PROBIOTICS: - The New York Academy of Sciences

Present
P ROBIOTICS:
From Bench to Market
Friday, June 11, 2010
The New York Academy of Sciences Conference Center
New York, NY
SCIENTIFIC ORGANIZING COMMITTEE
Tri Duong, PhD
Texas A&M University
Howard Young, PhD
Marguerite Klein, MS
National Cancer Institute, National
Institutes of Health
Office of Dietary Supplements,
National Institutes of Health
Kathy Granger, PhD
The New York Academy of Sciences
Mary Ellen Sanders, PhD
Brooke Grindlinger, PhD
Dairy and Food Culture Technologies
The New York Academy of Sciences
PROGRAM DESCRIPTION
This 1-day conference brings together both established and young career
scientists and clinicians from industry, academia, and government, as well as
other health professionals interested in the field of probiotics, in an open forum
for the discussion of the emerging science of mechanisms behind the possible
benefits of probiotic microorganisms in promoting human health and combating
disease, and the role of basic science in bringing a probiotic product to market.
The overall goal is to increase communication and collaboration in order to
advance probiotic research and public health.
Presenters will discuss:
•Benefits and limitations of probiotic microorganisms in promoting human health and combating disease
•Mechanisms responsible for beneficial effects of probiotics
•Relevant models for studying the efficacy and safety of probiotic
microorganisms
•Regulatory challenges in linking clinical studies to substantiation of
structure/function or health claims for probiotic products
The program includes a combination of a keynote address, plenary lectures,
poster presentations, and data blitz presentations. Please also tune in to
our pre-event podcast about probiotics, entitled More Than A Yogurt Cup
(www.nyas.org/morethanyogurt). The proceedings of this conference will be
published as an online meeting report in the Annals of The New York Academy
of Sciences, and a comprehensive meeting report with a selection of speakers’
slides and audio will be made available online as a multimedia report or eBriefing.
ACKNOWLEDGEMENT OF SUPPORT
This event is sponsored by an unrestricted education grant from
The Dannon Company, Inc.
Funding for this conference was made possible in part by 1 R13 AI 088836 - 01
from the National Institute of Allergy and Infectious Diseases. The views expressed
in written conference materials or publications and by speakers and moderators do
not necessarily reflect the official policies of the Department of Health and Human
Services; nor does mention of trade names, commercial practices, or organizations
imply endorsement by the U.S. Government.
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WELCOME
The New York Academy of Sciences is very pleased to welcome you to our
conference Probiotics: From Bench To Market. Our goal is to provide the most
conducive environment for a lively and informed exploration of: (i) the emerging
science of mechanisms responsible for the beneficial effects of probiotics, particularly with respect to the gastrointestinal, nervous, and immune systems; (ii) innovative approaches to substantiation of health effects of probiotic strains; and (iii)
US regulatory experiences in the conduct of probiotic clinical research, the results
of which may be used to substantiate beneficial health effects of probiotics.
This event is sponsored by an unrestricted education grant from
Dannon Company Inc.
In addition to the conference Agenda and Abstracts, this registration packet
includes a Program Evaluation Questionnaire. Please return your Program
Evaluation Questionnaire to the registration staff at the conclusion of
today’s conference, or mail it to the address listed on the form.
The New York Academy of Sciences. Founded in 1817, the New York Academy
of Sciences is the third oldest scientific society in the United States. The Academy
is an independent, 501(c)(3) nonprofit, scientific and educational organization
with a membership of more than 24,000 scientists, physicians, engineers, students,
and educators in 140 countries. Serving as a neutral nexus between industry,
academia, government, and the general public, the Academy’s goal is to advance
the understanding of science, technology, and medicine, and to stimulate new
ways to think about how scientific research is applied in society. Members of
the Academy receive free or reduced registration to our more than 100 events
per year, a subscription to The New York Academy of Sciences Magazine, and
access to our exclusive online content, including: the Annals of The New York
Academy of Sciences; our catalog of hundreds of Academy eBriefings, consisting
of multimedia presentations and summaries of cutting-edge Academy symposia;
and career information and resources.
As an attendee of this conference, you will receive a 1-year membership to the
New York Academy of Sciences. We encourage you to become active members
of our community. Your membership and participation will help support programs
that promote public understanding of science, help young researchers jump
start their careers, and connect scientists to address pressing needs in science,
technology, and public health. You can look forward to building networks and
exchanging ideas with leaders like yourself. For more information about your
NYAS membership, please visit www.nyas.org or email [email protected].
We look forward to an exciting day of discussion and hope this conference
meets your expectations. Please do not hesitate to notify our staff of any
questions or concerns.
Brooke Grindlinger, PhD
Director, Life Sciences
The New York Academy of Sciences
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FACULTY DISCLOSURES
All faculty participating in this activity are required to disclose to the audience
any significant financial interest and/or other relationship with the manufacturer(s)
of any commercial product(s) and/or provider(s) of commercial services discussed
in his/her presentation and/or the commercial contributor(s) of this activity.
Philippe Caradec, MS
Employee
• The Dannon Company, Inc
Duane Charbonneau, PhD
Employee
• Procter & Gamble
Research Support
• Procter & Gamble
Kevin A. Donato, BSc
None
Colin Hill, PhD
None
Marguerite Klein, MS
None
Emeran A. Mayer, MD
Research Support
• The Dannon Company, Inc.
