflammation (POI) Study
Priya Janardhana1, Nisha Acharya1, Durga S. Borkar1, Vivien M.
Tham2, John V. Parker3, Aleli C. Vinoya3, Aileen Uchida3, Erica
Browne1. 1Ophthalmology, Proctor Foundation, UCSF, San Francisco,
CA; 2Pacific Vision Institue of Hawaii, Honolulu, HI; 3Kaiser
Permanente Hawaii, Honolulu, HI.
Purpose: To assess the validity of International Classification
of Diseases, 9th Edition (ICD9) codes used in the Pacific Ocular
Inflammation Study, a population-based study using the electronic
medical records of Kaiser Permanente Hawaii to investigate the
epidemiology of ocular inflammatory disease in the Hawaiian islands.
Methods: The electronic record system of Kaiser Permanente Hawaii
was searched for any visit from January 1, 2006 to December 31,
2007 that referenced an ICD9 code that might be associated with a
diagnosis of uveitis. The inclusive list of diagnosis codes used for this
study was adapted from two prior uveitis incidence and prevalence
studies using administrative data. A subset of these ICD9 codes
included all diagnoses codes pertinent to herpes zoster ophthalmicus
(HZO). Subsequently, all charts from this electronic query were
individually reviewed by a uveitis specialist (NRA) to confirm a
diagnosis of uveitis, as well as HZO.
Results: Of the 873 patients identified as possibly having uveitis
by ICD9 codes, 224 cases were confirmed as uveitis patient after
medical record review. In our study, the most accurate ICD9 codes
in identifying uveitis cases were herpes simplex iridocyclitis,
54.44 (75%), histoplasmosis retinitis, 115.92 (100 %), panuveitis,
360.12 (92%), disseminated chorioretinitis, 363.13 (100%), pars
planitis, 363.21 (100%), Harada disease, 363.22 (100%), recurrent
iridocyclitis, 364.02 (96%), and granulomatous uveitis, 364.1 (81%).
Sixty-nine patients had an accurate uveitis diagnosis using ICD9 code
364.04, secondary iridocyclitis noninfectious. However, using this
ICD9 code also contributed 122 patients with an inaccurate diagnosis
of uveitis after chart review and only had an accuracy of 36%. Using
ICD9 codes specific to HZO yielded 152 patients through electronic
search. After chart review, 138 had a confirmed diagnosis of HZO.
Overall, HZO codes had an accuracy of 91%.
Conclusions: With the increased use of data from electronic
medical records for research, it is important to validate whether
ICD9 diagnoses are accurate. These results suggest that in Kaiser
Permanente Hawaii, using ICD9 codes alone to capture uveitis
diagnoses is not always accurate. Chart review, as was done in
this study, can help further elucidate accurate diagnoses. However,
electronic search for ICD9 codes alone can be an accurate method for
identifying cases of HZO.
Commercial Relationships: Priya Janardhana, None; Nisha
Acharya, None; Durga S. Borkar, None; Vivien M. Tham, None;
John V. Parker, None; Aleli C. Vinoya, None; Aileen Uchida,
None; Erica Browne, None
Management and Outcomes of Uveitis in a Tertiary Eye Center
Over 30 Years
Chang Sup Lee1, Asima Bajwa1, James Patrie2, Wenjun Xin2, Ashvini
Reddy1. 1Ophthalmology, University of Virginia, Charlottesville, VA;
2
Biostatistics, University of Virginia, Charlottesville, VA.
Purpose: To report the long-term, clinical outcomes of patients with
uveitis managed in a tertiary medical center at University of Virginia
over a 30 year period.
Methods: Retrospective, observational study of patients with uveitis
seen at the University of Virginia from 1984-2014. Descriptive
statistics and, where appropriate, Wilcoxon Rank Sum test and
Pearson’s Exact Chi-Square test were used to analyze demographics,
laterality, anatomic location, etiology, total number of visits, change
in best-corrected visual acuity (BCVA), management, intraocular
pressure (IOP), and cataract.
Results: The study included 644 eyes of 491 patients. 153 patients
(31.2%) had bilateral disease and 213 (43.4%) were male. Mean age
was 51.7±1.1 (SE) years at presentation. The mean number of visits
per patient was 11.2±14.8 (median 6.0; range, 1.0–155).
The mean BCVA was 0.54±0.03 logMAR at initial presentation and
0.52±0.04 logMAR (P=0.002) at last follow-up. Change in mean
visual acuity from presentation to last follow-up was not statistically
significant for anterior (0.44±0.04 to 0.45±0.04, P=0.058), posterior
(1.07±0.13 to 0.99±0.13, P=0.197) and panuveitis (0.43±0.05 to
0.45±0.08, P=0.216). For patients with intermediate uveitis, the
mean BCVA significantly improved by the final visit (0.61±0.17 and
0.27±0.07, P=0.038). Severe vision loss (>1.0 logMAR) was rarely
seen with traumatic uveitis and HLA-B27-associated anterior uveitis.
Local steroids were given to 365 patients (74.6%) and systemic
steroids to 133 (27.3%). Antimetabolites were used in 52 (10.6%)
patients and anti-tumor necrosis factor agents in 17 (3.5%).
Intravitreal injection was given to 54 patients (11.1%); subtenon
injection was given to 23 (4.7%). Vitrectomy was performed in 46
patients (9.4%). Mean initial IOP was 15.8±6.4 mmHg, and mean
final IOP was 14.9±5.0 mmHg. 116 (23.7%) patients received
medical treatment for ocular hypertension (IOP>21 mmHg), and 43
(8.8%) patients underwent glaucoma surgery. 129 (26.4%) patients
underwent cataract surgery.
Conclusions: In this large series of patients with uveitis, mean
BCVA improved from initial presentation to last follow-up, and this
improvement was statistically significant. Patients with intermediate
uveitis had a better final visual acuity than those with anterior,
posterior or panuveitis. The majority of patients were managed
with local or systemic steroids, and many developed glaucoma and
cataract requiring treatment.
Commercial Relationships: Chang Sup Lee, None; Asima Bajwa,
None; James Patrie, None; Wenjun Xin, None; Ashvini Reddy,
None
Pediatric uveitis in a reference Centre in Mexico.
BEATRIZ VALADEZ BLANCO, Miguel Pedroza Seres. Uvea,
INSTITUTO DE OFTALMOLOGIA FUNDACION CONDE DE
VALENCIANA, Mexico, Mexico.
Purpose: Uveitis (intraocular inflammation) is an important cause of
blindness in México, 5% to 10% of the cases occurs in children. We
performed this study to know about the relative occurrence of uveítis,
the disease characteristics and its causes in pediatric population.
Methods: Observational and retrospective clinical study was
performed, we analyzed the data of 8 years (January 2007 to August
2014) from 357 patients with uveitis in a reference centre in Mexico
City and included 286 patients diagnosed from 0 to 18 years of
age, with more than one visit and specific diagnosis. Data retrieved
included age of diagnosis, gender, uveitis diagnosis, anatomic
location, and laterality. Detailed clinical information regarding the
course of uveitis included visual acuity, treatments, and if they were
already treated. The Standardization of Uveitis Nomenclature criteria
was used to report the clinical data.
Results: Out of the 276 patients with uveitis 64.69% were male and
35.31% female with a male to female ratio of 1.8 to 1, median age
at diagnosis was 10.4 years + 3.8 years, 61.54% had bilateral ocular
involvement. Intermediate uveitis was the most common diagnosis
61.54%, followed by posterior uveitis 22.38%, anterior uveitis
14.69% and panuveitis 1.40%. The underlying cause for uveitis was
evaluated as non-infectious 70.63%, infectious 21.27% and idiopathic
2.10%. The most common etiology was pars planitis for intermediate,
parasite infestation for infectious association and the systemic disease
association was juvenile idiopathic arthritis diagnosed in 1.39%
of these children, The prevalence of legal blindness was 24.1% at
baseline and at the end of treatment 20.27%. We treated 40.91% with
corticosteroids (topical, periocular, intraocular, oral or intravenous),
38.81% received immunosuppressive drugs.
Conclusions: The results from this cohort show the spectrum of
disease in pediatric patients. There are no many studies about uveitis
in mexican population, far less in children, with this study we can
determine the clinical characteristics of uveitis in this population that
when compared with other studies there are different outcomes, like
variations in the types of uveitis; we found a high rate of pars planitis.
Commercial Relationships: BEATRIZ VALADEZ BLANCO,
None; Miguel Pedroza Seres, None
Ocular complications of pediatric uveitis at a reference center in
Mexico city.
Olegario Ivan Castro Vite. uvea, Instituto de Oftalmologia Conde de
Valenciana, mexico DF, Mexico.
Purpose: To determine the rate of complications in pedriatic patients
with uveitis at a reference center.
Methods: Retrospective review of medical records of children with
uveitis diagnosed before the age of 18 years, from 2007 to 2014 in
the Department of Uveitis and Ocular Immunology at Institute of
Ophthalmology Conde de Valenciana. Age, etiological diagnosis and
development of ocular complications were evaluated.
Results: From a total of 357 patients, 276 patients were included in
the analysis. We excluded patients that had only one visit and loss of
follow-up.
Main types of uveitis were: pars planitis in 175 patients (63.4%),
toxoplasmosis in 45 patients (16.3%), herpes keratouveitis in 16
patients (5.8%), toxocariasis in 12 patients (4.3%) and idiopathic
non-granulomatous anterior uveitis in 10 patients (3.6%). One
hundred sixty-five patients (59.8%) developed one or more of the
following complications: cataract in 78 patients (19.8%), band
keratophaty in 40 patients (10.15%), epiretinal membrane in 26
patients (6.6%), glaucoma in 19 patients (4.8%) and macular edema
in 17 patients (4.3%).
Complications in the group of pars planitis (175) were: cataract in
45 patients (25.7%), band keratophaty in 22 patients (12.57%) and
epiretinal membrane in 16 patients (9.14%). The most common
complication of toxoplasmosis (45) was choroidal neovascular
membrane in 2 patients (4.4%).
In the herpes keratouveitis group (16) 5 patients (31.25%) had
corneal scarring. From the 12 patients with toxocariasis, 2 (16.6%)
developed glaucoma and 1 (8.3%) retinal detachment.
Conclusions: We found that more than 50% of pediatric patients with
uveitis developed ocular complications in the course of their disease,
one of the main reasons is because we received patients time long
after they were treated with several previous inadequate medications,
and because of the chronicity of uveitis in pediatric population.
Commercial Relationships: Olegario Ivan Castro Vite, None
Clinical features of syphilitic uveitis in an ophthalmologic
reference center in Mexico City
Rosalva Y. Bobadilla, Ricardo Blas Medina, Daniela Castro Farias,
Elsa Maria Flores Reyes, Miguel Pedroza-Seres. Uveitis and Ocular
Inmunology, Hospital Conde de Valenciana, Coacalco, Mexico.
Purpose: To describe demographic data of patients with diagnosis of
ocular syphilis in the department of uveitis and ocular immunology
at Hospital Conde de Valenciana, an ophthalmologic reference center
in Mexico
Methods: we analize all the electronic records from January 2004
to November 2014, selecting those diagnosed with ocular syphilis.
Epidemiological data included: age at diagnosis, gender, affected
eye, follow-up period, type of uveitis, laboratory test used, treatment,
visual acuity and intraocular pression in the first and last visit, and
ocular complications.
Results: Of the 4493 patients seen in the service of uveitis and ocular
immunology from January 2004 to November 2014, 50 (1.11%)
cases were diagnosed with ocular syphilis. Thirty three (66 %) were
female and 17 (34%) men. The average age was 56.34 years (16- 87
years). Of the 50 patients, 7 (14 %) had disease only in the right eye,
12 (24 %) in the left eye, and 31 (62 %) had both eyes afected. The
clinical manifestations were: scleritis in 2 (4%) patients, anterior
uveitis in 3 (6%) patients, intermediate uveitis in 1 (2%) patient,
posterior uveitis 0 (0 %) patients and panuveitis in 44 (88%) patients.
The initial and final visual acuity was classified into 5 groups,
excellent, good, regular, bad and no functional. With T -student test
0.603.The intraocular pressure of 82 eyes was averaged in 14.47
mmHg, and the averaged of the last visit was 13.66 mm Hg, all by
Goldman tonometry .Treatments applied were: benzathine penicillin
intravenously in 9 (18%) patients, intramuscular benzathine penicillin
in 33 (66%) patients, erythromycin in 6 (12%) patients and 2 (4%)
that did not returned. Within the complications more frequently
reported were: glaucoma in 13 (26%) patients, cataract in 12 (24%),
macular edema in 5 (10%) patients, ocular hypertension in 2 (4%),
macular hole in 2 (4%), band keratopathy in 2 (4%), tractional retinal
detachment in 2 (4%) patients.
Conclusions: Uveitis caused by syphilis is a pathology observed less
frequently due to public health programs, however when present,
clinical suspicion, appropriate interrogatory and laboratory test
are important to make the diagnosis, specially in patients without
previous systemic diagnosis that have bilateral panuveitis.
Commercial Relationships: Rosalva Y. Bobadilla, None; Ricardo
Blas Medina, None; Daniela Castro Farias, None; Elsa Maria
Flores Reyes, None; Miguel Pedroza-Seres, None
Fuchs Syndrome: A cross sectional study in a tertiary university
center in Argentina
Juan Pablo Fernandez1, Matias Portela1, Mariana Ingolotti1, Anahi
Lupinacci1, Cristobal A. Couto2, Mario J. Saravia1, Bernardo A.
Schlaen1. 1Ophthalmology, Hospital Universitario Austral, Buenos
Aires, Argentina; 2Oftalmologia, Universidad de Buenos Aires,
Buenos Aires, Argentina.
Purpose: To describe clinical features of patients with diagnosis of
Fuchs Syndrome in Argentina
Methods: Patients with diagnosis of Fuchs Syndrome who were seen
at Hospital Universitario Austral between June 2009 and October
2014 were included. Data recorded included, age, sex, presence
of keratic precipitates, anterior chamber cells, iris atrophy, and
complications.
Statistical analysis was carried out using excel 2012. Chi square and
Fisher exact tests were used as appropriate.
Results: Thirty three patients (12 females, 21 males) with diagnosis
of Fuchs Syndrome were included. This represented 8.04% of the
patients with diagnosis of uveitis who were seen during this period.
