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What Comes out May be Better than
what Goes in ?
Human Microflora in Mucosal Health
and Disease:
Therapeutic implications
Pediatric Grand Rounds.
Women and Children’s Hospital
Buffalo. NY
July 13. 2012
Pearay L Ogra. MD.
Division of Infectious Diseases
What Goes In:
We are What We Eat !
Focal points of the presentation.
•
Microbiome and its evolutionary adaptation to
mucosa and other mammalian tissues
• Role of Microbiome in mucosal immunity
• Disease states associated with altered Microbiome
• Therapeutic applications of Microbiome
modulation.
Life's Unforgettable experiences:
New Mexico 1990
El -Qanturio De Chimayo, New Mexico
El- Qanturio De Chimayo, New Mexico
1
Earth
~El- Qanturio
De Chimayo, New Mexico
The faithful come regularly to eat
the Deep Red Dirt of the
Earth of Chimayo
Eating Dirt:
1. Pica( 24 months;15-500mg/day )
2. Ingestion During pregnancy
3. Urban Environment
4. Living conditions, Famines
5. Traditions, rituals
6. Inhaled agents
Stanek EJ,Calabrese EJ, et al Environ. Health. Prespect.103:276-285,1995
Modern Controls over
what comes out
Modern Controls Over
what Goes in
Sanitation
Sterilization
Processed Foods
food hygiene
Scrubs
Hand sanitizers
Isolation
air flow controls
Antiseptics
Antibiotics
What Comes out:
Clean up the mess.
WC, Toilets
Septic tanks
Sewage and its disposal
Diapers and their disposal
Toilet cleaners
Blankets - laundry
Deodorants
Shampoos - soaps - mouth washes
Hand Sanitizers: Door Knob Cleaners
2
Post -natal Bacterial contribution
to human Cellular biomass
Microbial soil Ecosystem in 20th
century
Contributing source
No. of cells
Human : from fertilized egg
• About 4600 species of prokaryotic
microorganisms/gram natural soil
• 700-7000 grams of biomass per cubic
meter of soil
• Overall density of microbes on earth 1029
Cell types
>200
>10 trillion
Non human:
Bacterial flora
>500
>100 trillion
Other non human;
Viral, fungal, parasitic cells
>billions
>millions
10:1
For every 1 human cell,
there are at least 10 non
human cells in and on
human body
Non Human :Human
Cell Ratio
Callahan, Emerging Infect Dis 9:1016, 2003
Tannock 1995
Possible Evolutionary functions of
Neurologic and Immunologic systems
Manage
Inter and intra cellular
communications in Host
(> 10 trillion human cells)
• Central Nervous
System
• Spinal cord
• Enteric Nervous
system
Enteric Nervous
System (ENS)
Extends
Manage
Host cells communication
with Microbiome mass
(>100 trillion) in the Host
• Central-systemic
Immune system
• Common mucosal
immune system
• Enteric Immune
system
From Furness et al. (1991)
Intestinal Primary Afferent Neurons( IPANs) of Myenteric
Plexus: Microbiome related disease association
Microbial Load in Different
Human Body Surfaces
(After Wolfgang Kunze 2012)
Depression?
From
Oesophagus
To Rectum
Site
Skin
Naso-oro-pharynx
Lungs
No. Organisms
102-3/mm2
108-10/ml
0
Stomach
<103ml
Small intestine
103-5/ml
Large intestine
Genital tract (vagina, lower
urinary tract)
1010-11/g (feces)
108-10/ml
Alderberth I, et al. In Development of Gastrointestinal Tract. Sanderson, et al
(eds), p. 279, 1999
3
Intestinal Microbiota
Microbial composition of the small intestine
microbiota
Ileostomy effluent microbiota
Prominent presence of:
• >1014 bacteria representing over
Streptococcus spp.
Veillonella spp.
Clostridia spp.
Escherichia spp.
1100 species with over 160 species
per individual
• Gut Microbiota contains more
genes than the entire Host
(Human) genome
_________________
Neish AS Gastroenterol.136:65-80,2009
Qin J. Nature 464:59-65,2010
Carien Booijink et al. 2010 EMI, van den Bogert, 2011 AEM, Zoetendal, 2012 ISME J
Neonatal Microbiome
• Mucosal surfaces sterile at birth,
colonization initiated immediately after
birth
• Colonization with physiologic flora
complete after about one week.