Consultant
• The Dannon Company, Inc.
Tri Duong, PhD
None
Dan Merenstein, MD
Consultant
• General Mills
Cara R. Fiore, PhD*
None
Mansour Mohamadzadeh, PhD*
None
Cary P. Frye, BS
Employee
• International Dairy Foods Association
Arthur Ouwehand, PhD
Research Support
• Danisco
Glenn Gibson, PhD
None
Kathy Granger, PhD
None
Brooke Grindlinger, PhD
None
Patricia L. Hibberd, MD, PhD
Research Support
• Probiotic Lactobacillus
provision (In-kind)
4 • Amerifit Brands
Yehuda Ringel, MD*
Research Support
• Salix Pharmaceuticals, Danisco USA, General Mills, SmartPill, Procter & Gamble
Consultant
• Salix Pharmaceuticals, Procter & Gamble, Wyeth, Ganeden Biotech, Inc.
Guy Rousseau, PhD
Research Support
• Institut Lalleman (Rosell)
Mary Ellen Sanders, PhD
Consultant
•Numerous dietary
supplement and food
companies conducting
business in the probiotic
industry. Confidentiality
agreements preclude
disclosure of company names.
Employee
• Dairy and Food Culture Technologies
Justin L. Sonnenburg, PhD
Consultant
• Ganeden Biotech, Inc.
Patrick Veiga, PhD
Employee
• Danone Research
Howard Young, PhD
None
An * after the speaker’s name indicates that the speaker intends to discuss unlabeled
uses of commercial product, or an investigational use of a product not yet approved for
this purpose. The speaker will disclose this information during his/her presentation.
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AGENDA
7:30 – 8:30 am: Registration, Breakfast, and Poster Set-up
8:30 – 8:45 am: Opening Remarks
Brooke Grindlinger, PhD, The New York Academy
of Sciences
Howard Young, PhD, National Cancer Institute
Tri Duong, PhD, Texas A&M University
SESSION I: BASIC MECHANISMS OF ACTIONS
Moderated by Tri Duong, PhD and Howard Young, PhD
This session will provide an overview of the current state of our understanding
of the mechanisms responsible for probiotic benefits with particular focus on
the effects of probiotics on the gastrointestinal, nervous and immune systems.
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8:45 am:
Keynote Lecture
Probiotics: Myths vs. Facts
Mary Ellen Sanders, PhD,
Dairy and Food Culture Technologies
9:15 am:
The Effect of Probiotics on Intestinal Function
and Symptoms
Yehuda Ringel, MD, University of North Carolina at
Chapel Hill
9:45 am:
Impact of Probiotics on the Central Nervous System?
Emeran A. Mayer, MD, University of California,
Los Angeles
10:15 am: Probiotic Impact on the Immune System
Mansour Mohamadzadeh, PhD, Northwestern University
10:45 – 11:15 am: Coffee Break
11:15 am: Understanding and Altering the Intestinal Microbiota
Justin L. Sonnenburg, PhD, Stanford University
11:45 am: Bacteriocin Production as a Probiotic Trait to
Combat Infection
Colin Hill, PhD, University College Cork, Ireland
12:15 pm:
Models for Studying Efficacy in Probiotics
Glenn Gibson, PhD, University of Reading,
United Kingdom
12:45 – 2:00 pm: Lunch, Poster Session and Journalist Briefing
SESSION II: DATA BLITZ PRESENTATIONS
Moderated by Mary Ellen Sanders, PhD
This session aims to highlight innovative approaches to substantiation of health
effects of probiotic strains. Members of the industrial and academic scientific
community will be selected from submitted abstracts to present new data relevant to supporting health benefits of commercial or research probiotic strains.
2:00 pm:
Development of Bifidobacterium Longum Infantis 35624 for a Probiotic Supplement
Duane Charbonneau, PhD, Procter & Gamble Company
2:15 pm:
Lactobacillus Rhamnosus GG Attenuates Interferon-γ and Tumor Necrosis Factor-a – Induced Epithelial Dysfunction Kevin A. Donato, HonBSc, Research Institute, Hospital for Sick Children
2:30 pm:
Study of the Interplay Between Gut Microbiota and Ingested Beneficial Bacteria in Irritable Bowel Syndrome Subjects with Predominant Constipation
Patrick Veiga, PhD, Danone Research
2:45 pm:
Exacerbation of DSS-Induced Colitis by Localized Delivery of IFN-b Secreted by Lactobacillus Acidophilus
Howard Young, PhD, National Cancer Institute
3:00 pm:
Administration of Probiotic Bifidobacterium Lactis 420 Reverses Diabetic Status in Mice Under High-Fat Diet Arthur Ouwehand, PhD, Health & Nutrition, Danisco
3:15 pm:
Probiotics Inhibit Behavioral Signs of Depression After a Myocardial Infarction in a Rat Model
Guy Rousseau, PhD, Centre de Biomedecine, Hopital
du Sacre-Coeur de Montreal
3:30 – 4:00 pm:
Coffee Break
SESSION III: FROM CLINICAL TRIALS TO MARKET
Moderated by Marguerite Klein, MS
The session will provide description and discussions of US regulatory experiences in the conduct of probiotic clinical research, the results of which could
be used to substantiate health effects.
