Diet and Environment: Early Determinants of Immune Response

Diet and Environment: Early Determinants of Immune Response
November 8, 2018 Diet and Environment: Early Determinants of Immune Response Jose M. Saavedra, MD Associate Professor of Pediatrics, Johns Hopkins University School of Medicine & Medical and Scientific Director, Nestle Nutrition 1 1

Bacteria in the GI Tract: Complex Ecosystem Resident and Ingested
November 8, 2018 Bacteria in the GI Tract: Complex Ecosystem Resident and Ingested species Stomach 104 CFU/g Candida albicans Helicobacter pylori Lactobacillus Streptococcus Duodenum CFU/g Bacteroides Candida albicans Lactobacillus Streptococcus Jejunum CFU/g Bacteroides Candida albicans Lactobacillus Streptococcus Ileum CFU/g Bacteroides Clostridium Enterobacteriaceae Enterococcus Lactobacillus Veillonella Colon CFU/g Bacteroides Bacillus Bifidobacterium Clostridium Enterococcus Eubacterium Fusobacterium Peptostreptococcus Ruminococcus Streptococcus

Surface Areas of Exposure to the Environment
November 8, 2018 Surface Areas of Exposure to the Environment Skin sq m Respiratory mucosa sq m Intestinal mucosa sq m Gut epithelium mm Bacterial gut content up to 1012 / ml Tightly regulated mucosal immunity is needed to maintain health

GALT is the largest immune organ
November 8, 2018 GALT Gut associate lymphoid tissue (GALT) comprises 70-80% of immunologic cells in the body GALT is the largest immune organ Most of the immune cells of the infant are associated with the intestine- to help protect the infant from potential pathogens in the intestinal lumen

Germ-free vs. Colonized Gut
November 8, 2018 Germ-free vs. Colonized Gut The intestinal mucosa of animals who are born sterile and receive sterile food and are raised in a sterile environment do not develop an intestinal flora- and the intestinal mucosa is atrophic, has less cell turnover, less enzyme activity, less secretion of antibodies (IgA in particular), anf the mucosa wall is thin. Animals raised in a normal environment and receive non-sterile food, develop a normal intestinal flora; and a normal barrier function, to protect the host.

Role of Intestinal Microbiota
November 8, 2018 Role of Intestinal Microbiota Germ free animals Atrophic mucosa Germ free animals + normal flora Bacterial translocation leading to death Animal raised in sterile environment; when exposed to bacteria, develop infection and die A healthy flora is critical to train the immune system, and to maintaining GI barrier function, and survival

Antigens Microflora Th0 Intestinal Lumen Epithelium Intestinal Mucosa
November 8, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Optional Speaker Notes: This slide, and the three subsequent slides, provide in simplified form a pictorial representation of antigens and intestinal bacteria in the intestinal lumen. Their critical role in the activation of T-cells is described. T-cells are a major component of gut-associated lymphoid tissue (GALT). Undifferentiated (Th0) will normally differentiate, once activated through antigen presentation, into Th1 or Th2 cells. 7

Antigens Microflora Th0 Th2 Th1 Intestinal Lumen Epithelium
November 8, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Th2 Th1 Optional Speaker Notes: Certain types of stimuli from the lumen can activate T cells to differentiate into T-regulatory cells. Both of the Th1 and Th2 cell subsets are critical for maintaining health. However, a balanced response between Th1 and Th2 is critical to decrease the chances that the immune system will overreact. Each subset generates and responds to different types of cytokines, which are cellular mediators of these T-cell subsets. 8

Antigens Microflora Th0 Th2 Th1 TNF-α IL-4 Cytokines IFN-γ IL-5 IL-2
November 8, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 Optional Speaker Notes: Th1 cells, which are associated with TNF-alpha, interferon gamma, and IL2 are critical for cellular immunity and for pathogen protection, including the formation of IgM and IgG. Th2 cells, and their respective cytokines, including interleukins 4, 5 and 10, are important for adequate development of humoral immunity including formation of IgE and IgA antibodies. T cell differentiation is necessary for normal immune function and absolutely necessary for normal host defense. Cytokines Cellular Immunity Pathogen protection IgM, IgG response Humoral Immunity IgE, IgA response Response 9