• Microbial diversity and quantitation exhibit
marked fluctuations during 1st year.
• Established Microbiota surprisingly stable,
unique and specific to each individual.
________
Gross L. PLoS Biol. 5,e 177. 2007
Bjorksten B. Springer seminars immunopathol. 25:257-270,2004
Maternal Influences on Development of Neonatal Immune
Response and Oral Tolerance
Mother
Neonate
Diet and inhalation
Ag. uptake
Ag. – Microbiome
interaction
maternal mucosa
Transfer Via
Lactation
products: Milk
Immune
Response
Transfer to
Neonate
- Ag. Free, Ig Complexes , IgA
Complexes
- Immune modulators of tolerance TGF - ß, IL 10, Vitamin A
- Prebiotics, oligosaccharides,
glycoproteins
-Gut growth factors – EGF, TGF ß
Oral
Tolerance
Establishment of
gut microbiome;
Dietary Uptake
Breast feeding
Ogra 2012
4
Bacterial Induced Inflammation
• Colonization and or local infection with bacteria is the
rule, but development of disease is the exception.
• > 4.5 billion infections with Pylori; induction of cancer in a
tiny fraction of infected subjects.
• Acute inflammation associated with termination of
infection
• Chronic inflammation : increased association with disease
often related to chronic or overexpression of
1) Cytokines ,chemokines ,promoters of angiogenesis
or cell growth
2) Host DNA damage
3) NF -kβ , STAT(STAT3) activation
4) Activation of innate Immune system:
MyD88,TLR4 overexpression
________________
Grivennikov SI et al cell 140:883-899,2010
Interspecies Host-Microbial
interactions
Beneficial to Host
Early life exposure to microbes critical
against Inflammation
• Normal mice associated with significant decline in
Inflammatory cellular response (NKT) In Lung and
Colon with reduced susceptibility to RAD and IBD.
• High levels of NKT in Germ free mice with
extremely high susceptibility to RAD and IBD.
• Susceptibility reversed in the 2nd generation by
colonization of pregnant mice, or by use of AntiNKT antibody in the young. But not by colonization
or use of anti NKT in the germ free adult.
NK T- Natural killer T cells. RAD- reactive airway.
IBD- Inflammatory Bowel disease
Olszak T,et al. Science on Line March ,2012
•
•
•
Detrimental to Host
•
•
•
•
•
Interspecies symbiosis: Beneficial
Role of Microflora in mucosal homeostasis
z
z
z
z
z
z
z
Communicate with intestinal host cells
Induce altered gene expression
Influence gut development and function
Increase angiogenin production by Paneth cells and
induce angiogenesis
Metabolize food into energy : Influence fructose ,
Vitamins production: Induce fermentation and removal
of non-digestible dietary residues, cellular debris, and by
carbohydrate fermentation induced short chain fatty
acids
Regulation and modeling of host Immune responses
Regulation of emotional behavior and CNS development
Based on studies with β-Thetaiotamicron and other commensals :
Metabolism of food into energy
Modeling of host immune system and its
responses
Regulation of Immune response, gene
expression and disease prevention
Drive for chronic inflammation and
Disease Production:
Obesity; autoimmunity; cancer
Acute infection-inflammation
Autism spectrum disorder
_______________________
Microbial interaction with Mucosal
lymphoid tissue and immune responses
•
Different Bacterial compositions between developed and
developing economic settings
•
Auto-immune disorders more common in under developed
economic settings
•
Certain bacteria more effective in protection against
autoimmunity and Tolerance induction
•
Qualitative alterations of Microbiota effect tolerance
induction and sensitization for immune response: (
Bifidobacteria and Lactobacillus sp.)
•
Impact of Maternal Microbiota; delivery methods, incidental
microbial encounters, diet,
Breast feeding
•
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Commensals shape mucosal
responses
•
Establishment of intestinal Microbiota regulated by the host’s
Immune response. In Turn Intestinal Mucosal Microbiota
shapes host’s adaptive immune response.
•
All symbiotic bacteria not alike in action, All commensal
are not uniformly commensal
Segmented filamentous bacteria (SFB):greater impact on
Th17,(increased Th17 response after colonization
with SFB)
•
Retinoic acid :Inhibition of Th17 development; increased
induction of gut homing molecules
_________________
Gaboriau –Routhiau V et al. Immunity 31:677-689,2009
Mucida D et al. Science 317:256-260,2007
Host benefits of Mucosal Intestinal
Microbiome
• Non Toxigenic form of Bacteroides
Fragilis produce polysaccharide(PSA)
• PSA protection against development
of experimental colitis.