Average age was 41 ± 13.34 years. Bilaterality was seen in 5 patients
(15.15%). Characteristic keratic precipitates were seen in 23 out of
27 eyes (85.19%). Ten out of 33 eyes (30.3%) had 2+ or more of
anterior chamber cells. Eight out of 24 eyes (33.33%) had 2+ or more
of vitreous haze. Twenty out of 30 (66.6%) eyes had cataract. Eleven
out of 20 eyes (55.5%) underwent cataract surgery. Eyes with 2+ or
more vitreous haze had a statistically significant greater proportion
of cataract occurrence (Fisher exact test: 0.04). Eleven out of 38 eyes
had ocular hypertension (28.95%).
Conclusions: Fuchs syndrome is common in our country. Greater
degree of vitreous inflammation seems to be associated with greater
proportion of cataract occurrence.
Commercial Relationships: Juan Pablo Fernandez, None; Matias
Portela, None; Mariana Ingolotti, None; Anahi Lupinacci, None;
Cristobal A. Couto, None; Mario J. Saravia, None; Bernardo A.
Schlaen, None
Clinical and Epidemiologic characteristics of Fuchs
Heterochromic Iridocyclitis in Hispanic population
ETHEL B. GUINTO ARCOS, Miguel Pedroza-Seres, Janet S. SilvaOrtiz. UVEA AND INFLAMMATORY DISEASES, INSTITUTO
DE OFTALMOLOGIA CONDE DE VALENCIANA, Mexico City,
Mexico.
Purpose: Report clinical features and epidemiologic characteristics
of the disease in a Hispanic population attending a Uveitis Service in
an Ophthalmology Center in Mexico
Methods: Retrospective case series study.We reviewed
electronicrecords from patients attending Uvea and Ocular
Inflammation Department at Instituto de Oftalmologia Fundacion
Conde de Valenciana in Mexico City from January 1st 2001
to December 1st 2014 with diagnosis of Fuchs Heterochromic
Iridocyclitis. Records had to comply with clinical data based on
criteria of Kimura. Other causes of infectious or noninfectious uveitis
were excluded
Results: We reviewed a total of 209 electronic records.We enrolled
142 eyes of 136 patients with the diagnosis of Fuch’s Heterochromic
Iridocyclitis who completed a minimum of 6 months follow
up.The mean follow up was 16±14.5 months (range 6- 60 months).
Males predominated (72/136,52.9%).The most common form
of presentation was unilateral (95.6%).The age at presentation
was 35.8±11.6 (range 12–64) years. On the clinical findings,
25.4%(36/142) showed heterochromia.All patients showed fine
stellate filamentary keratic precipitates and 70.4% (100/142) showed
mild (1-2+ cellularity and flare) anterior chamber inflammation.
Iris atrophy was seen in 67.6% (96/142) eyes. Koeppe and Bussaca
nodules were seen in 36.6%(52/142).Iris vessels were present in
3.5%(5/142).Vitreous opacities were found in 90 eyes (63.4%).
31 eyes presented elevated intraocular pressure (21.8%). 120 eyes
(84.5%) had developed cataracts, of which 38.5% (43/120) were
posterior subcapsular cataracts and 31.7% total opacities (38/120).
Best corrected visual acuity was hand movement in the majority
of cases (33.1%) at the moment of first consultation.93 eyes (65%)
underwent cataract surgery.Best corrected visual acuity was 0.5 or
better in 120/142(84.5%) of eyes at the final follow up
Conclusions: Based on the predominant clinical findings, unilateral
cataracts, fine keratic precipitates, subtle iris atrophy and mild
anterior chamber inflammation along with vitreous opacities
could lead to the diagnosis of Fuch’s Heterochromic Iridocyclitis
in our population. As a predominantly brown eyed population,
heterochromia is not the most prevalent feature. Recognition of this
disease in any patient of any complexion is important. This is the
first report of clinical and epidemiological features of the disease in
Hispanic Mexican population
Commercial Relationships: ETHEL B. GUINTO ARCOS, None;
Miguel Pedroza-Seres, None; Janet S. Silva-Ortiz, None
Hypertension in uveitis: a case series study
Mariana Ingolotti1, Bernardo A. Schlaen1, Mario J. Saravia1, Juan
Pablo Fernandez1, Matias Portela1, Cristobal A. Couto2, Anahi
Lupinacci1. 1Hospital Universitario Austral, Pilar, Argentina;
2
Hospital de Clinicas, Capital Federal, Argentina.
Purpose: To present a case series of uveitis with ocular hypertension
and analyze the etiologies and mechanisms.
Methods: A retrospective cross-sectional study between June 2009
and October 2014 was performed. Patients with diagnosis of any
type of uveitis and IOP over 21mmHg were included. Collected data
from patients included age at presentation, gender, acute or chronic
infection, anatomic classification, etiology of the uveitis, IOP at
presentation, mechanism of IOP increase and treatment received.
Data was recorded in Microsoft Excel 2011. Odds Ratio and chi
square analysis were chosen for statistical analysis.
Results: A total of 413 patients with uveitis diagnosis were recruited.
One hundred and twenty-four (30%) patients with elevated IOP
were encountered. The anatomic distribution was anterior (52%),
diffuse (28%), posterior (18%) and intermediate (2%). The most
frequent etiologies among the anterior uveitis were herpetic (31%),
idiopathic (22%) and Fuchs iridocyclitis (14%); among the posterior
toxoplasmosis (54%) and VKH (40%) in the diffuse cases. The
most common mechanism of IOP increase was the use of corticoid
therapy (71 cases). The proportion of hypertensive uveitis was 30,6
%. Hypertensive uveitis was most commonly infectious (chi 2: 44.75
p<0.01). Chronic uveitis more frequently developed hypertension
than acute presentation (chi 2 14.10 p<0.01). One hundred and four
cases received medical treatment whereas 27 needed a surgical
intervention as well.
Conclusions: Ocular hypertension in uveitis is a common finding
that requires close follow-up and treatment. hypertensive uveitis is
more common in cases of infectious etiology.
Commercial Relationships: Mariana Ingolotti, None; Bernardo
A. Schlaen, None; Mario J. Saravia, None; Juan Pablo Fernandez,
None; Matias Portela, None; Cristobal A. Couto, None; Anahi
Lupinacci, None
62/77 patients (81%) had unilateral uveitis, and 60 patients (80%)
had insidious onset. Anterior uveitis was the most common anatomic
classification (39 patients, 51%) followed by panuveitis (20 patients,
26%), and posterior uveitis (18 patients, 23%).
The most common infectious etiology was herpetic anterior uveitis
(37 patients, 48%) followed by toxoplasma uveitis (14 patients,
18%). The most prevalent viral pathogen was herpes zoster (VZV)
(21 patients, 27%) followed by herpes simplex (HSV) (20 patients,
26%). Acute retinal necrosis (ARN) was diagnosed in 14 patients
(18%). Aqueous humor was analyzed in all 14 patients with ARN
and was positive in 7 patients (50%). Of the 14 patients with ARN,
4 tested positive for cytomegalovirus (CMV) and 3 for VZV. Only 5
patients had classic CMV retinitis (6%). 3 patients (4%) had fungal
endophthalmitis, one had syphilitic chorioretinitis (1%), and one had
tuberculous uveitis (1%).
The age distribution among different types of uveitis is shown in
Table 1. On presentation, 43 patients (56%) had a VA better than
20/40 and 17 (22%) had a VA worse than 20/200. VA at last f/u was
better than 20/40 in 39 patients (51%) and worse than 20/200 in 22
patients (29%).
Table 2 shows the VA at last f/u among the different types of uveitis.
16 (21%) and 10 (13%) of the eyes required cataract and vitrectomy
surgery, respectively. 14 of the eyes (18%) were on IOP-lowering
medications and four (5%) needed glaucoma surgery.
Conclusions: The most common type of infectious uveitis seen
over the study period was herpetic anterior uveitis secondary to
VZV or HSV, found to be most prevalent in patients above 60 yrs
old. This finding is comparable to other American epidemiologic
studies. Ocular toxoplasmosis and ARN were also common causes
of infectious uveitis. PCR of intraocular fluid yielded an etiologic
diagnosis in 50% of ARN cases. 51% of patients had a VA better than
20/40 at last f/u which could be secondary to prompt referral and
appropriate treatment.
Infectious Uveitis in Virginia
Zeina A. Haddad2, Asima Bajwa2, James Patrie1, xin wenjun1, Ashvini
Reddy2. 1University of Virginia, Charlottesville, VA; 2Ophthalmology,
University of Virginia, Charlottesville, VA.
Purpose: To report the causes, clinical features, and outcomes of
infectious uveitis seen at the University of Virginia (a tertiary care
center) from 1984-2014.
Methods: Retrospective review of 491 uveitis patients. Descriptive
statistics were used to report and analyze demographic features,
diagnoses, visual acuity (VA), laboratory findings, and outcomes.
Results: 77/491 pts (16%) had infectious uveitis (mean age 58 yrs,
71% F, 77% Caucasian). Mean f/u was 5 yrs (range 4 d–30 yrs).
Commercial Relationships: Zeina A. Haddad, None; Asima
Bajwa, None; James Patrie, None; xin wenjun, None; Ashvini
Reddy, None
Characteristics, Management, and Outcomes of Traumatic
Uveitis in Virginia
John Prenshaw, Asima Bajwa, James Patrie, xin wenjun, Ashvini
Reddy. Ophthalmology, University of Virginia, Charlottesville, VA.
Purpose: To report the clinical findings, management, and outcomes
of patients with post-traumatic uveitis seen at a tertiary referral center
over a 30 year-period.
Methods: Retrospective review of patients with ocular inflammation
following blunt trauma seen at the University of Virginia from
1984 to 2014. Descriptive statistics were used to report patient
historical information, clinical findings, therapy, and outcomes.
When applicable, the paired and unpaired T-test was used to compare
subgroups.
Results: A total of 57 patients (59 eyes) were included. Age at
presentation ranged from 6 to 84 years (mean, 42.8 years). Thirtyfour patients (60%) received topical steroids, 2 (3.5%) received
systemic steroids, 5 (8.7%) required cataract surgery, 5 (8.7%)
required glaucoma medications, and 1 (1.7%) required glaucoma
surgery. Average duration of follow up was 4.48 years. Mean visual
acuity (VA) at presentation of 20/50 (logMAR 0.424) improved to
20/40 (logMAR 0.284) by the final visit, and this difference was
statistically significant (P =0.015). Poor final VA (worse than 20/50)
was associated with black race (P = 0.04), but not age at presentation
(P = 0.405) or gender (P= 0.095). Mean intraocular pressure (IOP)
did not change significantly (15.4 mmHg to 15.1mmHg, p=0.681)
over the length of follow-up. Compared to 434 patients (585 eyes)
with non-traumatic uveitis managed over the same time period, mean
final VA 20/60 (0.50 LogMAR) and IOP (14.9mm Hg) were not
significantly different (P = 0.0686 and P = 0.7697, respectively).
Conclusions: Conclusions:
Traumatic uveitis is commonly encountered and carries a visual
acuity prognosis that is not significantly different from other
forms of uveitis, though black race was associated with poorer
outcomes. Intraocular pressure was not significantly different than
in nontraumatic uveitis eyes and tends to be well-controlled. Most
patients are managed with topical steroid therapy. Glaucoma surgery
is rarely needed.
Commercial Relationships: John Prenshaw, None; Asima Bajwa,
None; James Patrie, None; xin wenjun, None; Ashvini Reddy,
None
Characteristics, Management, and Outcomes of Non-infectious
Post-Procedural Uveitis in a Tertiary Care Center
Eric Liss1, Asima Bajwa1, James Patrie2, Wenjun Xin2, Ashvini
Reddy1. 1Ophthalmology, University of Virginia, Charlottesville, VA;
2
Biostatistics, University of Virginia, Charlottesville, VA.
Purpose: To report the characteristics, management, and outcomes
of noninfectious, post-procedural uveitis seen in a tertiary care center
over a 30-year period.
Methods: A retrospective chart review was performed on a database
of 492 eyes diagnosed with uveitis over a 30-year period. From this
larger cohort, 39 eyes from 36 patients were identified as having noninfectious, post-procedural uveitis (defined as ocular inflammation
following intraocular surgery, laser, or intravitreal injection).
Descriptive statistics were used to analyze and characterize the type
of procedures involved, anatomic location of inflammation, medical
management, surgical management, and visual acuity (VA).
Results: Of the 39 eyes identified, 19 (48.7%) were diagnosed
post-cataract extraction and IOL placement, 4 (10.3%) were postintravitreal injection, 6 (15.4%) were post-cornea surgery, 3 (7.7%)
were post-laser procedures, 6 (15.4%) were post-retina surgery, and 1
(2.6%) was following glaucoma surgery. No patients were diagnosed
with infection. There were 33 cases of anterior uveitis (84.6%)
and 6 cases of panuveitis (15.4%). All patients required treatment
for inflammation, with 34 (87.2%) using topical steroids, 3 (7.7%)
using systemic steroids, 9 (23.1%) using intravitreal or sub-tenon’s
injection, and 2 (5.2%) taking a systemic anti-metabolite agent. Mean
baseline VA was 0.759 logMAR (20/115 Snellen) and mean final
VA was 0.849 (20/140 Snellen) with a p value of <0.05. Final VA
was decreased by one line or more in 17 (43.6%) eyes. Additional
interventions included cataract extraction in 28 (71.8%) eyes, medical
management of increased IOP in 11 (28.2%) eyes, and glaucoma
surgery in 2 (5.1%) eyes.
Conclusions: Post-procedural uveitis can present following
intraocular surgery, laser therapy, or intravitreal injection, and
patients may require long-term steroids or steroid-sparing therapy for
control. It is also associated with secondary issues such as cataract
formation and IOP elevation. In this series, post-procedural uveitis
was associated with a clinically and statistically significant decrease
in final visual acuity relative to presentation.
Commercial Relationships: Eric Liss, None; Asima Bajwa, None;
James Patrie, None; Wenjun Xin, None; Ashvini Reddy, None
Epidemiological characteristics of Vogt-Koyanagi-Harada
syndrome in a mexican population
Daniel Rangel-O’Shea, Pablo J. Guzman-Salas, Miriam G. ArellanoGanem, Miguel Pedroza-Seres. Instituto de Oftalmologia Conde de
Valenciana, Mexico City, Mexico.
Purpose: Report the number of patients with Vogt-Koyanagi-Harada
syndrome attending a Uveitis Department and analyze different
epidemiological characteristics of this disease.