Antigens Microflora Th0 TReg Th2 Th1 TNF-α IL-4 Cytokines IFN-γ IL-5
November 8, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 TReg Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 Optional Speaker Notes: T regulatory (Treg) cells possess characteristics of both the Th1 and Th2 responses, and release cytokines such as transforming growth factor beta (TGF beta), which can inhibit Th1 or Th2 over expression, playing a role in the development of tolerance, autoimmunity, and allergy. In summary, undifferentiated (Th0) will normally differentiate once activated through antigen presentation into Th1 or Th2 cells. The balance between these T lymphocyte and cytokine pathways plays a role in modulating the immune response between proinflammatory and tolerance promoting mechanisms. Cytokines TGF-β Immune Tolerance Cellular Immunity Pathogen protection IgM, IgG response Humoral Immunity IgE, IgA response Response 10

Antigens Microflora Th0 TReg Th2 Th1 TNF-α IL-4 Cytokines IFN-γ IL-5
November 8, 2018 Antigens Microflora Intestinal Lumen Epithelium Intestinal Mucosa Antigen Presentation Activated T cell Th0 TReg Th2 Th1 TNF-α IFN-γ IL-2 IL-4 IL-5 IL-10 Optional Speaker Notes: T regulatory (Treg) cells possess characteristics of both the Th1 and Th2 responses, and release cytokines such as transforming growth factor beta (TGF beta), which can inhibit Th1 or Th2 over expression, playing a role in the development of tolerance, autoimmunity, and allergy. In summary, undifferentiated (Th0) will normally differentiate once activated through antigen presentation into Th1 or Th2 cells. The balance between these T lymphocyte and cytokine pathways plays a role in modulating the immune response between proinflammatory and tolerance promoting mechanisms. Cytokines TGF-β Immune Tolerance Allergy Asthma Atopic dermatitis Exaggerated Response Autoimmunity Chron disease 11

Chronic Disease Prevalence
November 8, 2018 Chronic Disease Prevalence in the Last 50 Years & the Hygiene Hypothesis This sharp decrease in infectious diseases has been associated with a similar increase in various types of autoimmune or immunologic disorders. Incremental increases in both Th1 (auto-immune type) mediated and Th2 (allergic type) mediated diseases have been documented. The association between a decrease bacterial exposure and an increase in abnormal immunologic conditions has led to the “hygiene hypothesis” which suggests that the lack of immunologic experience by the host could lead to a disordered immune response, and in turn, to an increase in immune related conditions. Bach JF. N Engl J Med. 2002;347: 12 12

Bacteria Support Gut Barrier and Balance Immune Response
November 8, 2018 Bacteria Support Gut Barrier and Balance Immune Response Gut microflora help support gut barrier function: ↑ Mucin production ↓ Permeability Gut microflora help support the adaptive immune response: Generate IgA activity (humoral) Balance in T helper cell subclasses (cellular) Research over the last years has demonstrated that intestinal bacteria help support gut barrier and immune function by mechanisms listed on the slide. A number of clinical trials, as well as review articles have identified and summarized the effects of microflora on gut immunity and barrier function. Data from several of the trials with infants will be presented on subsequent slides. Isolauri E., et al. Am J Clin Nutr 2001;73(suppl):444S-450S. Saavedra JM. Nutr Clin Pract 2007; 22: 13 13

Development of Intestinal Microbiota

November 8, 2018 Intestinal Flora and Feeding Type (%) Bifidobacteria are generally predominant in breast fed infants Breast fed Formula fed Bifidobacteria Bacteroides Coliforms Harmsen HJM et al.JPGN 2000;30:61

The intestine is sterile at birth
Where do bacteria come from?