• PSA induced IL10 ,induction of
FOXP3+ Tregs via TLR 2 dependent
mechanisms
_______________
•
Mazmanian SK Nature 453:620-625,2008
Role of Intestinal flora in Allergic disorders.
The Body-The Brain and The Bugs
Prolonged treatment with lactobacillus rhamnosus
induced
• Acquisition of Physiologic Microbiome in mucosa with
the first few days-weeks after birth.
• High diversity of DNA sequences of Bacterial genome
and a variety of bacteria in the gut flora associated with
significant protection against atopic eczema and food
allergies and reduced levels of serum IgE
• Diversity greater in healthy children at one month of age
and in children growing up on live stock farms with
cattle
• Protobacteria , Bifidobacteria ,some gram negative
bacteria ,Bacteroides species more commonly
associated with protection against allergy development
1.
region dependent alterations in GABA B1b mRNA in brain
with
2. Concomitant reduction in expression in hippocampus,
amygdala and locus coeruleus
3. L. rhamnosus (JB-1) reduced GABA Aa2 in hippocampus
and reduced stress induced corticosterone and anxiety
and depression related behavior.
4. Vagus nerve is the major modulating constitutive
communication pathway between bacteria exposed to
the gut and Brain
----------------------------------------------------------------------
________________
•
Bravo JA. PNAS 107:1-6,2011. Rao AV. Gut pathol.1:6-8,2009
Gut Microbiota and Sensitization
for IgE responses
• Ig E sensitized children exhibit Over
representation of Actinobacteria at 4 months of
age and Firmicutes sp. At 1 year of age at high
taxonomic level.
•
•
Bifidobacter longum overrepresented at 1 year
and enterococcus at 4 months of age.
Consistent patters of microbial colonization and Ig
E sensitization and impaired immunologic
development in infancy.
____________________________
Vebo HC et al Clin.Vaccine. Immunol. 10:1128: 2011
Abrahamson TR. et al. J.Allergy Clin.Immunol. 2011.DO1:10.1016 jad 2011.
Interspecies Host-Microbial
interactions
Beneficial to Host
•
•
•
Metabolism of food into energy
Modeling of host immune system and its
responses
Regulation of Immune response, gene
expression and disease prevention
Detrimental to Host
•
Drive for chronic inflammation
Disease Production:
•
Obesity ; autoimmunity; cancer
•
Acute infection-inflammation
•
Autism spectrum
______________________
•
Wu S. et al. Nature Med. 15:1016-1022,2009
6
Interspecies Symbiosis: Not always
Beneficial or Symbiotic.
• Heterocyclic amines(HCA). Not digested in intestine, but
fermented by colonic bacteria and converted to DNA
toxic electrophilic derivatives-high risk for cancer.
• Hydrogen sulfide. Propionic Acid and other Metabolites
generated by gut Microbiome. DNA damage; toxic for host
cells.
• Microbial Ethanol production and Human
Disease*
__________________
Huycke MM .Gaskins HR. Exp. Biol .Med 229:586-597,2004
Wallace BD. Et al. Science 330:831-835,2010
*Baker S et al 2012(unpublished)
Changing Spectrum of immunity.
infection-environmental interactions:
18th through 20th centuries
Environmental triggers assocaited with alteration
of Human Microbiome and Expression of disease
Environmental triggers:
Vitamin D Deficiency, Delayed exposure to viruses,
Molecular mimicry, Obesity-diet, Altered gut permeability,
Antibiotics, Commercial formula feeds
Disease Expression:
Allergy
Inflammatory Bowel Disease
Multiple Sclerosis
Diabetes mellitus
Other forms autoimmune disorders
Depression
Malignancy
Autism Spectrum disorder.