Methods: Retrospective case series study in an uveitis department
with 103 patients with Vogt-Koyanagi-Harada syndrome from
2001 to 2013. We reviewed medical records from patients at
Instituto de Oftalmologia “Conde de Valenciana“ in Mexico. We
collected information of: gender, age, visual acuity –before and after
treatment-, ocular manifestations, neurological signs, auditory signs,
cutaneous changes, number of inflammatory episodes and diagnosis
criteria.
Results: We analyzed 103 patients, with average age of 37.71±12.76
years, with 80 females and 23 males.
Average inicial visual acuity was 0.96±1.01 logMAR on right eye
and 0.92±0.93 logMAon left eye, Average final visual acuity was
0.48±0.63 logMAR on right eye and 0.55±0.77 logMAR on left
eye.Final visual acuity improved on 79 patients after treatment.
Number of acute episodes were diferent on patients, 30 patients
had only 1 episode of inflamation, 32 patients had two episodes, 23
patients had 3 episodes and 18 patients has more than three episodes.
Ocular findings were variable, 44(42.71%) patients showed serous
retinal detachment, 38 (36.89%) patients showed macular edema,
85 (82.52%)patients showed sunset fundus, 66 (64.07%) showed
optic disc swelling, 21 (20.38%) showed cutaneous changes, 31
(30.09%) had auditory signs and 37 (35.92%) had neurological signs.
27 ( 26.21%) patients had criteria for probale VKH, 9 (8.73%) for
incomplete VKH and 4 (3.88%) for complete VKH.
Conclusions: This is the first study in Mexico, with this number of
patients, describing VKH epidemiologic data.
The information obtained help us to understand better this patology in
mexican population and could predict future clinical course.
Commercial Relationships: Daniel Rangel-O’Shea, None; Pablo
J. Guzman-Salas, None; Miriam G. Arellano-Ganem, None;
Miguel Pedroza-Seres, None
Choroidal involvement in presumed ocular tuberculosis: Report
from a population in a low endemic area
Ioanna Triantafyllopoulou1, Julio J. González-López1, Bhaskar
Gupta2, Farzana Rahman1, Peter Addison1, Mark C. Westcott1,
Carlos Pavesio1, Rupesh Agrawal1, 3. 1NIHR Moorfields Biomedical
Research Centre, Moorfields Eye Hospital NHS Foundation Trust and
UCL Institute of Ophthalmology, London, United Kingdom; 2Royal
Devon and Exeter NHS trust, Exeter, United Kingdom; 3National
Healthcare Group Institute Tan Tock Seng Hospital, Singapore,
Singapore.
Purpose: We performed a retrospective, observational study to
describe the epidemiology, clinical manifestations, treatment and
outcome of choroidal involvement in TB in a tertiary care hospital in
a low endemic area.
Methods: Seventy-seven patients with presumed TB associated
choroidal lesions who underwent Quantiferon-TB Gold In Tube
(QFT) test were included. Patients without choroidal involvement
or with less than 6 months of follow-up were excluded. Treatment
failure was defined as inability to taper oral corticosteroids to less
than 10mg/day or topical steroids to less than twice a day, inability
to stop oral immunosuppressive agents or persistence or recurrence
of inflammation within the first six months of completion of
antitubercular therapy (ATT). For the patients not on ATT, failure was
defined by the inability to taper medications as above.
Results: Mean age was 45.5±15.7 years. Fourty-four (57.1%) were
male, and 51 (66.2%) presented with bilateral disease. Thirty-nine
patients were of Asian descent, 21 Caucasians and 17 Africans.
Multifocal choroiditis was the most frequent clinical presentation
(24 patients-31%), followed by serpiginous-like choroiditis (16
patients-21%), choroidal granuloma (16 patients-21%) and unifocal
choroiditis (11 patients-14%). QFT was negative in 9 (12%), and
indeterminate in 3.
Fifty patients received ATT, 58 oral corticotherapy and 16 oral
immunosupresants. ATT was given for 6 months to 22 patients, for
9 months to 5 and for 12 months to 23. Sixteen patients developed
cystoid macular oedema at any point during the follow-up period.
Sixteen developed glaucoma, 2 developed choroidal neovascular
membranes, and 8 required cataract surgery.
Binary logistic regression analysis correcting by age, sex and ATT
revealed that a positive QFT decreased the risk of treatment failure
(OR=0.09; p=0.020) and oral corticosteriods increased that risk
(OR=17.87; p=0.017). No statistical association was found between
ATT and failure rate(p=0.483) in the logistic regression model.
Conclusions: Multifocal choroiditis, choroidal tuberculoma and
serpiginous-like choroiditis were the most common presentations.
Treatment failure rates (i.e inablility to taper steroids) were
equivalent between ATT and non ATT treated groups. Patients with
positive QFT showed treatment failure less frequently, while those
receiving oral corticotherapy had an increased risk of failure.
Commercial Relationships: Ioanna Triantafyllopoulou, None;
Julio J. González-López, None; Bhaskar Gupta, None; Farzana
Rahman, None; Peter Addison, None; Mark C. Westcott, None;
Carlos Pavesio, None; Rupesh Agrawal, None
Diagnostic Criteria And Clinical Manifestations Of Presumed
Latent Tuberculosis-Related Uveitis In A Bacille Calmette-Guerin
Vaccinatinated Community
Ozlem Gurses2, 1, Eda Karaismailoglu3. 1Ophthalmology, Middle
East Technical University, Oran ankara, Turkey; 2ophthalmology/
uveitis, dunyagoz hospital, Ankara, Turkey; 3Biostatistics, Hacettepe
University Medical Faculty, Ankara, Turkey.
Purpose: The wide range of clinical manifestations of presumed
latent tuberculosis-related uveitis (TRU) make its diagnosis difficult
in an endemic community. We described the ocular manifestations
of patients with TRU, and we evaluated the correlation between
skin induration value of tuberculin skin test (TST) and tuberculosis
antigens tube value of QuantiFERON®-TB Gold (QFT) test in a
Bacille Calmette-Guerin
(BCG) vaccinated community.
Methods: This was a prospective 1-year study in a tertiary referral
center. 85 patients,47 (55.3 %) female diagnosed with TRU were
included. Mean (standard deviation, SD) age was 52.9 (13.6) years.
TST, QFT and pulmonary X-ray were performed. Other possible
etiologies of uveitis were ruled-out. Standard anti-tuberculosis
therapy (ATT) was started, and response to ATT was monitored.
Statistical analysis was performed by
using SPSS for Windows 13.0.1 (SPSS Inc., Chicago, IL, USA)
Pearson correlation coefficient (r) was used for analysis. p < 0.05 was
considered as significant.
Results: 43 patients (50.6%) had bilateral involvement. The most
common ocular manifestation was anterior uveitis (78.8 %) followed
by vitritis, panuveitis, papillitis, vasculitis, chorioretinitis and
scleritis. The mean (SD) value of TST was 16.53 (6.05) mm and
the mean (SD) value of QFT was 8.06 (4.53) IU/ml. Pulmonary
X-ray results were normal. All the patients responded to ATT. No
statistically significant correlation was found between TST and QFT.
(r= 0.105, p=0.498)
Conclusions: There is no pathognomonic clinical manifestation
of TRU in an endemic, BCG vaccinated community. Presence of
bilateral anterior uveitis, positive results for both TST and QFT,
and positive response to ATT support the diagnosis of TRU in an
endemic, BCG vaccinated community.
Commercial Relationships: Ozlem Gurses, None; Eda
Karaismailoglu, None
Prevalence and Risk Factors of Epiretinal membrane in the
Multicenter Uveitis Steroid Treatment (MUST) Trial
Lyndell L. Lim1, 2, Francis Abreu8, Elizabeth A. Sugar8, Alyce Burke3,
Michael M. Altaweel4, P. Kumar Rao7, Janet T. Holbrook3, Susan
G. Elner6, Richard Stawell1, 2, John H. Kempen5. 1Centre for Eye
Research Australia, University of Melbourne, Melbourne, VIC,
Australia; 2Royal Victorian Eye and Ear Hospital, Melbourne, VIC,
Australia; 3Center for Clinical Trials, Bloomberg School of Public
Health, Johns Hopkins University, Baltimore, MD; 4Ophthalmology
and Vision Sciences, University of Wisconsin, Madison, WI;
5
Ophthalmology Biostatistics & Epidemiology, Scheie Eye Institute,
University of Pennsylvania, Philadelphia, PA; 6Ophthalmology,
Kellogg Eye Center, University of Michigan, Ann Arbor, MI;
7
Ophthalmology and Visual Sciences, Washington University School
of Medicine, St Louis, MO; 8Department of Biostatistics, Bloomberg
School of Public Health, Johns Hopkins University, Baltimore, MD.
Purpose: To report baseline prevalence and associated risk factors
of epiretinal membrane (ERM) in patients with severe active non-
infectious intermediate, posterior or panuveitis recruited into the
Multicenter Uveitis Steroid Treatment (MUST) Trial.
Methods: All participants underwent a standardized interview,
systemic examination and ophthalmic examination. Baseline OCT
images were graded at the Reading Center according to the presence
and severity of ERM, plus any other associated ERM complications
such as macular traction. Generalized estimating equations were
used to fit logistic regression models to assess risk factors while
accounting for between eye correlation in patients with bilateral
uveitis.
Results: Of the 479 eyes with uveitis in the MUST Trial, 435 eyes
(91%) from 243 individuals had OCT images that were gradable for
ERM at randomization. Time from uveitis onset > 5 years, posterior
synechiae, visual acuity (VA) < 20/100, prior IOP-lowering surgery,
cataract, active uveitis, and any systemic disease were associated with
increased risk of inability to assess ERM with OCT.
Of the 435 gradeable eyes, 126 (29%) had an ERM. In a
multivariable analysis, having diabetes (OR=2.21, p=0.043), age >
50 years (OR = 2.95, p < 0.001), the presence of retinal vasculitis
(OR=2.68, p=0.013), macular edema (OR=1.87, p=0.013) and uveitis
activity (OR = 1.83, p = 0.050) were associated with the presence
of ERM. VA worse than 20/100, cataract [including prior cataract
surgery], and Vogt-Koyanagi Harada disease were associated with
increased ERM prevalence; however the association was abrogated
by adjustment for other risk factors in the final multivariable model.
Conclusions: Detection of ERM by OCT in patients with uveitis
may be limited by the optical impact of uveitic complications such
as cataract and posterior synechiae. ERM is a common complication
of intermediate, posterior and panuveitis, and was associated with
increasing age, diabetes, macular edema and retinal vasculitis at
randomization. Additional analysis of longitudinal data is needed to
estimate the incidence of ERM and determine the residual impact of
ERM on VA (once uveitis activity has been treated) and its outcome
over time.
Commercial Relationships: Lyndell L. Lim, None; Francis Abreu,
None; Elizabeth A. Sugar, None; Alyce Burke, None; Michael M.
Altaweel, None; P. Kumar Rao, None; Janet T. Holbrook, None;
Susan G. Elner, None; Richard Stawell, None; John H. Kempen,
None
Support: NEI Grant U10EY014655, NEI Grant U10EY014660,
NEI Grant U10EY014656, Bausch and Lomb provided support in
the form of donation of fluocinolone implants for those patients
randomsied to this therapy who otherwise would not have access to
these implants.
Purpose: To evaluate the risks and quality of life outcomes of
fluocinolone acetonide implant therapy versus systemic corticosteroid
therapy supplemented with immunosuppression when indicated for
intermediate, posterior, and panuveitis.
Methods: 255 subjects with intermediate, posterior, or panuveitis
(479 eyes) randomized to systemic treatment or implant were
followed for 54 months. Local and systemic potential complications
of the therapies and self-reported health utility, vision-related
and generic health-related quality of life (QoL) were studied
prospectively.
Results: Over 54 months, phakic eyes developed cataract and
required cataract surgery more often in the implant group (hazard
ratio (HR)=2.2, p=0.003 and HR=4.0, p<0.0001). IOP elevation
measures occurred more frequently in the implant group (range
of HR’s=3.7-5.6, p<0.0001), and glaucoma occurred more
frequently (26.3% vs. 10.2%, HR=3.0, p=0.0002). In contrast,
potential complications of systemic therapy including measures
of hypertension, hyperlipidemia, diabetes, bone disease, and
hematological and serum chemistry indicators of immunosuppression
toxicity did not differ significantly between groups. Indices of quality
of life initially favored implant therapy by a modest margin, but
summary measures of health utility and vision-related or generic
health-related QoL were minimally different by 54 months. The SF36 physical component summary score favored implant by a small
margin (3.17 on a scale of 100, p=0.01). Mean QoL results were
favorable in both groups.
Conclusions: Fluocinolone acetonide implant therapy is associated
with a clinically important increased risk of glaucoma and cataract
with respect to systemic therapy. These complications potentially
can be addressed surgically. Despite regular follow-up and available
treatment, the implant group had a 16% excess risk of glaucoma,
suggesting that careful monitoring and early intervention is
warranted to prevent progression. A treatment regimen of systemic
corticosteroid and immunosuppressive therapies following consensus
recommendations was well tolerated with minimal toxicities. Selfreported QoL measures initially favored implant therapy, but over
time the measures converged, with generally favorable QoL in both
groups.
Commercial Relationships: Michael M. Altaweel, None; John
H. Kempen, None; Lea T. Drye, None; Janet T. Holbrook, None;
Douglas A. Jabs, None; Elizabeth A. Sugar, None; Jennifer E.
Thorne, None
U10EY014656
Risks and Quality of Life associated with Fluocinolone Acetonide
Intraocular Implant Versus Systemic Anti-inflammatory Therapy
for Intermediate, Posterior or Panuveitis: 4.5 year results of The
Multicenter Uveitis Steroid Treatment Trial and Follow-up Study
Michael M. Altaweel1, John H. Kempen2, Lea T. Drye3, Janet T.
Holbrook4, 3, Douglas A. Jabs5, Elizabeth A. Sugar4, Jennifer E.
Thorne3, 6. 1Ophthalmology & Visual Science, Univ of WisconsinMadison, Madison, WI; 2Center for Preventive Ophthalmology and
Biostatistics, Scheie Eye Institute, Philadelphia, PA; 3Center for
Clinical Trials, Johns Hopkins Bloomberg School of Public Health,
Baltimore, MD; 4Departments of Epidemiology and Biostatistics,
Johns Hopkins Bloomberg School of Public Health, Baltimore, MD;
5
Ophthalmology and Medicine, Mount Sinai School of Medicine,
New York, NY; 6Ophthalmology, Johns Hopkins University School of
Medicine, Baltimore, MD.