4:00 pm:
Introductory Comments
Marguerite Klein, MS, Office of Dietary Supplements, NIH
4:15 pm: What are the Major Regulatory Challenges to Linking
Clinical Studies to Substantiation of Structure/Function or Health Claims on Probiotic Foods?
Cary P. Frye, BS, International Dairy Foods Association
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4:30 pm:
Current CBER/US FDA Regulatory Issues Using Live Biotherapeutic Products
Cara R. Fiore, PhD, Office of Vaccine Research and
Review, US FDA
4:45 pm:
Probiotic Foods: Developing and Implementing Quality Cinical Trials
Dan Merenstein, MD, Georgetown University
Medical Center
5:00 pm:
Therapeutic Probiotics: Designing and Implementing Quality Clinical Trials
Patricia L. Hibberd, MD, PhD, Tufts University School
of Medicine
5:15 – 6:15 pm:
PANEL DISCUSSION:
•
•
•
•
•
•
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Cary P. Frye, BS, International Dairy Foods Association
Cara R. Fiore, PhD, Office of Vaccine Research and Review, US FDA
Dan Merenstein, MD, Georgetown University
Medical Center
Patricia L. Hibberd, MD, PhD, Tufts University School
of Medicine
Philippe Caradec, MS, The Dannon Company, Inc.
Marguerite Klein, MS, Office of Dietary Supplements, NIH
6:15 – 6:45 pm:
Closing Statements
Marguerite Klein, MS, Office of Dietary Supplements, NIH
6:45 – 8:45 pm:
Networking Reception
ABSTRACTS
Speaker abstracts are listed in order of presentation.
SESSION I: BASIC MECHANISMS OF ACTIONS
KEYNOTE LECTURE
Probiotics: Myths vs. Facts
Mary Ellen Sanders, PhD, Dairy & Food Culture Technologies
It used to be that many misconceptions and unproven assertions characterized
the field of probiotics. As the science – and perhaps also regulatory scrutiny has advanced, it seems that the myths perpetuated in the field have yielded
to a more science-based approach to communications on probiotics. Efforts
by several scientific organizations, including the American Gastroenterology
Association, World Gastroenterology Organisation, International Scientific
Association for Probiotics and Prebiotics, International Life Sciences Institute
and National Institutes of Health, to provide guidelines and scientific perspective
to consumers and healthcare providers has perhaps helped in this regard. But
still some myths persist. This lecture will identify some common myths about
probiotics and provide the scientific perspective on them.
Some of the myths that will be explored:
Myth: Probiotics balance your microflora
Fact: Some evidence exists that certain probiotics can accelerate a return to
normal after microbiota perturbation, but the most common impact of probiotics
on your microflora is an increase in the genus/species of the probiotic being fed.
Myth: Human origin of probiotics is essential
Fact: Although probiotic strains initially isolated from humans may have unique
attributes related to efficacy, several efficacious probiotics were not originally
isolated from humans.
Myth: Probiotics must be bile tolerant and survive intestinal transit to be effective
Fact: Survival at the target site of action in the body is likely important; however,
isolation of the probiotic from feces may not be an important attribute of probiotics targeted for non-intestinal sites such as the oral cavity or the stomach.
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The Effect of Probiotics on Intestinal Function and Symptoms
Yehuda Ringel, MD, University of North Carolina at Chapel Hill School of
Medicine
Indirect evidence suggests that the intestinal microbiota is important in maintaining
normal gastrointestinal function and that alterations in the intestinal microbiota
can play a role in the pathogenesis of various GI disease conditions and GI
symptoms. Epidemiological studies have shown that acute GI infection (e.g.,
acute gastroenteritis) can lead to the development of long-lasting abnormalities
in gut motor function (e.g., post infectious gastroparesis), symptoms (e.g., post
infectious irritable bowel syndrome, functional dyspepsia), or inflammation (e.g.,
post infectious inflammatory bowel diseases). Up to one third of patients who
recover from intestinal infection continue to have chronic GI symptoms for more
than 3 months and meet the criteria for IBS. This suggests that in some individuals acute infection can lead to ongoing intestinal damage resulting in altered GI
function and chronic abdominal symptoms that can persist even after the acute
insult/infection is cleared.
Further support for the significance of the intestinal microbiota comes from
physiological studies in animals and humans. Studies in germ free animals have
shown that lack of normal intestinal flora results in accelerated gastric emptying,
delayed intestinal transit, reduced migratory motor complexs and long-lasting
dysmotility and altered sensation. These studies demonstrate the important role
of intestinal microbiota in preserving and maintaining normal GI function.
Furthermore, microbiological studies using advanced molecular biology techniques have demonstrated that the GI microbiota in patients experiencing certain
GI disease conditions and chronic GI symptoms is distinct from the microbiota
harbored within the intestine of asymptomatic individuals with healthy GI function.
These epidemiological, physiological and microbiological data associating the
disruption in intestinal microbiota and the development of altered GI function and
chronic GI symptoms have led to the development of novel approaches targeting
the intestinal microbiota for prevention and/or treatment of these abnormalities.