Birth Promote Establishment of “healthy” intestinal flora
Vaginal delivery Breast feeding Promote Establishment of “healthy” intestinal flora Predominance of bifidobacteria An infant is born with a sterile gut lumen, which rapidly becomes colonized. Vaginal delivery and exposure of the infant to maternal bacteria during birth helps determine which microorganisms may be the first to colonize the newborn gut. Breastfeeding, which contains both bacteria as well as growth factors (galacto-oligosaccharides) for certain species of bacteria, promotes a balanced and healthy intestinal flora which is predominant in certain species of bacteria, particularly bifidobacteria. Birth by C-section, which is a sterile procedure, feeding of formula that is also sterile, early antibiotic treatment and environments contaminated with microorganisms that would not typically be a part of maternal flora or a home environment, all hinder the establishment of a healthy balance of intestinal flora. The establishment of a healthy intestinal flora is ultimately critical in the development of an adequate gut barrier function and adequate immune response. Development of adequate gut barrier function Development of adequate immune response

The intestine is sterile at birth
Vaginal delivery is NOT a sterile procedure Breast ,milk is NOT a sterile fluid

Bifidobacterial Counts in Breast Milk
Log 10 number of bifidobacteria per ml Results from a recent analysis of breast milk samples indicates that breast milk contains bifidobacteria, and specific Bifidobacterium species that may promote a healthy microflora development during infancy. In this study, breast milk samples were collected by manual expression from 20 mothers of one month old breast fed infants. Molecular techniques for analysis (PCR) were applied to samples, in triplicate, prior to calculating a mean value for bifidobacteria species identified. Mothers were interviewed about their previous month’s consumption of probiotic containing preparations. The average bifidobacteria level was 2.64 log cells. In three samples, levels were below the detection limit of the methodology used. However, Bifidobacterium longum was identified in all other samples. Bifidobacterium animalis (of which B. lactis is a sub-species) was the second most prevalent species, detected in 74% of cases. There were no differences between bifidobacteria levels in breast milk samples from mothers that had consumed probiotic containing products (n=13), and those that had not (n=7). Given that bifidobacteria are a strictly anaerobic genus, it is unlikely that bifidobacteria detected were derived from the skin. The authors suggest that these results, in conjunction with other reports of lactic acid bacteria identified in breast milk, point to breast milk, per se, as the source of the bifidobacteria. Mean = 2.64 log cells; 95% CI: Gueimonde M., et al. Neonatology 2007;92:64-66.

Bifidobacterium species in breast milk
November 8, 2018 Bifidobacterium species in breast milk Bifidobacterium species-composition in breast-milk (20 samples by PCR using species-specific primers Gueimonde M et al ESPGHAN 2007

Birth Promote Hinder Establishment of “healthy” intestinal flora
C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Promote Hinder Establishment of “healthy” intestinal flora Predominance of bifidobacteria In review, an infant is born with a sterile gut lumen, which rapidly becomes colonized. A number of factors promote or hinder the establishment of a “healthy” intestinal flora in which bifidobacteria predominates. The probiotic bifidobacteria has been shown to benefit the host by promoting a “healthy” intestinal flora that is associated with gut barrier function and immune response. The establishment of a healthy intestinal flora is ultimately critical in the development of an adequate gut barrier function and adequate immune response. What Development of adequate gut barrier function Development of adequate immune response

Morbidity in Children Born via Cesarean Delivery (Odds Ratio)
November 8, 2018 Morbidity in Children Born via Cesarean Delivery (Odds Ratio) Gastroenteritis 1.31 1.24 1.38 Asthma 1.31 1.23 1.4 Gastroenteritis + Asthma 1.74 1.36 2.23 1 2 Decreased risk Increased risk Hakansson S et al. Clin Exp Allergy 2003; 33:

Food Allergy to egg confirmed by testing at age 1 – 2.
November 8, 2018 Influence of Cesarean Delivery on Relative Risk of Childhood Food Allergy * CI ( ) CI ( ) Several studies have shown a significant association between the occurrence of allergy and infants born by C-section. Data presented in this slide are specific for children with confirmed reactions to egg allergy testing between one and two years of age. Results from this study, of 2803 children from a population-based birth cohort, show the increased odds ratio of allergy among children born vaginally to mothers with no history of allergy, compared to an increase of more than twofold in infants born vaginally to mothers with a history of allergy. A more than sevenfold increase in risk of allergy was reported for children born to mothers with a history of allergy of whom were also delivered by C-section. These relationships remained significant after adjusting for confounding variables including maternal smiling, short-term breast-feeding, older siblings, and others. Results from this study indicate that Cesarean delivery is associated with subsequent food allergy, especially among predisposed children, supporting the theory that factors interfering with colonization may play an important role in the development of food allergy. THE BACTERIA THAT AN INFANT RECEIVES IN THE FIRST FEW MINUTES OF LIFE CAN AFFECT THEIR IMMUNE SYSTEM FOR YEARS *P<0.01; adjusted for covariates. Food Allergy to egg confirmed by testing at age 1 – 2. Eggesbo M., et al. J Allergy Clin Immunol 2003;112: 23

“Modern” Lifestyle Has Decreased Exposure to Bacteria
November 8, 2018 “Modern” Lifestyle Has Decreased Exposure to Bacteria Lower microbial exposure Cesarean sections Sterile formulas and foods Decrease in naturally fermented foods Increased hygiene measures Urban life Antibiotics Altered Intestinal microbiota Environmental changes in the industrialized world, as well as changes of life style in western societies, and medicine, have reduced the extent of microbial contact at an early age. These changes affect the initial establishment of the microflora. The hygiene hypothesis, first proposed by Strachan in 1989, offered, in part, an explanation to the increasing prevalence of atopic conditions. The idea was based on epidemiological observations, suggesting an inverse correlation between family size and the prevalence of allergic rhinitis. Today, an extended hygiene hypothesis is emerging that suggests that the modern infant may lack interactions with specific bacteria that promote the development of an anti-inflammatory and tolerogenic immune environment. The initial composition of gut microflora, through exposure to bacteria, may be considered a key determinant in the development and regulation of immune factors. Inadequate immune response Rautava S., et al. JPGN 2004;38: Rook GA. Immunol Today 1998;19: 24

Birth Promote Hinder Establishment of “healthy” intestinal flora
C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Promote Hinder Establishment of “healthy” intestinal flora Predominance of bifidobacteria In review, an infant is born with a sterile gut lumen, which rapidly becomes colonized. A number of factors promote or hinder the establishment of a “healthy” intestinal flora in which bifidobacteria predominates. The probiotic bifidobacteria has been shown to benefit the host by promoting a “healthy” intestinal flora that is associated with gut barrier function and immune response. The establishment of a healthy intestinal flora is ultimately critical in the development of an adequate gut barrier function and adequate immune response. What Development of adequate gut barrier function Development of adequate immune response

Intestinal Flora: A Balanced Ecosystem
November 8, 2018 Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Pseudomonas Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli The intestinal microflora is a complex and balanced ecosystem, composed of 100s of species of bacteria that are important for the development of gut immune function. No single bacteria is necessarily completely harmful or completely helpful. Some bacteria could be potentially harmful and are generally considered “less beneficial;” these include pseudomonas, staphylococci and clostridia. Other bacteria are known to have potentially beneficial effects to the host, including inhibition or competition for potential pathogens, aiding in digestion and absorption, providing trophic factors for the epithelium, involvement in vitamin synthesis, and support of gut barrier function and modulation of the gut immune response of gut-associated lymphoid tissue (GALT). Amongst the potentially beneficial bacteria are multiple species of the genus Lactobacillus and the genus Bifidobacterium. Lactobacilli and bifidobacteria are resident in most human hosts in varying numbers. Streptococci Eubacteria Bifidobacteria Bacteroides Adapted from: Gibson GR. J Nutrition 1995;125: 26