Interim Analyses of the SAVE-2 Study: Sirolimus as a
Therapeutic Approach for UVEitis: A Phase 2, Open-label,
Randomized Study to Assess the Safety, Tolerability, and
Bioactivity of Two Doses of Intravitreal Injection of Sirolimus in
Patients with Non-infectious Uveitis
Yasir J. Sepah1, Mohammad A. Sadiq1, Mohamed K. Soliman1, 2,
Mohamed Ibrahim1, Mostafa S. Hanout1, Salman Sarwar1, Aniruddha
Agarwal1, Diana V. Do1, Quan Dong Nguyen1. 1University of
Nebraska Medical Center, Omaha, MA; 2Ophthalmology, Assiut
University, Assiut, Egypt.
Purpose: To report the interim analyses of the efficacy of 2 different
doses of intravitreal (IVT) sirolimus in eyes with non-infectious
posterior, intermediate, or panuveitis in the SAVE-2 Study at the
primary endpoint.
Methods: SAVE-2 is a randomized, phase 2, open-label study
conducted at 3 clinical centers in the United States. At least 28, but
not more than 32, subjects are to be enrolled. Key inclusion criteria
were: 1) diagnosis of non-infectious uveitis; 2) active uveitis, defined
as having at least 1+ vitreous haze (NEI scale) and/or at least 1+
vitreous cell count (SUN scale); 3) best-corrected ETDRS visual
acuity of 20/400 or better in the study eye. Eligible subjects were
randomized into one of two treatment arms in a ratio of 1:1. Group
1 received IVT 440 mg of sirolimus in study eyes on Days 0, 30, 60,
90, 120, and 150; group 2 received 880 mg of sirolimus on Days 0,
60, and 120. Fellow eyes are also eligible to receive sirolimus (of
opposite dose to that of study eye) in SAVE-2. Primary endpoint of
the study is at M6. Starting at M6, both study and fellow eyes can be
retreated based on retreatment criteria with originally assigned doses.
Patients are followed until M12.
Results: 25 subjects have been randomized in SAVE-2 and are
included in the analysis. Baseline characteristics are shown in Table.
Vitreous haze (VH) decreased (M6 compared to baseline) by 1 step
or more in 81.8% and 92.9% of patients in group 1 (low dose) and 2
(high dose), respectively at M6 (p=0.564). VH decreased by 2 steps
or more in 63.6% and 50% of patients in groups 1 and 2, respectively
at M6 (p=0.695). Mean change in VA for subjects who completed
M6 visit was +3.66 and -2.91 ETDRS letters in group 1 and 2,
respectively. Among subjects with macular edema at baseline (n=13),
the mean change in foveal thickness was -89.42 mm in group 1 and
+81.5 mm in group 2 at M6.
Conclusions: Both low and high doses of IVT sirolimus are found to
decrease vitreous haze in eyes with non-infectious uveitis. Low dose
(440 mg) sirolimus administered monthly may be more efficacious in
reducing uveitic macular edema than high dose (880 mg) administered
every 2 months.
Table – Baseline Characteristics of Subjects in the SAVE-2 Study
Commercial Relationships: Yasir J. Sepah, None; Mohammad
A. Sadiq, None; Mohamed K. Soliman, None; Mohamed
Ibrahim, None; Mostafa S. Hanout, None; Salman Sarwar, None;
Aniruddha Agarwal, None; Diana V. Do, None; Quan Dong
Nguyen, Santen (S)
Support: Unrestricted educational grant SANTEN
Long-Term Safety of Intravitreal Sirolimus for the Treatment of
Non-infectious Uveitis (NIU) of the Posterior Segment: 12-Month
Results from SAKURA Study 1
Pauline T. Merrill1, Yang Yang2. 1Ophthalmology, Rush University,
Chicago, IL; 2Santen Inc., Emeryville, CA.
Purpose: The SAKURA trial is a Phase III, randomized, multicenter,
24-month, multinational study assessing the safety and efficacy of
intravitreal sirolimus as monotherapy for the treatment of active
NIU of the posterior segment. In the 6-month double-masked period
of SAKURA Study 1, bimonthly injections of intravitreal sirolimus
preserved subjects’ best corrected visual acuity while significantly
improving vitreous haze (VH) scores. Here, we report the long-term
safety of intravitreal sirolimus during the first 12 months of treatment
(the double-masked period combined with the open-label period).
Methods: Subjects with active NIU of the posterior segment were
randomized in 1:1:1 fashion to receive 44 μg, 440 μg, or 880 μg
injections of intravitreal sirolimus, administered every 2 months (Day
1 and Months 2 and 4; double-masked period). Primary efficacy was
assessed at Month 5. At Month 6, subjects eligible to receive further
intravitreal sirolimus received 880 mg injections every 2 months
(Months 6-10; open-label treatment period) under the then current
amendment.
Results: Of the 346 randomized and treated subjects, 287 entered the
open-label period and completed the VH assessment at Month 12. Of
these, 211 received at least 1 injection of intravitreal sirolimus. The
most common reasons for premature discontinuation prior to Month
12 were subject withdrawal (n=16), adverse event (n=12), and loss
to follow-up (n=11). The adverse events in the open-label period
were similar to those reported in the double-masked period. Over
the first 12-month treatment period, the most common serious ocular
adverse events (≥2%) were worsening of uveitis (7.2%), worsening
of choroiditis (4.0%), cataract (3.8%), non-infectious endophthalmitis
(2.9%), medication residue (2.3%), and increased intraocular pressure
(2.0%). The incidence of post-injection endophthalmitis was 1.2%,
all with the 880 μg dose (1 culture positive, 3 culture negative).
Conclusions: Intravitreal sirolimus was associated with a low
incidence of serious ocular adverse events over 12 months in this
diverse population of subjects with NIU of the posterior segment. The
types of adverse events in the open-label period of SAKURA Study 1
were similar to those observed in the double-masked period.
Commercial Relationships: Pauline T. Merrill, Abbvie (C), Abbvie
(F), Santen (C), Santen (F); Yang Yang, Santen Inc. (E)
Relationships between HIV-related Neuroretinal Disorder and
Measures of Vision-Specific Quality of Life among People with
AIDS
Davin Ashraf1, Kevin P. May2, Gary N. Holland1, Mark L. Van
Natta2, Albert Wu2, 3, Jennifer E. Thorne4, Douglas A. Jabs5, 6. 1Ocular
Inflammatory Disease Center, UCLA Stein Eye Institute and the
Department of Ophthalmology, David Geffen School of Medicine
at UCLA, Los Angeles, CA; 2Department of Epidemiology, Johns
Hopkins University Bloomberg School of Public Health, Baltimore,
MD; 3Department of Health Policy and Management, Johns Hopkins
University Bloomberg School of Public Health, Baltimore, MD;
4
Department of Ophthalmology, Wilmer Eye Institute at Johns
Hopkins, Baltimore, MD; 5Department of Ophthalmology, Icahn
School of Medicine at Mount Sinai, New York, NY; 6Department of
Medicine, Icahn School of Medicine at Mount Sinai, New York, NY.
Purpose: Some HIV-infected individuals have evidence of optic
nerve or retinal dysfunction, even with good visual acuity, that
manifests as decreased contrast sensitivity (CS), and is termed
neuroretinal disorder (NRD). HIV-related NRD is a risk factor for
vision impairment, blindness, and mortality, but its effect on visionspecific quality of life (QOL) has not been explored.
Methods: We performed a cross-sectional study of participants
in the Longitudinal Study of the Ocular Complications of AIDS
(LSOCA) at initial completion of the National Eye Institute 25-item
Visual Function Questionnaire (VFQ-25) who met the following
inclusion criteria: no evidence of ocular opportunistic infection or
cataract and best corrected visual acuity (BCVA) of 20/40 or better.
Those with contrast sensitivity <1.50 logCS in either eye were
considered to have NRD. QOL was compared between individuals
with and those without NRD, with adjustment for age, BCVA,
CD4+ T-lymphocyte count, and interval since AIDS diagnosis. The
eleven VFQ-25 subscales and composite score were scored from 0 to
100, with higher scores representing better QOL. The relationships
between NRD and VFQ-25 scores, and between logCS and VFQ-25
scores, were assessed using multiple linear regression and Spearman
correlation, respectively.
Results: A total of 811 individuals met study criteria, 39 (4.8%)
of whom had NRD. After adjustment, individuals with NRD had a
significantly lower mean VFQ-25 composite score than those without
NRD (79 vs. 87, respectively, p=0.0006). NRD was also significantly
associated with lower mean scores in the following VFQ-25
subscales: near activities (78 vs. 86, p=0.009); distance activities
(85 vs. 91, p=0.04); social functioning (89 vs. 96, p=0.001); mental
health (76 vs. 87, p=0.0007); dependency (81 vs. 94, p<0.0001);
and color vision (90 vs. 97, p<0.0001). Among those with NRD, the
correlation between logCS and VFQ-25 composite score was 0.35
(p=0.03).
Conclusions: HIV-related NRD is associated with reduced visionspecific QOL among people with AIDS. Among those with NRD,
decreasing contrast sensitivity is associated with lower VFQ-25
composite scores.
Commercial Relationships: Davin Ashraf, None; Kevin P. May,
None; Gary N. Holland, Genentech (C), Novartis International AG
(C), Santen Pharmaceutical (C), Xoma (US) LLC (C); Mark L. Van
Natta, None; Albert Wu, None; Jennifer E. Thorne, Gilead (C),
National Eye Institute (F), National Institute of Allergy and Infectious
Diseases (F); Douglas A. Jabs, Applied Genetic Technologies, Inc.
(S), Novartis Pharmaceutical Corp. (S), Santen Pharmaceutical (C)
Support: U10 EY 08052, U10 EY 08057, U10 EY 08067
Comparison of the expression of TGFβ2 activating molecules in
conjunctival inflammation
Laura Soriano-Romani1, 2, Laura Contreras-Ruiz3, Laura GarciaPosadas1, 2, Antonio Lopez-Garcia1, 2, Sharmila Masli3, Yolanda
Diebold1, 2. 1Ocular Surface Group, IOBA - University of Valladolid,
Valladolid, Spain; 2CIBER-BBN (Biomedical Research Networking
Center on Bioengineering, Biomaterials and Nanomedicine),
Valladolid, Spain; 3Ophthalmology, Boston University School of
Medicine, Boston, MA.
Purpose: Increased expression of TGFβ2 is reported in the
conjunctiva of dry eye patients despite the decline in TGFβ2expressing goblet cells suggesting a lack of anti-inflammatory
activity of TGFβ2 in the context of conjunctival inflammation. While
integrins expressed in inflamed tissue are unable to activate this
isoform of TGFβ, Thrombospondin-1 (TSP1) is known to activate it
efficiently via ligation of its receptor CD36. Our aim was to compare
expression of molecules associated with TGFβ2 activation during
murine conjunctival inflammation and assess their correlation with
inflammatory conjunctival epithelial apoptosis.
Methods: Human conjunctival tissue from cadaveric donors, primary
human conjunctival epihelial, stromal cells and murine conjunctiva
were immunostained for CD36, TSP1 or latent TGFβ2. Inflamed
conjunctival tissues were obtained from scopolamine-injected
C57BL/6 (WT) mice induced to develop Experimental Dry Eye
(EDE) with 5 days of desiccating conditions and TSP1 deficient
(TSP1-/-) mice, which spontaneously develop Sjögren’s syndrome
associated conjunctival inflammation with age. Immunostaining
intensities were compared with ImageJ analysis. Apoptosis was
assessed by detecting activated caspase-3/7 using CellEvent detection
kit (Life Technologies).
Results: Both CD36 and TSP1 were detectable in human
conjunctival tissue as well as primary conjunctival epithelial and
stromal cells just as in normal WT mouse conjunctiva that lacked
caspase-3/7 positive cells. However, epithelial cells positively
stained from caspase-3/7 were detected in conjunctiva derived from
both EDE and TSP1-/- mice indicative of apoptosis in line with
local inflammation. Increased immunostaining of latent TGFβ2 was
detected in TSP1-/- as compared with WT mice, supporting lack of its
activation in inflamed murine conjunctiva. Interestingly, compared to
WT conjunctiva increased TSP1 and reduced CD36 immunostaining
was detected in EDE mice. Conversely increased CD36
immunostaining was seen in TSP1-/- conjunctiva in comparison to
WT mice.
Conclusions: The absence or reduced expression of one of the
molecules, CD36 or TSP1, involved in TGFβ2 activation supports
pro-inflammatory conditions in the conjunctiva that lead to apoptotic
cell death of conjunctival epithelial cells. Therapeutic strategies
directed towards restoring activation of latent TGFβ2 may help treat
chronic conjunctival inflammation.
Commercial Relationships: Laura Soriano-Romani, None; Laura
Contreras-Ruiz, None; Laura Garcia-Posadas, None; Antonio
Lopez-Garcia, None; Sharmila Masli, None; Yolanda Diebold,
None
Support: FEDER-CICYT Grant MAT2013-47501-C02-1-R (YD),
Regional JCyL Scholarship/European Social Fund Program (LSR),
FPI Scholarship Program (LGP), NEI grant EY015472 (SM)
Clinical features of glaucoma in cytomegalovirus corneal
endotheliitis
Hideaki Yokogawa1, Akira Kobayashi1, Natsuko Yamazaki1, Yoshiaki
Saito2, Kazuhisa Sugiyama1. 1Ophthalmology, Kanazawa University
Graduate School of Medical Science, Kanazawa, Japan; 2Saito Eye
Clinic, Kanazawa, Japan.
Purpose: Recently, diagnostic criteria of cytomegalovirus (CMV)
corneal endotheliitis was established by the Japan Corneal
Endotheliitis Study Group. However, clinical features of ocular
hypertension / secondary glaucoma associated with the disease
had not been well understood. We performed a retrospective,
observational clinical study to reveal manifestations of glaucoma
associated with CMV corneal endotheliitis.
Methods: Eighteen eyes of 18 patients with CMV corneal
endotheliitis (14 eyes with typical CMV endotheliitis and 4 eyes with
atypical CMV endotheliitis) were enrolled this study. We analyzed the
clinical manifestations highlighting the glaucoma status, including
onset of glaucoma, fellow eye glaucoma, intraocular pressure,
gonioscopic findings, visual field, and glaucoma surgery.
Results: Mean age was 70.4±11.5 years. Seventeen cases (94.4%)
were male. All 18 eyes had glaucoma history which had been
treated for long duration (9.6±9.0 years). Eight eyes (44.4%) had
been diagnosed as Posner-Schlosmann syndrome by previous
ophthalmologist. Nine cases (50.0%) had the fellow eye glaucoma.