Early clinical trials using probiotics have shown mixed results and exhibit considerable methodological limitations. However, in the last 5 to 7 years, a number
of studies that have employed sound methodologies provided more solid data
regarding the possible benefit(s) of using certain probiotics in the management
of certain GI malfunctions and symptoms. Several studies have shown improved
general well being and health-related quality of life while others have shown beneficial effect(s) on overall (composite) or specific GI symptoms. Few studies have
assessed the effect of the probiotic intervention on intestinal physiology and
even fewer have investigated the mechanism(s) of this by attempting to correlate
the clinical effects with relevant physiological factors/mediators.
Though the data emerging from these recent studies is not conclusive the positive
information accumulating from these recent studies emphasizes the need for further
research in this area. This will lead to a better understanding of the role of manipulation of the intestinal microbiota with probiotics as a possible mode of intervention
in maintaining GI health and managing common GI disorders/symptoms.
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Impact of Probiotics on the Central Nervous System?
Emeran A. Mayer, MD, Kirsten Tillisch, MD, David Geffen School of Medicine
at University of California, Los Angeles
While bidirectional interactions between the digestive tract and the nervous
system are well established as an important mechanism in the regulation of
gut function in health and disease, a role for the gut flora (microbiota) in these
interactions has only been recently implicated. The evidence for such interactions comes almost exclusively from preclinical studies in rodent models. The
brain can influence the microbiota indirectly (via changes in motility, secretion
and permeability) or directly via signaling molecules released from cells in the
lamina propria (enterochromaffin [EC] cells, neurons, immune cells) into the
gut lumen. Communication between the microbiota and the central nervous
system can occur via multiple signaling mechanisms, which include signaling via toll-like and nod-like receptors, via G-protein coupled receptors on the
luminal surface of gut epithelial cells, and via direct modulation of immune cells
in the lamina propria when intestinal permeability is increased. Modulation of
gut cytokine production by microbiota may indirectly result in CNS modulation.
Enterochromaffin cells represent an important bidirectional transducer between
the gut lumen and the nervous system. Vagal afferent innervation of EC cells
provides a direct pathway for EC cell signals to neuronal circuits, which may
play an important role in pain and immune modulation, background emotions
and other homeostatic functions. Dysregulation of the bidirectional interactions
between the gut flora and the nervous system may be involved in the pathophysiology of acute and chronic GI disease states, including functional and
inflammatory bowel disorders.
Probiotic Impact on the Immune System
Mansour Mohamadzadeh, PhD, Northwestern University, Feinberg School
of Medicine
Effective vaccines combined with adjuvants potentiate antibody avidity and
T cell longevity, particularly in immune suppressive individuals. A new generation of vaccines is being developed using specific species of probiotic
Lactobacillus (L.) species (L. acidophilus and L. gasseri). Data show that
employed Lactobacillus species not only optimally activate dendritic cells (DCs)
but also deliver targeted anthrax protective antigen (PA) or tumor-associated
antigens (TAAs) to mucosal DCs via 12-mer peptides derived from a bacteriophage library. Orally delivered immunogenic fusions by lactobacilli confer robust
immune protection against anthrax or tumor challenge. This vaccine effort is
accomplished via novel adjuvants, evaluation of vaccine effectiveness against
a deadly pathogen or cancer, and controlling gene expression in probiotics that
can be orally consumed at high levels resulting in natural delivery of a “targeted”
antigen to mucosal DCs. Additionally, to regulate Imbalance in the mucosal
immune mechanisms leading to induction of the detrimental signals characterized in humans as inflammatory bowel disease (IBD), the phosphoglycerol
transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis
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in L. acidophilus (NCK56) was deleted. NCK2025 deficient for LTA not only
decreased IL-12/TNFα by DCs but also significantly enhanced IL-10 and controlled co-stimulatory DC-functions resulting in diminished CD4+ T cell activation. Treatment of mice with NCK2025 deficient for LTA significantly mitigated
DSS-induced colitis and potently reduced established colitis via a mechanism
involving IL-10 and intestinal CD4+Foxp3+Tregs. Thus, directed cell surface
modification of L. acidophilus may establish a potential strategy for the treatment of inflammatory intestinal disorders.
Understanding and Altering the Intestinal Microbiota
Justin L. Sonnenburg, PhD, Stanford University School of Medicine
A dense and relatively complex microbial ecosystem dwells within the human
intestine. This intestinal microbiota is composed of trillions of microbes that
influence our biology in diverse ways. Several diseases, including obesity and
inflammatory bowel diseases, have been associated with shifts in microbiota
composition. The question of whether disease-associated alterations in the
microbiota are a cause or symptom of disease is difficult to address due to the
difficulty in manipulating this ecosystem in a predictable manner. With a goal
of understanding how changes in microbiota function and composition directly
impact host biology we are pursuing a mechanistic understanding of microbiota
function. Specifically, we are exploring how the microbiota functionally adapts
to perturbations in the intestinal environment, such as changes in host diet,
microbial community composition, and host genotype. To pursue these aims,
we study germ-free (gnotobiotic) mice colonized with simplified, model microbial
communities, apply systems approaches (e.g., functional genomics) and use
genetic tools for the host and microbes to gain mechanistic insight into emergent
properties of the host-microbial superorganism. Our long-term goals are to create a knowledgebase and the necessary tools to permit rational manipulation of
our resident microbes and to aid in incorporating the intestinal microbiota into
the emerging paradigm for personalized genomic medicine.