Intestinal Flora: A Balanced Ecosystem
November 8, 2018 Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Pseudomonas Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli Lactobacilli and bifidobacteria are resident in most human hosts in varying numbers. The potential modification of the intestinal flora by increasing the amount of lactobacilli and bifidobacteria in the intestine, particularly in the small bowel, has led to the concept of probiotics: the INGESTION of microorganisms for the purposes of a benefit to the host. Eating beneficial bacteria (probiotics) to balance the intestinal flora and benefit the host is now well recognized Streptococci Potential Probiotic Bacteria (When Ingested) Eubacteria Bifidobacteria Bacteroides Adapted from: Gibson GR. J Nutrition 1995;125: 27

Birth Promote Promote Hinder Probiotic bifidobacteria
C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Probiotic bifidobacteria Promote Promote Hinder Establishment of “healthy” intestinal flora Predominance of bifidobacteria In review, an infant is born with a sterile gut lumen, which rapidly becomes colonized. A number of factors promote or hinder the establishment of a “healthy” intestinal flora in which bifidobacteria predominates. The probiotic bifidobacteria has been shown to benefit the host by promoting a “healthy” intestinal flora that is associated with gut barrier function and immune response. The establishment of a healthy intestinal flora is ultimately critical in the development of an adequate gut barrier function and adequate immune response. What Development of adequate gut barrier function Development of adequate immune response

November 8, 2018 Probiotics Nonpathogenic, live microorganisms in the food supply that, when consumed in adequate amounts, are capable of conferring a health benefit to the host Bifidobacteria Lactobacilli Yeasts (S. boulardii) Optional Speaker Notes: There is currently no formal or regulatory definition for probiotics or prebiotics. In general, probiotics are considered non-pathogenic live organisms that yield a benefit to the host. The most commonly used genera of probiotic bacteria include bifidobacteria and lactobacilli. Some would consider certain yeasts, like Saccharomyces boulardii, a probiotic. Prebiotics are substrates that selectively stimulate the growth of certain bacteria. Most prebiotics are non-digestible carbohydrates in the diet. The most common forms of prebiotics in the diet of infants and children include galacto-oligosaccharides (GOS) that are found in breast milk, and fructo-oligosaccharides (FOS) extracted from the chicory root, and other oligosaccharides found in fruits and vegetables. Most prebiotics are considered so based on the degree to which they enhance the growth of bifidobacteria in the host. “Bifidogenicity” is the most important characteristic that an oligosaccharide requires to be considered a prebiotic. 29 29

Probiotic Characteristics:
November 8, 2018 Probiotic Characteristics: Be nonpathogenic Be resistant to destruction by technical processing Be resistant to destruction by gastric acid and bile Adhere to or transiently colonize intestinal epithelial tissue Provide a measurable benefit to the host By definition, a probiotic should be non-pathogenic. Although any organism can be pathogenic in nature, the best-studied probiotics are expected not to cause disease in the host. Probiotics need to be viable in the product in which they are consumed. Therefore, they need to be resistant to the technical processing prior to consumption. Probiotics should remain viable after passage through the stomach and the small bowel, and therefore need to be resistant to gastric acid and bile digestion. Until recently, some considered “being of human origin” as a necessary characteristic for a bacterium to be considered a probiotic. That is no longer the case. In general, to have an effect, a probiotic bacterium needs to transiently colonize the intestinal epithelium. Once the intestinal flora of the host is well-established, the oral intake of bacteria will modify the composition of the flora and may have the various immunologic effects; however once consumption stops, the quantities of the specific probiotic species being ingested will decrease and most often disappear. Therefore, for maintaining its probiotic effects, most of these bacteria need to be consumed regularly and consistently. Most importantly, a probiotic bacterium can only be called a probiotic if a benefit to the host has been demonstrated. Not all bacteria that are ingested and survive digestion are necessarily probiotics. Teitelbaum JE and Walker WA. Ann Rev Nutr 2002;22: 30 30

November 8, 2018 So, Can we benefit the infant by feeding probiotic bacteria, and lead to clinical benefits ? IMPORTANT Not all bacteria in the diet are probiotics Not all probiotics are created equal Efficacy and safety are species - specific