All 18 eyes had received antiglaucoma agents and topical steroid
treatment with (6 eyes) or without (12 eyes) aciclovir / valaciclovir.
At initial visit, intraocular pressure was 21.5±10.9mmHg, and all
sixteen eyes excluded 2 post-glaucoma surgery or keratoplasty eyes
had open angle. Normal visual field or normal optic disc was noted
in 5 eyes (27.8%), and visual field defects were early stage (MD>6dB) in 4 eyes (22.2%), middle stage (-6dB>MD>-12dB) in 2 eyes
(11.1%), and late stage (MD<-12dB, Kosaki classification) in 7 eyes
(38.9%). After we made diagnosis of CMV corneal endotheliitis, we
treated all cases with anti-CMV drug including systemic ganciclovir
/ valganciclovir, topical ganciclovir, and topical corticosteroids. Five
eyes (27.8%) required glaucoma surgeries, including trabeculectomy
(3 eyes), and 360-degree trabeculotomy (2 eyes).
Conclusions: All cases with CMV corneal endotheliitis had
glaucoma history. In cases with refractory glaucoma due to PosnerSchlosmann syndrome or anterior uveitis, CMV corneal endotheliitis
should be concerned as one of the differential diagnosis.
Commercial Relationships: Hideaki Yokogawa, None; Akira
Kobayashi, None; Natsuko Yamazaki, None; Yoshiaki Saito, None;
Kazuhisa Sugiyama, None
Support: Grant-in-Aid for Scientific Research KAKENHI, Japan
(No. 25462705).
Cytomegalovirus as a cause of acute endothelial cell loss in
immunocompetent patients with hypertensive anterior uveitis
Jin A Choi, Ku Sub Kim, Chan Kee Park. Ophthalmology, Catholic
university of Korea, Suwon, Korea (the Republic of).
Purpose: Virus has been known to play a role in the idiopathic
anterior uveitis associated with ocular hypertension. In this study,
we investigated the clinical characteristics of patients with anterior
hypertensive uveitis and compared the characteristics between
patients in cytomegalovirus (CMV)-positive group and those in
CMV-negative group in their aqueous humor sample.
Methods: Medical records of forty-two patients with hypertensive
anterior uveitis were analyzed retrospectively. All patients underwent
slit lamp biomicroscopy examination, specular microscopy,
gonioscopy, and serological test. Among 42 patients with
hypertensive anterior uveitis, an aqueous sampling was done in 22
patients, and their aqueous analyzed for viral deoxyribonucleic acid
by polymerase chain reaction.
Results: The average age of 42 patients with hypertensive anterior
uveitis 57.6 years and 29 (69.0%) of subjects were male. Twentytwo patients (52.4%) underwent glaucoma surgery, and the average
corneal endothelial cell counts were 1,908 cells/ mm2. Among 22
patients having an aqueous sampling, 6 patients showed the CMVPCR positive, whereas 16 patients showed the CMV-PCR negative.
The CMV-positive group were significantly younger (CMV-positive
vs.CMV-negative: 47.5 ± 14.8 yrs vs. 67.6 ±11.8 yrs; P = 0.006)
and more myopic compared with the CMV-negative group (-3.6
± 4.2 vs. 0.0 ± 1.6D; P = 0.031). The frequency of glaucoma
surgery was similar between groups (66.0% vs 66.0%, P = 0.701).
However, 66.7% of CMV-positive group had glaucoma tube shunt
surgery, whereas majority of CMV-negative group (80%) underwent
trabeculectomy as a glaucoma surgery. Interestingly, the corneal
endothelial cell counts were significantly lower in CMV-positive
group, compared with CMV-negative group (1245 ± 560 cells/mm2
vs 1981 ± 387 cells/mm2; P = 0.009). In the CMV-positive group,
systemic ganciclovir therapy was used in 2 patients. After a 1month
of gangiclovir therapy, the aqueous CMV-PCR titer was dramatically
decreased in both patients. However, extensive corneal endothelial
cell loss was found after 5 months later, despite the ganciclovir
therapy.
Conclusions: CMV was found to be one of the etiologic factors
in patients with hypertensive anterior uveitis. Special cautions are
needed for patients with CMV-positive hypertensive anterior uveitis,
considering its adverse effect on corneal endothelium.
Commercial Relationships: Jin A Choi, None; Ku Sub Kim, None;
Chan Kee Park, None
Keratic precipitate morphology in uveitic eyes of various
etiologies using RTVue-100 fourier-domain corneal anterior
module OCT system
Noriyasu Hashida, Shizuka Koh, Takeshi Soma, Kohji Nishida.
Ophthalmology, Osaka University Graduate School of Medicine,
Suita, Japan.
Purpose: To identify the morphologic appearance of keratic
precipitate (KP) with RTVue-100 fourier-domain corneal anterior
module optical coherence tomography (OCT) system (RTVue)
(optovue, USA) for the diagnosis of uveitic eyes of various
etiologies.
Methods: RTVue OCT scan was performed on consecutive 23
eyes of 19 patients with different types of uveitis to investigate the
morphologic appearance of KPs. The study included sarcoidosis in
5 eyes, herpetic iridocyclitis in 8, Fuchs heterochromic iridocyclitis
(FHI) in 3, idiopathic granulomatous uveitis in 2, tubulointerstitial
nephritis and uveitis in 1, and masquerade syndrome with primary
vitreoretinal lymphoma in 4. RTVue was used to analyze the
differences in types of KPs between various uveitic groups.
Results: Mean age of the patients was 58.0 ± 19.9 (range, 23-96)
years, and 11 (52.6 %) were female. Bilateral involvement was
observed in 4 cases (21.0 %). In all cases, the slit-lamp examination
revealed various pattern such as whitish and/or brownish KPs. Those
KPs also showed various morphologies such as vaguely-outlined,
round, and dendriform appearance. In RTVue examination, intensity
of KPs demonstrated various patterns. Morphologic features of KPs
also showed various patterns such as globular, dome-shaped, sawedged, rectangle-shaped appearance protruded from the retrocornea.
Especially, stippled, small and dendriform KPs were observed in FHI
cases. In contrast, round, big and high intensity KPs were observed
in cytomegalovirus (CMV) positive iridocyclitis cases. In herpetic
iridocyclitis cases, KPs were diffusely scattered over the corneal
endothelium, but, through the course of antiviral treatment, they
aggregated each other and deposited dispersedly. By the limitation of
number of cases, no significant difference was observed, however;
RTVue images in various type of disease showed characteristic and
specific morphological patterns.
Conclusions: The morphology of KPs in various uveitic eye diseases
showed characteristic images in slit-lamp and RTVue examination.
RTVue examination is non-invasive and repeatable methods for
diagnosis of uveitis by evaluating the KPs morphologies.
Commercial Relationships: Noriyasu Hashida, None; Shizuka
Koh, None; Takeshi Soma, None; Kohji Nishida, None
Clinical Trial: UMIN000010096
Dendritic cells in non-infectious anterior uveitis
Micheal O’Rourke1, 2, Mary Canavan2, Ursula Fearon2, Conor C.
Murphy1. 1Ophthalmology, Royal College of Surgeons in Ireland,
Dublin 2, Ireland; 2St Vincent’s University Hospital, Dublin, Ireland.
Purpose: Innate immunity is triggered when toll-like receptors
(TLRs) on antigen presenting cells become activated leading to
subsequent activation of inflammatory cascades. Dendritic cells
(DC) are professional antigen presenting cells (APCs), which can
be divided into 2 major subsets – myeloid (mDC) and plasmacytoid
(pDC). TLRs promote maturation of APCs by the production of
pro-inflammatory cytokines and up-regulation of co-stimulatory
molecules. This study compared APC percentage, activation
status and intracellular cytokine production of mDC and pDC
in the circulation of AU patients to healthy controls (HCs). The
inflammatory cell profile in inflamed aqueous humor (AqH) of AU
patients was also carried investigated.
Methods: Circulating DC were defined as HLADR+, Lineageand further subdivided as myeloid (CD11c+) or plasmacytoid DC
(CD123+). CD40, CD80 and CD83 cell surface expression was used
to assess activation and maturation status of each subtype. After cell
permeabilisation, intracellular cytokine staining was carried out for
IL-10 and TNFa under basal, TLR4 (LPS), TLR7/8 (Resiquimod)
and TLR9 (CpG) stimulated conditions. To examine the local
inflammatory response, approximately 250uL of inflamed AqH from
active AU patients was centrifuged to obtain a cell pellet and stained
for CD45, HLA DR and CD11c.
Results: AU patients (n=5) had a decrease in circulating mDC and
pDC compared to healthy controls (HC) (p<0.05). CD40 expression
on mDC in AU patients was increased (p<0.05) with no differences
in CD80 and CD83. There was no difference in IL10 or TNFa
production under basal conditions. However, pDC showed hyporesponsiveness to TLR4 stimulation with lower IL10 and TNFa
production in AU compared to controls (p<0.05). Inflamed AqH cells
were CD45+ with approximately 1% being HLA DR+ CD11c+.
Conclusions: These results provide evidence that DCs are recruited
to the eye from the circulation during AU with decreased numbers
in circulation and a population of DC present in the inflamed AqH.
Circulating DC may be tolerised to TLR4 stimulation with decreased
cytokine production on stimulation. Current work is profiling
cytokine concentration in AqH and the functional effect of inflamed
AqH on HC monocyte derived DC model co-cultured with T cells.
Commercial Relationships: Micheal O’Rourke, None; Mary
Canavan, None; Ursula Fearon, None; Conor C. Murphy, None
Circulating regulatory T cells as biomarkers for macular edema
associated with non-infectious uveitis
Blanca Molins1, 2, Jessica Matas2, Alex Fonollosa3, Victor Llorens2,
Marina Mesquida2, David Díaz-Valle4, Barbara Berasategui3,
Maite Sainz De La Maza2, Pilar Calvo5, Alfredo Adan Civera2, 1.
1
Ophthalmology, IDIBAPS, Barcelona, Spain; 2Hospital Clinic de
Barcelona, Barcelona, Spain; 3Hospital de Cruces, Bilbao, Spain;
4
Hospital Clinico San Carlos, Madrid, Spain; 5Hospital Universitario
Miguel Servet, Zaragoza, Spain.
Purpose: To evaluate the profile of circulating levels of regulatory T
cells (Treg) in patients with macular edema (ME) related to noninfectious uveitis and its relationship with central retinal thickness
(CRT), anatomical classification, and therapeutical management.
Methods: Twenty-one patients with ME associated with noninfectious uveitis and 10 healthy subjects from 3 tertiary referral
centers in Spain were included. Blood samples were obtained at
baseline (T0) when patients presented with ME (considered as CRT >
300 μm, measured by optical coherence tomography [OCT]) and also
when ME improved after treatment (T1, CRT <300 μm). Peripheral
blood mononuclear cells (PBMCs) were obtained by Ficoll gradient
from heparinized blood and Treg (CD3+CD4+Foxp3+CD25hi) levels
in PBMCs were determined by flow cytometry.
Results: Patients with ME at T0 had significantly lower Treg levels
than controls (2.08±0.24 % vs. 3.13±0.39 %, P<0.05). Resolution
of ME seemed to be accompanied by an increase in Treg levels,
although the difference did not reach statistical significance (T0
2.08±0.24 %, vs T1 2.98±0.83 %, P=0.247). Remarkably, patients
who received systemic immunomodulatory therapy (IMT) showed
a significant increase in Treg levels compared to those who received
local therapy (dexamethasone intravitreal implant, periocular
triamcinolone injection), (82±40 % improvement vs. -17±7 %,
P<0.05).
Conclusions: Our preliminary data suggest that Treg levels may
serve as biomarkers of ME associated with non-infectiuos uveitis,
as patients with ME showed reduced levels of Treg compared to
healthy subjects. Moreover, ME resolution appeared to correlate with
an increase in Treg levels, particularly in those patients receiving
systemic IMT.
Commercial Relationships: Blanca Molins, None; Jessica Matas,
None; Alex Fonollosa, None; Victor Llorens, None; Marina
Mesquida, None; David Díaz-Valle, None; Barbara Berasategui,
None; Maite Sainz De La Maza, None; Pilar Calvo, None; Alfredo
Adan Civera, None
Support: This work was supported by the Ministry of Science and
Innovation of Spain, “Instituto de Salud Carlos III”, “Fondo de
Investigación Sanitaria” (PI13/00217).
Characterization of Ophthalmic and Rheumatologic Features in
Patients with Psoriasis and Psoriatic Arthritis
Anton M. Kolomeyer1, Ashwinee Ragam4, Natasha V. Nayak2,
Christina Yu4, Sergio Schwartzman3, David S. Chu4. 1Ophthalmology,
UPMC, Pittsburgh, PA; 2Ophthalmology, NYEEI, New York, NY;
3
Rheumatology, HSS, New York, NY; 4Rutgers University, Newark,
NJ.
Purpose: To characterize and describe ophthalmic and rheumatologic
findings.
Methods: Retrospective chart review of ophthalmic and
rheumatologic manifestations in patients with psoriasis (n=6) and
psoriatic arthritis (n=15) from two tertiary care centers in the United
States specializing in autoimmune ophthalmic disease. Data was
collected on age, gender, ethnicity, associated autoimmune disease,
visual acuity (VA), intraocular pressure (IOP), type and grade of
inflammation, ocular and rheumatologic characteristics of disease,
systemic immunomodulating agents, and ocular therapy.
Results: Twenty-one patients were included (mean ± SD age was
51.1 ± 14.4 years; mean follow-up, 48.5 months; 76% were female;
and 86% were Caucasian). Six (29%) patients had an associated
systemic disease (three each with sarcoidosis and rheumatoid
arthritis). None of the eyes experienced a change in Snellen VA by
two or more lines. Mean ± SD initial and final IOP was 14.6 ± 4.4
mm Hg and 14.5 ± 4.1 mm Hg, respectively. Ocular manifestations
included anterior uveitis (n=9 [43%]), panuveitis (n=5 [24%]),
scleritis (n=4 [19%]), peripheral ulcerative keratitis (n=2 [9.5%]),
retinal vasculitis (n=1 [4.8%], and multifocal choroiditis (n=1
[4.8%]). Most common patterns of musculoskeletal involvement
were oligoarthritis (n=7 [33%]), polyarthritis (n=3 [14%]), and axial
(n=2 [9.5%]), while three (14%) had no articular involvement. All
patients required systemic immunomodulatory therapy, with 11
(52%) requiring more than one agent.