Bacteriocin Production as a Probiotic Trait to Combat Infection
Colin Hill, PhD, University College Cork, Ireland
A number of plausible mechanisms have been proposed to account for the
ability of certain probiotic strains to ameliorate infection in humans and animals.
These include improved barrier function, direct antagonism, immunomodulation and competitive exclusion. This presentation will focus on one of these
mechanisms, direct antagonism, mediated by the production of bacteriocins.
Evidence will be presented that unequivocally demonstrates bacteriocin production by a probiotic strain as the key anti-infective strategy employed by
Lactobacillus salivarius UCC118 to protect mice against Listeria infection.
The precise definition of a probiotic strategy offers several key opportunities in
probiotic research. Firstly, a more rigorous selection strategy can be employed
to select naturally occurring probiotics with a higher likelihood of efficacy
against selected target pathogens; secondly, existing probiotics could be
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manipulated to improve efficacy in a directed manner; and thirdly, the bacteriocins can be exploited as pharmabiotics: probiotic-derived molecules with
therapeutic possibilities. Lastly, the effect of bacteriocins on host flora other
than the target pathogens will be discussed, highlighting a possible benefit of
these natural substances over classical antimicrobials – a targeted host range.
An example of where bacteriocins have been used to target Clostridium difficile
without causing collateral damage to the commensal microbiota.
Models for Studying Efficacy in Probiotics
Glenn Gibson, PhD, University of Reading, United Kingdom
The ultimate test for probiotic and prebiotic efficacy is a well controlled blinded
human study. However, human trials that correlated modulation of the microbiota with proven indicators of health are costly. To better plan such interventions, several model systems are possible. Given careful planning, such models
can be used to determine potential mechanisms of effect. Most probiotics
and prebiotics need interaction with the gut microbiota to elicit their effects. It
is important therefore that reliable (molecular based) approaches are used to
characterize population changes. In vitro models can range from pure culture
studies, through to anaerobic batch culture fermenters and continuous culture
systems. Complex models such as the SHIME and TNO reactors are useful for
underpinning probiotic-mediated effects in simulations of the gastrointestinal
tract. One colonic model has been validated against gut contents from sudden
death victims and gives a close analogy to bacterial activities in different areas
of the hindgut (Microbial Ecology 1998; 35, 180-187). The system consists of
3 vessels, of increasing size and pH, aligned in series, such that a sequential
feeding of growth medium occurs. The advantage of such an approach is that
it can predict microbial events in different areas of the large intestine. These
laboratory systems are useful for predicting fermentation profiles. Animal models
are used to simulate immune function and other physiological processes such
as absorption. Intestinal cell lines are useful for uptake studies and microbial
binding experiments. Today, such approaches are leading to the better planning
of human trials and development of useful technologies such as proteomics and
metabonomics.
SESSION II: DATA BLITZ PRESENTATIONS
Development of Bifidobacterium Longum Infantis 35624 for a
Probiotic Supplement
Duane Charbonneau, PhD1, Liam O’Mahony, PhD2, Fergus Shanahan, MD3,
Barry Kiely, PhD4, Ray Grant, PhD1, Linda McKean, BS1, Yuli Song, PhD1.
1Procter & Gamble, Cincinnati, OH; 2Swiss Institute of Allergy and Asthma
Research, Davos, Switzerland; 3Alimentary Pharmabiotic Center University
College Cork, Ireland; 4Alimentary Health Ltd, Kinsale, County Cork, Ireland
Bifidobacterium longum subsp infantis 35624 (B. infantis 35624) was isolated
from an ileocecal biopsy of a healthy individual after removal of extraneous
loosely associated bacteria. This strain was selected for further study following
comparative assessments to benchmark strains. The evaluation criteria were
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based on antimicrobial efficacy, microbial community modification, biomarker
induction and efficacy in animal infection models. The impact of B. infantis
35624 on fecal floral composition and metabolism in healthy as well as Irritable
Bowel Syndrome (IBS) subjects was determined. Following daily consumption,
the greatest effects observed were increases in lactic acid bacteria, changes in
fecal bacterial composition as well as changes in short-chain fatty acid profiles.
In clinical trials, B. infantis 35624 improved certain symptoms associated with
IBS, although consistent effects on symptoms were not seen in all clinical studies. Using quantitative PCR analysis of stool, it was shown that GI colonization
by B. infantis 35624 was transitory in nature, and counts decreased within 2
weeks after dosing was suspended. Clinical studies that examined induction of
biomarkers appeared to produce results analogous to those observed in vitro
in that exposure to B. infantis 35624 increased the ratio of IL10 to IL12. These
results are consistent with the conclusion that B. infantis 35624 exerts an effect
both on the microbiome as well as the host.
Lactobacillus Rhamnosus GG Attenuates Interferon-γ and Tumor
Necrosis Factor-a – Induced Epithelial Dysfunction
Kevin A. Donato, HonBSc, Yu Jing Wang, Hon BSc, and Philip M. Sherman,
MD, FRCPC. Department of Laboratory Medicine and Pathobiology, University
of Toronto; Cell Biology Program, Research Institute, Hospital for Sick Children;
Toronto, ON, Canada
The intestinal epithelial tight junctions (TJ) form a protective barrier against luminal
contents and can be disrupted by infection or pro-inflammatory cytokines.