Microbial task division
Sugars PTS
Glucose
Mannose
Fructose
Sucrose
Emergence of new Human pathogens
GLYCOLYSIS/
GLUCONEOGENESIS
Streptococcus
Veillonella
Re emergence of previously controlled
Microbial agents
Emergence of Antimicrobial resistance
Clostridium
Formate
transporter
Pyruvate
Acetyl co‐A
THF
Butyrat
Lactate
Pyruvate
carboxylase
Acetyl co‐A
Formate
Acetyl co‐A
Butanoate
metabolism
L-lactate
dehydrogenase
Pyruvateformate lyase
Pyruvate
Folate
Acetyl‐P
L-Lactate
permease
TCA
cycle
Propanoate
metabolism
Acetate
Propionate
Nucleotide
biosynthesis
Approaches Proposed to Reverse Altered
Mucosal Microflora and Associated
Disease
• Introduction of Mucosa- friendly changes in
the Environment:
Artificial foods, organic agriculture.
Use of antibiotics and antimicrobials.
Breast feeding.
• Reestablishment of natural mucosal flora
Probiotics and Prebiotics.
Homologous and Autologous Bacteriotherapy.
Induction of Interspecies transient parasitism.
Kamikaze Bacteria.
Probiotics and Prebiotics
Probiotics
Bacterial preparations which impart clinically verified beneficial
health effects on the host when consumed orally.
Human origin, nonpathogenic properties, resist technologic
processing( viability in delivery vehicles), stability in bile
and acid, adhesion to target mucosa, persist and or
replicate within the GI tract, produce antimicrobial
peptides, and beneficially influence immune function and
metabolic activity
Prebotics
Non absorbable carbohydrates and other products which act by
promoting beneficial members of intestinal microbiota in a
manner that provides demonstrated heath benefits to humans
Salminen S and Isolauri. NAMI research group report. Brit. J. Nutr.80( supp 10: 147, 1998
7
lactobacilli in the small intestine?
Prominent community impact of probiotic intervention
Carien Booijink et al. 2010 EMI, van den Bogert, 2011 AEM, Zoetendal, 2012 ISME
ƒ Lactobacillus species are not dominant
in small intestine communities
ƒ Probiotic intervention can provide
temporal community shift
ƒ Prominent presence of Lactobacillus
ƒ Impact on mucosal biology ?
Probiotic product administration
Single serving ~ 109 cells
Effects of Yogurt on Gut
microbial flora.
• Bacteria in fermented dairy products alter
gene expression in human gut microbes and
later subsequent bacterial –host metabolic
pathways.
• Gene expression in gut microbes by yogurt
probiotics affects gene encoding metabolic
enzymes such as those involved in sugar
catabolism( xylo oligosaccharides)
McNulty G.et al. Scin.trans.Med. DOI 10:1126,2011
Fecal transplants- BacteriotherapyHuman probiotic infusions
• Veterinary Medicine- 100+ years: indigestion in
grazing animals(Cows) effectively treated by
feeding the sick with rumen fluid sucked out of
healthy cows stomach.
• 1958- intractable gut inflammation in 4 human
subjects resolved after fecal infusion from normal
healthy donors.
• 1980. first case of incurable colitis of
unknown etiology following a vacation in
Fiji –complete recovery after healthy fecal
infusion.
Clostridium Difficile: Disease Burden
• C. Difficile colitis
relapse rates 1st episode
15%
2nd episode 45%
3rd episode
65%
• Mortality
1.5-3.4%
• Estimated annual costs for
Hospital care
$ > 3 Billion
Brandt LJ Amer. J. Gastroenterol. 2012 doi.10.1038 /ajg
8
Fecal Bacterial therapy: current status
•
Fecal transplant
Possible Indications
>3 relapses
•
Antibiotic refractory disease
vancomycin resistant disease
Possible donors
•
1st generations relatives; spouses
Negative for HIV,HBV, OC&P
Recipient Status
•
Aggressive cleansing of existing flora;
6-24 hr. non frozen feces, homogenized in nonbacteriologic saline(200-700cc) administered
via colonoscopy(biopsy Channel)
Outcome
98% cure rate- with subsequent vancomycin therapy
Possible improvement in pre existing RA, Sinusitis
•
•
•
Swapping Germs: Should
Fecal Transplants
Become Routine for
Debilitating Diarrhea?
A potentially beneficial
but unusual treatment for
serious intestinal
ailments may fall victim
to regulatory difficulties
By Maryn McKenna |
Tuesday, December 6, 2011
|
26
Brandt LJ. Amer. J. Gastroenterol. 2012:march 27,doi,10.1038/ajg
Fecal bacteriotherapy:
Possible benefits
• Reduced risk of cultivating antibiotic
associated resistance in pathogenic
bacteria.