Conclusions: Psoriasis and psoriatic arthritis resulted in a wide
variety of chronic anterior and posterior segment inflammation, the
most common of which was anterior uveitis. The oligoarticular form
of psoriatic arthritis was the most likely to result in the development
of ophthalmic disease. Presence of associated systemic autoimmune
disease was common. The majority of patients required more than
one immunomodulatory agent to achieve inflammation control.
Commercial Relationships: Anton M. Kolomeyer, None;
Ashwinee Ragam, None; Natasha V. Nayak, None; Christina Yu,
None; Sergio Schwartzman, None; David S. Chu, None
Psoriatic uveitis : a potentially severe and sight-threatening
entity?
Céline Mebsout, Audrey Fel, Christine Fardeau, Phuc Lehoang,
Bahram Bodaghi. Ophtalmologie, Hôpital La Pitié Salpétrière Paris,
Paris, France.
Purpose: To describe the clinical characteristics and therapeutic
management of psoriatic uveitis.
Methods: A retrospective study of patients with psoriatic uveitis
referred to the Ophthalmology clinic of Pitié-Salpétrière Hospital.
Clinical characteristics, therapy, complications and severity of uveitis
were reviewed. Complications and the visual outcome have been
evaluated in this group.
Results: Nine patients were finally included with a mean followup of 10.4 years. The mean age at presentation was 38.6 years.
Among them, only 33.3% (3 of 9 patients) were HLA-B27 positive
and 66.7% (6/9) had psoriatic arthritis. Uveitis was bilateral in 6
patients (66.7%). Posterior involvement was noted in 55.6% (5/9) of
cases, (3 patients with vitritis, 3 with cystoid macular edema, 2 with
papillitis and 1 with retinal vasculitis). Secondary glaucoma occurred
in 2 patients (22.2%) with severe consequences. Twenty percent
of affected eyes (3 of 15 eyes) developed legal blindness (visual
acuity less than 20/400). The final visual acuity ranged from 20/20
to no light perception; with a mean visual acuity of 20/40. Uveitis
was considered severe in 7 patients (77.8%). Three patients (33.3%)
received long-term oral corticosteroids and 44.4% (4/9) required
intravenous pulses of methylprednisolone. Methotrexate therapy was
necessary in 6 patients (66.7%) and TNFα blockers were introduced
in 5 patients (55.6%). Two patients needed other immunosuppressive
agents (cyclosporine, azathioprine, mycophenolate mofetil, abatacept,
anakinra and tocilizumab). Dexamethasone intravitreal implant was
used in each eye of one patient for cystoid macular edema. Severe
complications of therapy (skin carcinoma and severe infections)
occurred in one patient.
Conclusions: In this case series, most of the patients had a severe
psoriatic uveitis. Larger multicentre studies are needed in order to
confirm these results and clearly identify this entity as a worrying
condition, requiring an appropriate therapeutic strategy.
Commercial Relationships: Céline Mebsout, None; Audrey Fel,
None; Christine Fardeau, None; Phuc Lehoang, None; Bahram
Bodaghi, None
Ocular Hypotension and Hypertension as Determinants of
Outcomes in Uveitis
Rabia Aman1, Asima Bajwa1, patrie james1, Wenjun Xin2,
Ashvini Reddy1. 1Dept of Ophthalmology, University of Virginia,
Charlottesville, VA; 2BioStatistics, University of Virginia,
Charlottesville, VA.
Purpose: To report outcomes associated with ocular hypotony and
hypertension in a cohort of uveitis patients managed over a 30 year
period.
Methods: Retrospective review of 461 patients (481 eyes) with
uveitis managed at the University of Virginia from 1984 – 2014.
Ocular hypotony and hypertension were defined as baseline
intraocular pressure (IOP) less than 8 mmHg or greater than
21 mmHg, respectively. Primary outcome measures were final
visual acuity and final IOP. Demographics, clinical findings, and
management were analyzed for statistical significance.
Results: Twenty-six eyes of 25 patients had baseline ocular
hypotony, which was not significantly associated with age (P=0.963),
race (P =1.00), gender (P=0.537), or anatomical classification of
uveitis (P=0.826). Of these patients, 18 (72%), 4 (16%), and 2 (8%)
were treated with local steroids, combination local and systemic
steroids, and antimetabolites, respectively. One patient received no
treatment. Final visual acuity of eyes with ocular hypotension was
20/150, which was not significantly different from normotensive
eyes (P=0.0748). Final IOP of eyes with baseline hypotony was
14.9 mmHg, which was not significantly different than that of
normotensive uveitic eyes (P=0.8829).
110 eyes of 85 patients had baseline ocular hypertension, which
was associated with anterior uveitis (76%, P=0.072), but not age
(P=0.9407), race (P =0.072) or gender (P=0.628?). 70 ocular
hypertension patients (82%) had been treated with topical steroid
during study (p=0.093). 27 patients (32%) were managed with
glaucoma medications, 3 patients (4%) glaucoma surgery, and
15 patients (18%),combination medical and surgical glaucoma
management. Final visual acuity of eyes with baseline ocular
hypertension was Snellen 20/90, which was not significantly different
than that of 356 normotensive uveitic eyes (P=0.2237). Final IOP
of eyes with baseline hypertension was 15.0 mmHg, which was
not significantly different than that of eyes with normal baseline
intraocular pressure (P=0.9868).
Conclusions: Neither baseline hypotony nor hypertension in
uveitis was associated with poorer visual acuity and lower final IOP
compared to patients with normal baseline IOP. Uveitic patients with
abnormal IOP can expect final vision and pressure similar to uveitic
patients who are normotensive at baseline.
Commercial Relationships: Rabia Aman, None; Asima Bajwa,
None; patrie james, None; Wenjun Xin, None; Ashvini Reddy,
None
Clinical characteristics of ocular syphilis in patients with and
without HIV infection
Vincent Cheng, Sun Young Lee, Narsing A. Rao. USC Eye Institute,
University of Southern California, Los Angeles, CA.
Purpose: To compare clinical and laboratory findings of ocular
syphilis between HIV positive and negative patients.
Methods: Medical records of patients diagnosed with ocular syphilis
with serologic confirmation from 2008 to 2014 were retrospectively
reviewed.
Results: Sixteen consecutive patients (10 HIV-positive vs 6 HIVnegative) with 29 eyes were included. All patients were male and
the mean age of onset was 43 (mean 42.65 ± 13.13). Regardless of
HIV status, ocular findings of ocular syphilis were variable including
anterior uveitis (4 eyes), posterior uveitis (8 eyes), panuveitis (13
eyes), isolated papillitis (4 eyes) and CN III and VII palsy (1 eye).
However, panuveitis was the most common feature (12/18 eyes,
67%) in HIV-positive patients whereas posterior uveitis was the
predominant feature (6/11 eyes, 55%) in HIV-negative patients.
Significantly higher serum rapid plasma reagin (RPR) titers were
found in HIV-positive patients (range 1:64-1:16,348 in HIV-positive
vs 1:2-1.8 except 1 patient with 1:2,048 in HIV-negative, p =
0.019). A higher proportion of HIV-positive patients tested positive
for cerebrospinal fluid fluorescent treponemal antibody absorbed
(CSF FTA-ABS) or venereal disease research laboratory (VDRL)
than HIV-negative patients (70% in HIV-positive vs 16% in HIVnegative). CD4 cell count in HIV-positive patients at onset was
typically ranged from 127 to 535 (mean 237 ± 142). These patients
responded to 10-14 days of intravenous penicillin with relatively
good visual outcome.
Conclusions: HIV status in patients with syphilis plays a role
in ocular manifestations, primarily presenting with pan uveitis
associated with positive CSF FTA-ABS or VDRL and high serum
RPR titers compared to non-HIV syphilis. These findings indicate
that HIV positive individuals with ocular manifestations of syphilis
should be treated for neuro-syphilis.
Commercial Relationships: Vincent Cheng, None; Sun Young
Lee, None; Narsing A. Rao, None
Support: An Unrestricted grant from Research to Prevent Blindness,
New York, NY 10022
Retinoschisis in Pars Planitis
Julia Malalis1, Pooja Bhat2, Sarah Escott1, Michael Shapiro2, 3, Debra
A. Goldstein1. 1Ophthalmology, Northwestern University, Chicago,
IL; 2Ophthalmology, University of Illinois at Chicago, Chicago, IL;
3
Retina Consultants Ltd, Des Plaines, IL.
Purpose: Retinoschisis is a well-recognized complication of pars
planitis, yet little data is available regarding its prevalence and
course. The purpose of this study is to determine the incidence,
presentation, and course of retinoschisis in patients with pars planitis.
Methods: Retrospective chart review was performed on all patients
with pars planitis meeting Standardization of Uveitis Nomenclature
disease definition seen by the Uveitis service of one of the authors
from July 2012 - September 2014.
Results: 34 patients (68 eyes) who met disease definition were
included. 21 patients were female (62%). The majority of patients
were Caucasian (n=23, 68%); the remainder were Hispanic (n=10,
29%) and Asian (n=1, 3%). 13 eyes (19%) developed retinoschisis.
In all cases, schisis was inferiorly located, posterior to the snowbank.
In 6 patients (86%) the schisis was bilateral. 4 patients with schisis
were Caucasian (57%), 2 were Hispanic (29%) and 1 was Asian
(14%). 4 patients were female (57%). Average follow-up of patients
with schisis was 7 years (3.7 - 9.6 years); average visual acuity of
eyes with schisis was 20/22 at last follow-up. 5 eyes of 5 patients
underwent pars plana vitrectomy. 3 had vitrectomy for disease control
with scleral buckle placement to reduce residual traction. In one eye,
the schisis did not progress despite active vitreous inflammation,
while the other developed schisis while inflammation was
uncontrolled. The third was only noted to have schisis at the time of
vitrectomy. Two eyes of two patients required pars plana vitrectomy
for retinal detachment with progressive schisis despite control of
uveitis. Silicone oil was used in these cases. 8 eyes with retinoschisis
remained stable without need for surgical intervention.
Conclusions: Retinoschisis is a common complication in patients
with pars planitis at a tertiary referral practice. It is typically
bilateral, inferior, and may develop in eyes with both controlled
and uncontrolled disease. Even in eyes that require surgical
management of progressive schisis, visual outcome can be favorable.
While inflammatory mediators may play a role in development
and progression of schisis, the presence of inflammation may not
correlate with progression of schisis. In all cases schisis was adjacent
to a snowbank, suggesting that mechanical forces may lead to
development of schisis and retinal detachment even in patients with
inactive disease.
Commercial Relationships: Julia Malalis, None; Pooja Bhat,
None; Sarah Escott, None; Michael Shapiro, None; Debra A.
Goldstein, None
Vaccine Associated Uveitis
Matthew Benage, Rick W. Fraunfelder. Department of
Ophthalmology, University of Missouri, Columbia, MO.
Purpose: To describe a series of case reports of uveitis following
vaccination with hepatitis A, hepatitis B, HPV, BCG, brucella, DPT,
herpes, influenza, measles, MMR, pneumococcal, smallpox, tetanus,
varicella, and zoster.
Methods: Reports from the National Registry of Drug-Induced
Ocular Side Effects (Columbia, Missouri), WHO, and the FDA were
collected on vaccine associated with uveitis between 1988 and 2014.
We also performed a Medline literature search using the keywords of
uveitis, or iritis, in combination with vaccines, hepatitis A, hepatitis
B, HPV, BCG, brucella, DPT, herpes, influenza, measles, MMR,
pneumococcal, smallpox, tetanus, varicella, and zoster vaccine.
Results: A total of 290 cases of uveitis following vaccine
administration were reported following the use of vaccinations.
199 cases were female and 77 cases were male; 12 did not disclose
gender. The mean age was 30 years (0.2-86). The mean number of
days until uveitis was reported after vaccination was 141 days (1 day6 years). 14 were still recovering, while 22 did not recover. 166 cases
did not report resolution status. The most prolific vaccine associated
uveitis is hepatitis B, with a total of 115 cases reported. Additionally,
forty-four cases of uveitis were reported following HPV vaccination;
five cases were reported following hepatitis A vaccination; twentyone cases of uveitis were reported following BCG vaccination;
twenty-seven cases of uveitis were reported following influenza
vaccination; and thirteen cases of uveitis were reported following
MMR vaccination. Thirty-five cases of uveitis were reported
following administration of multiple vaccines. Two cases of uveitis
were reported after smallpox vaccination and one case of brucella,
DPT, herpes, measles, pneumococcal, and tetanus were reported to be
associated with uveitis, respectively.
Conclusions: All commonly administered vaccinations are associated
with uveitis. Inflammation is temporary and resolves with topical
ocular steroids usually without long term damage to the eye. The
mechanism is unclear, however, various hypotheses have been
suggested. The proposed mechanisms are molecular mimicry
secondary to close resemblance of vaccine peptide fragments and
uveal self-peptides, delayed-type hypersensitivity with deposition of
immune complexes, and immune reaction to vaccination adjuvants.
Despite mechanistic uncertainty, clinicians are encouraged to be
aware of vaccine-associated uveitis for prompt diagnosis, treatment,
and reporting.
Commercial Relationships: Matthew Benage, None; Rick W.
Fraunfelder, None
Unusual Manifestations of Zoster Vasculitis
Andrew Mincey, Steven A. Newman. Ophthalmology, University of
Virginia, Charlottesville, VA.
Purpose: Herpes zoster has had a myriad of potential effects
involving the visual pathways including ophthalmoplegia, acute
retinal necrosis, and severe post herpetic neuralgia. The propensity
of this DNA virus to involve vessels makes other forms of vasculitis
possible.
Methods: Review of 2 unusual cases of zoster causing vascular
occlusive disease affecting the ophthalmic artery and branches of the
central retinal artery occlusion were reviewed.
Results: In one of these 2 unusual cases, a central retinal artery
occlusion occurred following typical zoster uveitis. The presence of
a cilioretinal artery sparred central fixation. The second patient was
misdiagnosed with giant cell arteritis. A substantial delay in diagnosis
was made because of failure to order fat sat with gadolinium. When
the MRI was repeated prominent enhancement was seen of the optic
nerve sheath.
Conclusions: Varicella virus has a propensity for involving the
vascular system potentially producing occlusive ischemic changes
involving both the optic nerve and the retina. Recognition can be
difficult if the zoster is not considered.
Commercial Relationships: Andrew Mincey, None; Steven A.