Abnormalities in TJ contribute to a variety of intestinal disorders, including
diarrhea and chronic inflammatory bowel diseases. Probiotics preserve intestinal TJ, but the mechanisms are not well characterized. We hypothesized that
probiotics preserve barrier function by interfering with pro-inflammatory cytokine
signaling. Polarized epithelial monolayers were inoculated apically with probiotic, Lactobacillus rhamnosus GG (LGG) 3 h prior to treatment of the basolateral
medium with IFN-γ overnight. The monolayers were then placed in fresh basal
medium ± TNF-α and transepithelial electrical resistance (TER) measurements
were taken over the time course of TNF-α stimulation. To complement TER findings, cells were processed for zona occludens-1 (ZO-1) immunofluorescence.
Basal tissue culture medium was collected after overnight TNF-α stimulation to
measure secreted chemokines (interleukin-8, eotaxin). For measures of TNF-α
signaling, both immunofluorescence and electromobility shift assay techniques
labeling the NF-κB p65 subunit were used to detect cytoplasmic to nuclear
translocation after 30 min of TNF-α stimulation. LGG ameliorated the deleterious
affects of interferon-γ and tumor necrosis factor-α stimuli on tight junction architecture and barrier function. LGG dampened the NF-κB signalling response as
evidenced by the reduction of NF-κB translocation and chemokine secretion.
Further research will characterize the mechanism by which LGG modulates additional cell signal transduction pathways. These findings provide insight to the
design of novel interventions that could be used in the prevention of both acute
and chronic inflammatory bowel diseases.
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Study of the Interplay Between Gut Microbiota and Ingested
Beneficial Bacteria in Irritable Bowel Syndrome Subjects with
Predominant Constipation
Raish Oozeer1, Karine Roy1, Julie Morris3, Jan Knol1, Anurag Agrawal2,
Pascale Rondeau1, Nathalie Goupil-Feuillerat1, Johan E.T. Van Hylckama
Vlieg1, Denis Guyonnet1, Peter J. Whorwell2, Lesley A. Houghton2, &
Patrick Veiga1. 1Danone Research, Centre Daniel Carasso, Palaiseau, France;
2Neurogastroenterology Unit, Translational Medicine – GI Sciences, University
of Manchester, Manchester, UK; 3Dept of Medical Statistics, University Hospital
of South Manchester NHS Foundation Trust, Manchester, United Kingdom
Previously, we showed in a randomized, double-blind, controlled, parallel group
study that a 4-weeks consumption of a fermented milk containing Bifidobacterium lactis DN-173010 and strains of Lactococcus lactis, Steptococcus thermophilus, Lactobacillus bulgaricus led to an improvement of IBS symptoms
in women with predominant-constipation (IBS-C) (Agrawal et al., 2008). Fecal
samples (n=34) from this study were analyzed with the aim to identify potential
changes in the gut microbiota (GM) induced by product consumption. Samples
acquired before and after the consumption of test product or a control product
(a non-fermented acidified dairy product) were analysed using phylogenetic
microarrays (HitChips) and qPCR. Intragroup HitChips analysis (confirmed
by qPCR) showed that the consumption of the test product did not affect the
global structure of the GM whereas some specific phylogenetic groups were
rearranged as a result of i) the recovery of the ingested bacteria and ii) the
rearrangement of the resident GM. In addition, fecal abundance of ingested
beneficial bacteria was shown to correlate with the abundance of specific gut
microbial components before the intervention. In conclusion, we showed that
the improvement of symptoms in the studied IBS-C population correlates with
GM rearrangement and that the resident bacteria can predict the susceptibility
of ingested beneficial bacteria to transiently integrate the gut microbial community. Future studies will be needed to establish whether the titers of live bacteria
in the gut microbial community are a key factor underlying the variability in the
efficacy of beneficial ingested bacteria.
Exacerbation of DSS-Induced Colitis by Localized Delivery of IFN-b
Secreted by Lactobacillus Acidophilus
Adelle McFarland, Ram Savan, Sagie Wagage, Augustina Addison,
Howard A. Young. Cancer and Inflammation Program Laboratory of
Experimental Immunology, Center for Cancer Research, National Cancer
Institute, Frederick, MD
There have been conflicting reports of the role of Type I interferons in gut inflammation and inflammatory bowel diseases. While in some patients with active
ulcerative colitis (UC), treatment with Type I interferons induced remission of
symptoms, other reports have documented the spontaneous occurrence of UC
in multiple sclerosis and chronic hepatitis C patients receiving IFN-beta therapy.
In order to evaluate the potential for IFN-beta as a therapeutic for IBD, we developed a delivery system involving probiotic bacteria. Treatment of healthy mice
with Lactobacilli secreting IFN-beta results in increased IFN-gamma positive
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cells within the gut as well as a reduction in the percentage of T-regulatory
cells. When mice were pretreated with Lactobacilli secreting IFN-beta prior to
induction of acute DSS colitis, we observed more weight loss and a worsening of intestinal thickening/shortening. In addition, there was an increase in the
percentage of Th17 cells, as well as an increase in the production of TNF-alpha
by colonic tissue. This study demonstrates that localized delivery of IFN-beta by
probiotic bacteria results in an exacerbation of DSS induced colitis in a preventative model.
