Abstracts

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.

The Effect of Probiotics on the Intestinal Function and Symptoms

Yehuda Ringel, MD, University of North Carolina at Chapel Hill

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.

Impact of Probiotics on the Central Nervous System?

Emeran A. Mayer, MD, Kirsten Tillisch, MD, David Gefffen School of Medicine at UCLA

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 dendritic cells (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 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, Stanford, CA

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 toaccount 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 which 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. Frstly, 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 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, UK

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 been validated against gut contents from sudden death victims and give 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.

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.

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.

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.