Newman, None
OPHTHALMIC AND SYSTEMIC MANIFESTATIONS
OF IgG4 RELATED DISEASE : ABOUT 6 PATIENTS IN A
SINGLE CENTER
Nathalie Butel1, 2, Mathieu ZMUDA2, Olivier Galatoire2. 1hôpital pitié
salpetriere, Boulogne, France; 2fondation Rotschild, Paris, France.
Purpose: We presented a french descriptive dataset of orbital, eyelid
and systemic manifestations in lymphoproliferative disorder of IgG4
related disease (IgG4-RD) in 6 patients. The main manifestation is
a non-specific inflammatory orbitopathy which is cortico-sensitive.
This recent syndrome, underknown, often makes a Masquerade
syndrome, responsible of misdiagnosis.
Methods: Retrospective and monocentric descriptive evaluation
conducted between 2012 and 2013 in a tertiary center in Paris,
including patients with orbital or eyelid impairment in IgG4-RD
according to the Kawa et al criteria.
Results: 6 patients (4 men, 2 women) were included. 2 patients
were childrens. Median age was 45 years (9-62 years). The average
diagnostic delay between the onset ocular symptoms and immuno
histo chemical confirmation was 18 months (2-72 months). Mean
follow-up was 32 months (2-72 months). 1 patient (age 9) had
a single eye-lid reached, while 5/6 patients also had systemic
involvement like parotidis,affected lymph node and tonsil, thyroiditis,
aortitis, skin involvement. In 2 cases we found an infraorbital nerve
enlargement. All patients had a permanent or transient clinical
improvement with corticotherapy.
Conclusions: IgG4-RD is rarely described in children, this study
shows that it is important to think about it in an orbital or eyelid
unexplained inflammatory disease, even in children. Involvement of
cranial nerves in IgG4-RD was once described in the literature. We
found 2 patients with infra orbital nerve enlargement which seems to
be a very typical manifestations in IgG4-RD.
Commercial Relationships: Nathalie Butel, None; Mathieu
ZMUDA, None; Olivier Galatoire, None
Assessment of macular pigment optical density in patients with
sunset glow fundus in Vogt-Koyanagi-Harada disease
Taro Seino, Kouhei Hashizume, Mana Nagasawa, Yasunori Nishida,
Daijiro Kurosaka. Ophthalmology, Iwate Medical University,
Morioka, Japan.
Purpose: To compare the macular pigment optical density (MPOD)
of eyes with sunset glow fundus in Vogt-Koyanagi-Harada (VKH)
disease with the MPOD of eyes without retinal disease.
Methods: The MPOD of 19 eyes with sunset glow fundus of VKH
was measured and compared with the MPOD of 25 eyes without
any signs of retinal disease. None of subjects was under carotenoid
supplementation. The MPOD was measured with Macular pigment
screener (MPS II) from Elektron Technology.
Results: Eyes with sunset glow of VKH have significantly lower
level of MPOD than eyes without retinal disease (0.486 ± 0.184
versus 0.625 ± 0.176, p = 0.016, Student’s t test). Thirteen eyes of
19 eyes were treated with initial steroid pulse therapy, 1000 mg of
intravenous methylprednisolone for 3 days. Eyes with VKH not
treated with initial steroid therapy have significantly lower level of
MPOD than eyes with VKH treated with initial steroid therapy (0.257
± 0.075 versus 0.592 ± 0.100, p < 0.001, Student t test), and there
was no significant difference between the level of MPOD of eyes
with VKH treated with initial steroid therapy and eyes without retinal
disease (p = 0.551, Student t test) There was no significant correlation
between MPOD and visual acuity in VKH patients (p = 0.064,
Pearson’s correlation coefficient).
Conclusions: Inflammation decreased MPOD in patients with VKH.
Initial steroid therapy prevented loss of MPOD in patients with VKH.
But relation between loss of macular pigment and visual function
remains unclear.
Commercial Relationships: Taro Seino, None; Kouhei Hashizume,
None; Mana Nagasawa, None; Yasunori Nishida, None; Daijiro
Kurosaka, None
Fundus autofluorescence imaging in Vogt-Koyanagi-Harada
disease from acute onset in a 12-month follow-up
CELSO MORITA, Viviane M. Sakata, Ever E. Rodriguez, Smairah F.
Abdallah, Carlos E. HIrata, Maria K. Oyamada, Joyce H. Yamamoto.
Ophthalmology, University of São Paulo, São Paulo, Brazil.
Purpose: Vogt-Koyanagi-Harada disease (VKHD), an autoimmune
aggression against melanocytes, is characterized by an acute
bilateral diffuse choroiditis with extraocular manifestations. Disease
inflammation and therapy are monitored based on clinical and
fundus imaging, i.e. fluorescein angiography and indocyanine green
angiography. Fundus autofluorescence imaging (FAF) may indicate
retinal pigment epithelium (RPE) functional and metabolic changes:
blue autofluorescence (BL-FAF) may indicate lipofuscin abnormality;
near-infrared light (NIR-FAF) may indicate melanin and melanin
compounds abnormalities. The present study aimed to evaluate
fundus autofluorescence imaging in patients with VKHD during a
12-month follow-up from disease onset.
Methods: Retrospective longitudinal study including 9 patients
(18 eyes) with VKHD, diagnosed according to Revised Diagnostic
Criteria, followed from disease onset for a minimum 12 months. All
patients were treated with methylprednisolone 3-day pulsetherapy
followed by oral prednisone (1mg/kg/day) with a slow 12-15 month
tapper. Fundus autofluorescence (BL-FAF and NIR-FAF) was carried
out as part of a multimodal fundus imaging analysis (Spectralis
HRA+OCT, Heidelberg Engineering, Germany) alongside functional
exams (RETI-port system; Roland Consult, Germany). Images
obtained at M0, M1, M3, M6 and M12 were analysed by two readers.
Autofluorescence changes were classified into hyper (Hy) and hypo
(Ho) changes and into the patterns: diffuse (D), plaque (PL), granular
(G) and reticular (R). The study was approved by the Institutional
Ethics Committee.
Results: Nine patients (7F/2M), with median age of 33 y/o and
median time to treatment of 12 days (3-46), were included. FAF at
M1 disclosed two distinct patterns, a mild granular Hy-FAF with
progressive return to normal (mild FAF) and a exuberant plaque and/
or diffuse Hy-FAF with or not plaque Ho-FAF (severe FAF) (Figure).
According to this classification, 8 eyes had mild FAF and 10 eyes
had severe FAF. Severe FAF group had thicker subfoveal choroidal
thickness at M1 (p=0.017); had more subretinal neovascular
membrane (p=0.034) and more deranged full-field electroretinogram
parameters (kappa=0.667; CI95%0.324-1.000) at M12.
Conclusions: FAF pattern at M1 can be a method for detection of
severe RPE abnormalities and may be useful for managing treatment.
FAF imaging-mild pattern
FAF imaging-severe pattern
Commercial Relationships: CELSO MORITA, None; Viviane M.
Sakata, None; Ever E. Rodriguez, None; Smairah F. Abdallah,
None; Carlos E. HIrata, None; Maria K. Oyamada, None; Joyce
H. Yamamoto, None
Support: FAPESP 2011/50936-7;2011/19194-4;2014/01222-0
Functional Correlations of Optical Coherence Tomography
Findings in Birdshot Chorioretinoopathy
Gokul Kumar, Jessica Shantha, Purnima Patel, Steven Yeh.
Ophthalmology, Emory University, Decatur, GA.
Purpose: To evaluate SD-OCT findings in patients with birdshot
chorioretinopathy and correlate them with visual acuity, kinetic
perimetry, and electroretinography.
Methods: We performed a retrospective analysis of patients with
clinical diagnosis of birdshot chorioretinopathy who had concurrent
OCT, kinetic perimetry, and ERG. All OCT scans were evaluated
for external limiting membrane (ELM) integrity, ellipsoid zone (EZ)
integrity, and presence of cystoid macular edema (CME). These were
compared to select functional measures – rod and cone function on
ERG, visual acuity, and visual field abnormalities.
Results: Thirteen encounters of eleven patients were analyzed. 12
(46.2%) OCT images showed ELM disruption; 14 (53.9%) images
showed EZ disruption; 9 (35.6%) images showed CME. 20 Goldman
visual fields (76.9%) showed abnormalities, including enlarged
blind spots and areas of relative scotomas. On ERG, 14 studies
had normal rod function (53.9%) and 3 had normal cone function
(11.5%). 7 studies had mild rod dysfunction (26.9%), 3 moderate
(11.5%) and 2 severe (7.7%). 6 studies had mild cone dysfunction
(23.1%), 15 moderate (23.1%), and 2 severe (7.7%). Visual acuities
were categorized as normal (20/20 Snellen acuity), mild impairment
(20/25-20/40), moderate impairment (20/50-20/150), or severe
(20/200 or below). 5 studies were normal (19.2%), 10 had mild
impairment (38.5%), and 11 had moderate impairment (42.3%). The
relationship between EZ disruption and visual acuity (p=0.023) and
the relationship between EZ disruption and cone function on ERG
testing (p=0.025) appeared statistically significant by Chi-square
test of independence. The relationship between ELM disruption
and presence of visual field loss (p=0.017) appeared statistically
significant by Fisher’s exact test. The relationship between ELM
disruption and visual acuity (p=0.051) and the relationship between
presence of CME and visual acuity (p=0.051) approached statistical
significance.
Conclusions: SD-OCT imaging of the fovea can be a useful adjunct
test in birdshot chorioretinopathy. Changes in ELM and EZ on OCT
may relate to important functional measures, including visual field
loss and visual acuity impairment. However, some functional changes
appear to be independent of the anatomical changes noted on foveal
OCT.
Commercial Relationships: Gokul Kumar, None; Jessica
Shantha, None; Purnima Patel, None; Steven Yeh, None
The efficacy and safety of immunosuppressive agents in the
treatment of necrotizing scleritis - A multi-center retrospective
analysis in Korea
Hyun Sun Jeon1, Mee Kum Kim2, 3, Joon-Young Hyon1, 3.
1
Ophthalmology, Seoul National University Bundang Hospital,
Seoungnamsi, Korea (the Republic of); 2Ophthalmology, Seoul
National University Hospital, Seoul, Korea (the Republic of); 3Seoul
National University College of Medicine, Seoul, Korea (the Republic
of).
Purpose: Necrotizing scleritis is the most severe form of scleritis,
however there have been only several small case reports regarding its
treatment. We performed a multi-center, retrospective, case series
study to investigate the efficacy and safety of immunosuppressive
agents (ISA) in the treatment of necrotizing scleritis.
Methods: Medical charts of fifty-two patients treated with ISA for
necrotizing scleritis from June 2002 to May 2012 at eleven tertiarycare centers were reviewed. Patient characteristics, clinical features,
risk factors, and treatment results were analyzed. The efficacy and
safety was evaluated and compared between cyclophosphamide
and other ISA (azathioprine, cyclosporine, methotrexate, and
mycophenolate mofetil). Efficacy was assessed by remission rate,
relapse rate, visual loss (more than 2 lines) rate and steroid sparing
rate. Safety was assessed by occurrence of adverse effects and
discontinuation of medication from its adverse effects.
Results: Of 52 patients, 50 who treated with ISA within 3 months
periods were included. The mean age was 65.8 ± 10.5 years.
Complete or partial remission was achieved in 90% of patients
by 6 months and 95% by 12 months. Patients who were initially
treated with ISA showed significantly better complete remission
rate (P=0.043). There were no significant differences in remission
rate, relapse rate, visual loss rate, and steroid sparing rate between
cyclophosphamide group and other ISA group. Incidence of adverse
effects were comparable between cyclophosphamide and other ISA
(61.9% vs. 41.4%, P=0.152), however, incidence of leukopenia,
hemorrhagic cystitis, and discontinuation of medication from adverse
effects were much higher in cyclophosphamide group (P<0.001,
P<0.001, P=0.05, respectively).
Conclusions: Initial treatment with ISA showed clinical benefit in
the treatment of necrotizing scleritis. Although overall incidences of
adverse effects were not different among agents, side effects related
medication discontinuation was much higher with cyclophosphamide.
Commercial Relationships: Hyun Sun Jeon, None; Mee Kum
Kim, None; Joon-Young Hyon, None
Support: This research was supported by a grant (12172MFDS231)
from Ministry of Food and Drug safety in 2012.
Long-term efficacy of interferon in severe uveitis associated with
Behçet’s disease
Eleonore Diwo1, David Saadoun1, Julie Gueudry2, Phuc Lehoang1,
Bahram Bodaghi1. 1Paris, Pitie Salpetriere Hospital, Paris, France;
2
Rouen Hospital, Rouen, France.
Purpose: To retrospectively assess the frequency of ocular relapse
and the possibility of long-term remission in patients who were
treated with interferon (IFN) alpha for severe uveitis associated with
Behçet’s disease (BD)
Methods: All patients with severe uveitis associated with BD and
referred to Pitié-Salpêtrière Hospital, France, between June 1994
and January 2010 and treated by interferon alpha, whatever the
treatment duration, were included in our retrospective cohort study.
All patients were treated with initial dosage of IFN alpha 3 million
IU thrice a week, increased to 6 million IU thrice a week in case
of relapse or discontinued in case of stable clinical examination.
All patients had active severe uveitis : refractory to at least one
conventional immunosuppressive drug and/or requiring high doses
oral corticosteroids (> 10mg per day). All patients whose compliance
with medication could not be established were excluded.The main
assessment criterion was the number of relapses per patient per year
before and after initiation of IFN and after discontinuation of IFN.
Best visual acuity, anterior segment inflammation, vitritis, presence
of vasculitis, macular edema, papillitis and retinitis were assessed at
initiation and at 2, 4 and 9 years after the initiation of IFN.
Results: Of 36 patients (67 eyes), 31 (86.1%) responded to IFN.
For responders, the mean relapse per person per year decreased
significantly from 1.39 to 0.0496 (p = 1.82*10^-10) during treatment
period. After cessation of IFN, possible for 21 patients, the mean
annual incidence of relapse remained at 0.057 relapses per person per
year. The mean period from the initiation of IFN to the date of the
last follow-up consultation was 8.19 years (range, 6 to 216 months).
Thirty three, 25 and 18 patients were followed respectively at 2, 4
and 9 years. Visual acuity was stable or improved for 90.3% of all
patients. For the group “9 years”, anterior segment inflammation
and vitreous haze were reduced during the first year. The prevalence
of macular edema and the prevalence of retinitis and papilledema
plummeted to zero at 4 years and 1 year after IFN initiation.
Conclusions: The incidence of relapses in severe uveitis associated
with Behçet’s disease decreases under IFN alpha treatment. This
treatment also seems to permit a long-term remission even after
discontinuation. It is an efficient treatment for vasculitis, cystoid
macular edema and papilledema.