Administration of Probiotic Bifidobacterium Lactis 420 Reverses
Diabetic Status in Mice Under High-Fat Diet
Arthur Ouwehand1, A. Waget2, P. Klopp2, Kaisa Olli1, Sampo Lahtinen1,
Didier Carcano3, Nina Rautonen1, Remi Burcelin2. 1Danisco Health and
Nutrition, Kantvik, Finland; 2Institut National de la Santé et de la Recherche
Médicale (INSERM), U858, Toulouse, France; 3Danisco Health and Nutrition,
Paris, France
Recent findings indicate that a high-fat diet (HFD) induces metabolic endotoxemia (elevated plasma lipopolysaccharides; LPS) leading to inflammation, and
is an early triggering factor of metabolic syndrome and type II diabetes. We
hypothesized that administration of a probiotic Bifidobacterium may reduce
endotoxemia and subsequently inhibit inflammation, improve glucose tolerance
and contribute to weight maintenance. C57Bl6 mice and CD14 knock-out mice
were fed with normal or high-fat diet to induce diabetic state. Bifidobacterium
lactis 420 was administered daily for four weeks. Diabetic status was measured
by IPGTT and hyperinsulinemic euglycemic clamp. RNA was extracted from
tissues for the determination of inflammatory cytokines. Body composition was
measured by ECO-MRI and bacteriological analyses were done by qPCR. LPS
was determined from blood. Epithelial integrity was measured with cell culture.
B. lactis 420 reduced fasting glycemia, improved glucose tolerance and reduced
insulin resistance and tissue inflammation. Weight gain, total fat mass, mesenteric adipose tissue weight and plasma LPS were lowered by the treatment.
Probiotic effect was blunted in knock-out mice lacking CD14 (LPS receptor),
suggesting a central role of LPS. Additional mechanistic studies showed that
B. lactis 420 improved epithelial barrier function and reduced bacterial translocation from gut into mouse tissues. Administration of B. lactis 420 may offer a
new strategy for treatment of metabolic syndrome, type II diabetes, and co-morbidities including obesity and chronic inflammation. The mechanism associated
with the positive effect of B. lactis 420 involves reduction of translocation of
gut-derived LPS into the host, thus reducing inflammation.
Probiotics Inhibit Behavioral Signs of Depression After a Myocardial
Infarction in a Rat Model
J. Arseneault-Bréard1, I. Rondeau1, K.Gilbert1, S.A. Girard1, R. Godbout2, T.A.
Tompkins3, G. Rousseau1. Departments of Pharmacology1 and Psychiatry2,
Université de Montréal, Montréal (Québec) Canada; Institut Rosell Inc.3,
Montreal (Quebec) Canada
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We have previously demonstrated that pre-treatment with probiotics reduces
apoptosis observed in the limbic system after myocardial infarction (MI). This
study tested whether probiotics could also attenuate post-MI depressive behavior.
Methods: MI was induced in anesthetized rats via a 40-minute transient occlusion of the left anterior coronary artery. Sham rats underwent the same surgical procedure without actual coronary occlusion. For the 7 days before MI and
between the 7th and 14th day post MI, half the MI and sham rats were given > 1
billion live bacterial cells of Lactobacillus helveticus R0052 and Bifidobacterium
longum R0175 per day dissolved in water while the rest of the rats received only
the vehicle. Depressive behavior was evaluated 14 days post MI using Social
Interaction, Forced Swimming and Passive Avoidance tests. Results: Vehicletreated MI rats showed the expected behavioral syndrome of depression.
Probiotics-treated MI rats displayed more Social Interactions and a better performance in the Forced Swimming and the Passive Avoidance tests compared
to vehicle-treated MI rats (p < 0.05). Probiotics had no impact on behavioral
performance in sham rats. Conclusion: Probiotics can interfere with the development of post-MI depressive behaviour. These results further suggest a role of
the gastro-intestinal tract in mediating this response.
SESSION III: FROM CLINICAL TRIALS TO MARKET
What are the Major Regulatory Challenges to Linking Clinical Studies
to Substantiation of Structure/Function or Health Claims on Probiotic
Foods?
Cary P. Frye, BS, International Dairy Foods Association
The U.S. Food and Drug Administration (FDA) has broad jurisdiction to regulate
the use of food labeling, including probiotic food claims. Label claims are subject to different FDA requirements depending on the types of claims made.
Health claims are statements that characterize the relationship of a substance in
a food to the reduction in the risk of a disease or health-related condition. FDA
may consider statements about outcomes of probiotic clinical studies to
be unapproved drug claims if they convey that the probiotic's intended use is
to diagnose, cure, mitigate, treat or prevent disease. Permitted health claims are
those that are authorized by FDA regulation, by a health claim notification that
is based on a recognized authoritative statement or an appropriately qualified
health claim where the quality and strength of the supporting science is not
conclusive. All health claims require FDA review. Currently there are no health
claims for probiotics. Structure/function claims are distinguished from health
claims by their focus not on disease prevention, but on maintaining or supporting normal structures or functions of the body. Such claims have been used on
food for many years and include statements such as "calcium helps build strong
bones." Structure/function claims can be made in food labeling if they are truthful and not misleading and are substantiated by competent and reliable scientific evidence -- essentially the same standard the Federal Trade Commission
(FTC) applies to advertising claims. No FDA approval or notification is required
for the use of structure /function claims on foods.