• Displacement of pathogenic bacteria from
the recipient gut mucosa
• Modulation of Inflammatory and immuno
regulatory responses and cytokine release
in mucosal tissues.
Following B. infantis
Increased Foxp3+, IL10+ Tregs:
Plasticity of Th17 regulatory T cells
Therapeutic use of Helminths in Human and
Animal models of Disease
• Animal models
Heligmosomoides
Allergy, IBD ,DM
Schistosoma mansoni
Strongyloides Stercolis
Fasciola hepatica
Trichenella spiralis
EAE, IBD, Arthritis, DM
Allergy
EAE
DM,EAE
Hymenolepsis diminuta
IBD, Arthritis
Rook GWW, Microbe 7:173-18u0,2012
Ova- Parasites or Fecal Bacterial
infusions and Human disease*
•
•
•
•
•
•
•
IBD
Metabolic syndrome
Chronic fatigue syndrome
Multiple Sclerosis
Autism spectrum disorders
Parkinson’s Disease
Clostridium difficile colitis
_________
Turnbaugh PJ. Science. Trans. Med. 1:6ra 14,2009
Borody TJ J. Clin. Gastroenterol. 38:475-483,2004
*Trichuris Suis
:Nectar Americanis
Fecal bacteriotherapy: Current
Limitations
• No case control or blinded
studies to document specific
effects
• Risk of infections
• Inadequate regulatory oversight
• Lack of data- based uniform
protocols for the procedure
• Costs and reimbursements
9
Microbial composition of gut Microflora:
the good, the bad and the Ugly
Kamikaze Bacteria
(based on Fuller R, Gibson G, Scan J. Gastroenterology Suppl, 22:28,1997)
• Acyl homoserine Lactones: quorum
sensing molecules generated by many
bacteria
( Pseudomonas aerogenosa).
• Escherichia Coli( E. Coli) engineered to
detect such molecules via release of
Pyocin S5,a protein antibiotic
• Lysis of Pseudomonas by Pyocin S5
• Lysis of E. Coli bacterial burst following
the release of Pyocin S5.
____________
•
Saeidi et al. Molecular System Biology 7: 521-525,2011
Microbial Ancestry of man-1
•
Paleolithic Period>10,000.BCE
Hunters-gatherers: Small groups < 100
Micro organisms present in Early Humans
for as long as >100,000 years
Helminths ; Toxoplasma
Mycobacteria; TB; Helicobacter; Salmonella
HAV
other Established Microbiome; Lactobacillus
1st Epidemiologic
Transition
Microbial Ancestry of man-2
•
Neolithic period: 3500 BCE:
Bronze Age: 1500 BCE : Iron Age:1700-1800 CE
Large Social groups- Animal Husbandry- Domestication of
Cats and Dogs- Increased oral fecal contact with Mud, Feces,
Water source
Major Microbial changes at 1st Epidemiologic Transition
Reorganization of and changes in Microbial
ecosystem
1) More Helminths, More orofecal contact,
More settled life style
2) Novel sporadic infections from Farm animals –
Rotavirus, Calicivirus, Influenza, Measles, Smallpox
Cholera, Plague, Typhus
2nd Epidemiologic Transition
Microbial Ancestry of man-3
•
Give yourself a break !!!
We have been around for over 2 billion years .
Modern Age: 1800- present
Continuing Social evolution: Large cities-Concrete-Tarmacless mud-increasing sanitation-less animal contact- more &
more hygiene-less breast feeding-more antibiotics and
antiseptics- immuno and chemotherapydeworming- increased long distance travelmarked changes in physical activity
Replacement of earlier flora with recent
Immigrant "Alien” arrivals:
Significant Alteration in Native Micro flora
Less: Helminths, Toxoplasma,Helicobacter,Salmonella,TB,
you just got here
You still don’t know much about us
learn to live with us , otherwise ?
↓
↓ ↓
Commensals from Mud and water, HAV
More: HIV and other restricted “Alien "animal flora
Next Epidemiologic Transition ??
10
To regard any form of
life merely as slave or
foe will one day be
considered poor
philosophy,
for all living things,
constitute an integral
part of the cosmic
order.
René Dubos
1901-82
Thank You ;
And be prepared for Change
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