Commercial Relationships: Eleonore Diwo, None; David
Saadoun, None; Julie Gueudry, None; Phuc Lehoang, None;
Bahram Bodaghi, None
Treating autoimmune retinopathy (AIR) with
immunosuppressive therapy: results of a retrospective singlecenter study.
Armin Maghsoudlou1, Naira Khachatryan2, 1, Ninani Kombo1,
3
, C Stephen Foster1. 1Massachusetts Eye Research and Surgery
Institutition(MERSI), Cambridge, MA; 2The Scheie Eye Institute,The
University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA; 3Ophthalmology, Yale School of Medicine, New
Haven, CT.
Purpose: The autoimmune retinopathy (AIRs) is characterized by
progressive vision loss, an abnormal Electroretinogram (ERG) and
circulating antibodies directed against retina proteins. This study
aimed to investigate the results of treatment of AIR patients with
immunosuppressive therapy.
Methods: This is a retrospective study of AIR patients who were
treated with systemic immunosuppressants. Treatment outcomes
were assessed by the following parameters: visual acuity (VA), mean
deviation (MD) and pattern standard deviation (PSD) parameters
in Humphrey visual field (HVF), implicit time and amplitude
parameters in Ganzfield Electroretinograpy (ERG) and serologic
findings for anti-retina and anti-optic nerve antibodies
Results: The study included 15 eyes of 8 participants with a mean
age at entry of 52.9 (± 14.0) years. Four study participants (63%)
were female. The mean follow up time was 2.3 years (± 1.2 years).
During the course of treatment, of all participants, one (13%) had
worsening in serology findings for both anti-retina and anti-optic
nerve antibodies, four (50%) improved (reduction or became
negative), and two (25%) completely resolved.
Of seven participants with ERG measurements, four (57%)
improved in one or both eyes. Of seven participants with HVF MD
measurements, five (71%) reported stable or improved MD in one or
both eyes. Of all participants, five (63%) reported stable or improved
VA in one or both eyes. Three study participants (37.5%) improved
by 3 or 4 parameters, three (37.5%) were stable or improved by 2
parameters only, and two (25%) worsened by all parameters.
Of those improved by 3-4 parameters, two participants were
treated with a combination of Rituxan with Prednisone and either
Cyclosporine or Cyclophosphamide, and one participant was treated
with a combination of Rituxan and Cyclophosphamide. Of those
stable or improved by 2 parameters only, two participants were
treated with Rituxan only and one participant with combination of
Rituxan and Velcade.
Conclusions: This study reported that the majority (n=6; 77%) of
the patients with AIR treated with immunosuppressants were stable
or improved by two or more parameters. Further studies with larger
sample size and longer follow up are needed to investigate long term
results of the treatment of AIR patients with immunosuppressive
therapy.
Commercial Relationships: Armin Maghsoudlou, None; Naira
Khachatryan, None; Ninani Kombo, None; C Stephen Foster,
None
Cataract surgery in patients with pars planitis and
immunosuppressive therapy
Tania Albavera-Giles, Juan Carlos Serna-Ojeda, Miguel PedrozaSeres. Instituto de Oftalmologia Conde de Valenciana, Distrito
federal, Mexico.
Purpose: To evaluate the characteristics and outcomes of
cataract surgery in patients with pars planitis who received
immunosuppressive therapy in a tertiary institution of ophthalmology
in Mexico.
Methods: We reviewed our database between January 2003 and
November 2014 of 374 patients with pars planitis, and from the
49 patients that received immunosuppressive therapy, we included
the patients with cataract surgery. A retrospective analysis was
performed, and the following data was collected: age at presentation,
age at cataract surgery, follow-up, visual acuity before, 1 week,
1 month and 6 months after the surgery, inflammation after the
surgery, immunosuppressive therapy used for each case, surgical
and postoperative complications and causes for failed visual
improvement.
Results: Sixteen patients were included, with a median age at
presentation of 10.5 years and with a median age at the moment of
the cataract surgery of 11 years (range 4-26 years). All the patients
had no inflammation before the surgery for at least 2 months. The
immunosuppressive therapy used for the patients were methotrexate
in 15 patients (93.7%) and azathioprine in 6 (37.5%), with 5 patients
requiring a combination of drugs. Thirteen patients received the
immunosuppressive therapy before the surgery for a median time of
8 months, and 3 patients received only previously systemic steroids,
and the immunosuppressors were administered after the surgery.
All the patients had phacoemulsification with intraocular lens
implantation in the capsular bag in 15 patients (93.7%) and 9 patients
(56.25%) required anterior vitrectomy. The visual acuity improved
from a median of 20/800 (range 20/60 to hand movements), to 20/100
(range 20/25 to 20/2000) after 6 months of follow up; 14 patients
(87.5%) improved two lines of vision or more and the other 2 patients
remained the same visual acuity. No improvement in visual acuity
was attributed to posterior segment manifestations or amblyopia.
The median follow up after the surgery was 32 months (range 4 to 91
months) and with immunosuppressive therapy was 18 months (range
2 to 56 months).
Conclusions: Phacoemulsification was the procedure for all the
patients in this study, with a high rate of anterior vitrectomy. Visual
acuity improved in 87.5% of patients with pars planitis treated with
immunosuppressive drugs who underwent cataract surgery.
Commercial Relationships: Tania Albavera-Giles, None; Juan
Carlos Serna-Ojeda, None; Miguel Pedroza-Seres, None
Characteristics of Non-Infectious Persistent Postoperative
Inflammation
Russell W. Read, Kinley Beck. Ophthalmology, University of
Alabama at Birmingham, Birmingham, AL.
Purpose: To determine the characteristics of patients diagnosed
with non-infectious persistent postoperative inflammation and to
determine if this diagnosis occurs more often in various subgroups
(race and gender.)
Methods: Retrospective chart review of all patients seen between
2007 and 2010 (inclusive) at a single tertiary care academic
uveitis center. Patients with non-infectious persistent postoperative
inflammation were identified and compared to patients with other
categories of uveitis. Characteristics were compared between groups
using t-test and chi-square test for continuous and categorical
variables, respectively.
Results: 732 patients were identified, 28 of which were diagnosed
with non-infectious persistent postoperative inflammation (PPI)
(3.8% of all patients). All but 3 cases followed cataract surgery and
lens implantation. Of the 3 non-cataract cases, 2 were following
trabeculectomy and one following SLT. All 28 had anterior disease,
so comparison to non-PPI patients was limited to those with
anterior uveitis (n = 485). The mean age at disease onset for PPI
was 62 years (range 10-74) versus 45 years (range 1-94) for nonPPI patients (p=0.00001). Of patients with PPI, 29% were male,
71% female as compared to 33% male and 67% female for non-PPI
patients (p = 0.64). Of PPI patients, African Americans comprised
61%, Caucasians 36%, and “Other” 4% versus for non-PPI, African
Americans comprised 41%, Caucasians 57%, and “Other” 2% (p =
0.079). PPI patients manifested bilateral disease in 25% of cases,
unilateral in 75% versus in non-PPI disease bilateral cases were 37%
and unilateral 63% (p = 0.19). Of PPI patients with bilateral disease,
86% were African American and 14% were Caucasian. Of PPI
patients with unilateral disease, 55% were African American and 45%
were Causcian. Stated conversely, of African American PPI patients,
35% had bilateral disease, 65% unilateral disease. Of Caucasian PPI
patients, 10% had bilateral disease, 90% unilateral disease (p = 0.15).
Conclusions: Persistent postoperative inflammation occurs most
commonly following cataract surgery and is anterior. Females are
more likely to be affected but not to a greater degree than in nonPPI anterior uveitis. A trend towards a higher frequency in African
Americans was found and African Americans were more likely to
have bilateral PPI, though not to a statistically significant degree.
Commercial Relationships: Russell W. Read, None; Kinley Beck,
None
Support: Research to Prevent Blindness Physician Scientist Award;
Unrestricted departmental grants from Research to Prevent Blindness
and the EyeSight Foundation of Alabama
Long-term surgical outcomes of acute retinal necrosis
Laura J. Kopplin1, Stephanie Cramer1, Steven Yeh2, Christina J.
Flaxel1. 1Casey Eye Institute, Portland, OR; 2Emory Eye Center,
Atlanta, GA.
Purpose: To assess the long-term visual, anatomic and surgical
outcomes in patients with acute retinal necrosis (ARN), including
rates of recurrent detachment following primary surgical repair. To
determine if prior intravitreal foscarnet therapy reduced the rate of
recurrent retinal detachment.
Methods: We conducted a single center retrospective chart review
from 2001-2012 of patients diagnosed with ARN. We assessed the
development of retinal detachment, types of surgical interventions
and the occurrence of recurrent retinal detachment after surgical
repair. We compared the rates of recurrent detachment between those
receiving and not receiving intravitreal foscarnet using Fisher’s exact
test.
Results: We identified 32 eyes from 27 patients with ARN (5 with
bilateral disease). Mean follow up was 51.8 months (range 7-206).
All subjects received systemic treatment with either intravenous
followed by oral acyclovir or oral valacyclovir alone and a subset
of eyes (50%) were treated with intravitreal foscarnet. Fifteen eyes
(46.9%) developed retinal detachments and 13 under went surgical
repair. Primary intervention consisted of pars plana vitrectomy with
silicone oil in 7 cases (53.8%), pars plana vitrectomy in combination
with scleral buckle and silicone oil in 4 cases (30.8%) and pars plana
vitrectomy with either scleral buckle (1 case) or cryotherapy (1 case).
Recurrent retinal detachment developed in 7 eyes (53.8%) occurring
35 days to 10 months after the primary retinal surgery. There was no
difference in the rate of recurrent detachment between eyes treated
with or without intravitreal foscarnet (p=0.74), although sample
size limits comparison. Three recurrent detachments happened
after removal of silicone oil (between 1 day and 3.5 months after
the procedure). At final follow up, all retinas that underwent repair
remained attached. Acuities at the last recorded visit ranged from
20/40 to no light perception; vision improved in 30.8% of patients
who underwent retinal repair.
Conclusions: We found a rate of retinal detachment secondary
to ARN similar to that previously reported. Recurrent retinal
detachment after primary surgical repair was a frequent complication
and the rate of recurrent detachment did not differ based on prior
treatment with foscarnet. Overall, visual prognosis was poor despite
surgical intervention.
Commercial Relationships: Laura J. Kopplin, None; Stephanie
Cramer, None; Steven Yeh, None; Christina J. Flaxel, None
Support: Institutional grant from Research to Prevent Blindness
531 Allergic conjunctivitis and ocular inflammation
Thursday, May 07, 2015 12:00 PM–1:45 PM
Gene expression profiles of orbital adipose and lacrimal gland
from subjects with sarcoidosis parallel some changes in blood
James T. Rosenbaum1, 2, Dongseok Choi1, Christina A. Harrington3,
David J. Wilson1, Hans E. Grossniklaus4, Patrick Stauffer1, Stephen
R. Planck1, 2. 1Casey Eye Institute, Oregon Health & Science Univ,
Portland, OR; 2Devers Eye Institute, Legacy Health System, Portland,
OR; 3Integrated Genomics Laboratory, Oregon Health & Science
Univ, Portland, OR; 4Ophthalmology, Emory University, Atlanta, GA.
Purpose: Gene expression profiling has the potential to assist
in the diagnosis and treatment selection for patients with orbital
inflammation. We compared gene expression levels in orbital
adipose and lacrimal gland of subjects with sarcoidosis with levels in
uninflamed control tissues and then compared those profiles to one
that we previously reported for blood.
Methods: Affymetrix U133 Plus 2 microarrays were used to
determine relative mRNA levels in formalin-fixed, paraffin-embedded
orbital biopsies of subjects with sarcoidosis (7 adipose, 5 lacrimal
gland) and from subjects with no known orbital pathology (6 adipose,
6 lacrimal gland). Lists of probe sets with significantly increased or
decreased signals in the sarcoidosis group were compared to similar
list previously obtained from blood collected from subjects with
sarcoidosis and healthy controls. The significance threshold was set at
>1.5-fold difference with a false discovery rate of <0.05.
Results: Significantly higher signals were obtained for 2457 probe
sets (transcripts from ~1349 genes) for adipose and for 3077 probe
sets (~2001 genes) for lacrimal gland from sarcoidosis subjects.
Significantly lower signals were obtained for 4050 probe sets
(~2769 genes) for adipose and 3320 probe sets (~2283 genes) for
lacrimal gland from sarcoidosis subjects. After comparing lists of the
differentially expressed transcripts for orbital adipose, lacrimal gland,
and blood, we identified 92 probe sets (~69 genes) that had increased
signals and found that these genes were enriched in binding sites for
the transcription factors, IRF-1, IRF-2, and NFκB. Decreased signals
were observed for 67 probe sets (~56 genes) in all three tissues from
sarcoidosis patients.
Conclusions: Orbital adipose and lacrimal gland tissues from
subjects with sarcoidosis can readily be distinguished from
uninflamed control tissues. The list of genes elevated in all three
tissues is consistent with previous reports from us and others that
activation of a JAK/STAT pathway, such as by interferon signaling,
is a common feature of sarcoidosis. These results support the idea
that an algorithm based on gene expression in peripheral blood might
assist in the diagnosis of sarcoidosis without a more invasive biopsy.
Commercial Relationships: James T. Rosenbaum, Genentech (C);
Dongseok Choi, None; Christina A. Harrington, None; David J.
Wilson, None; Hans E. Grossniklaus, None; Patrick Stauffer,
None; Stephen R. Planck, None
Support: NIH Grants EY020249, EY010572 and RR024140;
Research to Prevent Blindness
Lipid Mediator Pathways in the Pathogenesis of Bacterial
Keratitis
Thomas Carion2, Matthew Greenwood3, Karsten Gronert3, Elizabeth
A. Berger2, 1. 1Ophthalmology, Kresge Eye Institute, Detroit, MI;
2
Anatomy and Cell Biology, Wayne State University School
of Medicine, Detroit, MI; 3School of Optometry, University of
California, Berkeley, Berkeley, CA.
Purpose: Though prostaglandins and leukotrienes (LTs) primarily
derived from the COX-2 and 5-lipoxygenase (LOX) pathways are
essential potent mediators of inflammation, endogenous small proresolving molecules (SPMs) such as lipoxins/resolvins/protectins
act as robust agonists of resolution. To this

ARVO 2015 Annual Meeting Abstracts by Scientific Section/Group

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