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Current CBER/US FDA Regulatory Issues Using Live
Biotherapeutic Products
Cara R. Fiore, PhD, Center for Biologics Evaluation and Research,
Office of Vaccine Research and Review, US FDA
The US Food and Drug Administration (FDA), Center for Biologics Evaluation
and Research, Office of Vaccine Research and Review (OVRR) is responsible for
regulating vaccines, allergenics and live biotherapeutic products (LBPs) for use
in humans. LBPs are probiotics that are used to prevent, treat or cure a human
disease or condition, and, therefore are regulated as biological products (section
351(i) of the PHS Act, 41 U.S.C. 262(i)). Thus, lawfully marketed food products
may be considered biological products if they fall into this category of intended
use. An Investigational New Drug Application (IND) must be submitted to OVRR
to conduct a study to evaluate LBPs for such intended use. The primary goal of
early (Phase 1) studies is to assess clinical safety. IND submissions must contain sufficient information to assess the risks to subjects of the proposed studies. Information describing the manufacturing and composition of the product,
and the proposed clinical use, must be contained in the IND, or accurately referenced in the submission (Code of Federal Regulations; 21 CFR 312.23). Product
characterization information should include sufficient information to assure
identification, quality and purity of the LBP. Navigating the biologics regulatory
pathway often proves challenging to sponsors attempting to study LBPs, and
many IND submissions for LBPs fail to proceed to clinical studies due to lack of
sufficient information concerning product characterization. This presentation will
address common shortcomings of INDs for LBPs related to product characterization and strategies to avoid such pitfalls.
Probiotic Foods: Developing and Implementing Quality Clinical Trials
Dan Merenstein, MD, Georgetown University Medical Center
Dr. Merenstein is currently conducting his fifth randomized clinical trial on probiotics. Dr. Merenstein has studied DanActiveTM, ProbugsTM, and has conducted
three studies with a yogurt supplemented with Bifidobacterium animalis ssp.
lactis (B. lactis) BB-12. All studies except his current study were conducted with
healthy children. This current study, sponsored by the NIH, was peer evaluated
and received a funding score for a pediatric clinical trial. However, as the study
was to determine if probiotics can help decrease antibiotic-associated diarrhea,
the NIH required the investigator to seek FDA Investigational New Drug (IND)
approval before starting the trial. The FDA required a Phase I safety trial in
adults; as such, the NIH provided funding to conduct a Phase I safety study in
20 healthy adults receiving BB-12 yogurt and 20 receiving regular yogurt.
Dr. Merenstein’s previous studies did not require an IND, as the primary aims
were not to cure, treat, mitigate, prevent, or diagnose disease. Instead, the FDA
considers such outcomes as structure/function claims; for example, the outcomes in three previous studies were to prevent daycare absences and in one
study, to decrease parental report of loose stools.
During the presentation, Dr. Merenstein will address issues of implementing
clinical trials, with an emphasis on different elements involved in probiotic
clinical trials. He will also discuss the IND process from an academic University
perspective and discuss the timeline involved in working with the FDA.
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Therapeutic Probiotics: Designing and Implementing Quality
Clinical Trials
Patricia L. Hibberd, MD, PhD, Tufts University School of Medicine
Probiotics have a long history of traditional use, but when they are used to prevent, treat, mitigate or cure disease, they are being used therapeutically in the
United States. Investigators and manufacturers may need to comply with the
investigational new drug (IND) requirements of the FDA, particularly the Center
for Biologics Evaluation and Research (CBER). Manufacturers of probiotics
may need to take on the extensive responsibilities of owning a Drug Master File
(DMF) at the FDA and US investigators may need to take on a similar burden of
responsibilities to conduct studies under IND. A recent conference funded in
part by NIH recommended that probiotic investigators conduct Phase 1 studies
to provide the biologic basis of and justification for design of future probiotic
studies. With cooperative agreement grant support from NCCAM at the NIH,
our group has embraced this approach, but has had faced numerous challenges
and issues in achieving the goal of conducting the needed quality studies.
We will present specific issues that may need to be considered for investigators conducting IND studies of probiotics, and the opportunity to understand
the ethical aspects and science of the interactions between exogenous administration of probiotics, the human respiratory and gastrointestinal microbiome
and the immune response to the human microbiota. Our presentation includes
working with a Data and Safety Monitoring Board (required by NIH, but not
required by the FDA) as well as site monitoring organizations, more accustomed
to pharmaceutical studies, to ensure high quality of conducted clinical trials.
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NOTES
SPECIAL NEEDS
The New York Academy of Sciences complies with the public accommodation
requirements of the Americans with Disabilities Act and the rules and regulations
thereof. Please let us know if you require any assistance during the meeting.
20
NOTES
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23
Present
P ROBIOTICS:
From Bench to Market
Friday, June 11, 2010
The New York Academy of Sciences Conference Center
New York, NY
www.nyas.org/probiotics