Bifidobacterium lactis
November 8, 2018 Bifidobacteria Anaerobic, non motile, gram positive curved rods Produce acids: acetate and lactate Growth inhibited at pH of 5.5 Can survive intestinal digestion and appear in stool Constitute most of the microflora of breastfed infants Bifidobacteria have all the characteristics of a probiotic bacteria. Importantly, the genus Bifidobacterium constitute most of the microflora of breastfed infants. Amongst the bifidobacteria better studied for application as a probiotic is Bifidobacterium lactis. B. lactis is the short name for B. animalis subspecies lactis, previously also called B. bifidum, strain Bb12. Bifidobacterium lactis Nomenclature - B. lactis also: B. animalis sub-species lactis, B. bifidum, strain Bb12 32 32

November 8, 2018 Effects of B. lactis Supplementation on Gut Barrier Function and Immunity: Decreased gut permeability Increased tolerance-related cytokines Increased IgA secreting cells and secretory IgA B. lactis has also been shown in various studies to help support gut barrier function and immunity, including decreasing gut permeability, increasing cytokines associated with immunologic tolerance and increasing secretory IgA and IgA secreting cells. To determine the effect of B. lactis on gut mucosal barrier function, a recent case-controlled, blinded trial by Stratiki and colleagues (2007) measured intestinal permeability using the sugar absorption test. Seventy-five premature infants were randomized to a preterm formula supplemented with B. lactis, or to unsupplemented formula. The Lactulose/mannitol ratio in the B. lactis supplemented infants was significantly lower after 30 days than in the control group (p=0.003). Improved intestinal permeability has important health implications, especially in these high risk infants. Another recent double-blind, placebo-controlled trial assessed the influence of probiotic supplementation on cytokines associated with immunologic tolerance. Rautava and colleagues (2006), randomized 81 full term infants that were approximately 1 month old to a probiotic (L. rhamnosus GG and B. lactis) supplemented formula, or to unsupplemented formula. At 12 months, serum concentrations of the soluble innate microbial receptor (sCD14) were significantly higher in the probiotic supplemented infants than the control. Stimulation of CD14 by microbial products has been previously demonstrated to induce TGF-B production in other studies. At 12 months an approximate 6 fold increase in serum TGF-B2 was observed in the supplemented infants. Although not statistically significant, microbial stimulation in the intestine by probiotics could lead to augmented TGF-B2 responses both systemically, and on the mucosa as a result of enhanced signalling through CD14. Another finding in this study was that the number of cow’s milk-specific IgA secreting cells were significantly higher in the probiotic supplemented infants, compared to control. Results from an additional study evaluating B. lactis on IgA levels in young children (Fukushima, 1998) is on the next slide. Fukushima Y., et al. Int J Food Micorbiol 1998;42:39-44. Rautava S., et al. Pediatr Res 2006;60: Stratiki Z., et al. Early Hum Dev 2007 on-line at: 33 33

November 8, 2018 Clinical Benefits and Outcomes Associated with Probiotics in Infants and Children: Modification of intestinal microflora Treatment of acute diarrhea Prevention of acute diarrhea Decrease of antibiotic associated diarrhea Emerging evidence in Treatment and prevention of allergy Prevention of NEC Overall, it has been demonstrated that probiotics will modify intestinal microflora towards a more balanced composition, with a greater predominance of bifidobacteria and/or lactobacillus. As shown on the last few slides B. lactis supplementation to infants has resulted in increased counts in stool and increased number of infants being colonized with bifidobacteria. Specific probiotic bacteria have been effective in the treatment and prevention of acute diarrhea, and the greater part of the literature supports these benefits. Specific probiotics may decrease diarrhea associated with the intake of antibiotics, and there is emerging evidence for the benefits of specific probiotics in the treatment and prevention of allergy as well as the prevention of necrotizing enterocolitis. The next several slides discuss the risk factors for allergy, the relationship between allergy, immunity, and microflora and some clinical trials with probiotics in the treatment and prevention of allergy. 34 34

Probiotics for Prevention of Acute Diarrhea
November 8, 2018 Probiotics for Prevention of Acute Diarrhea * This study was the first to demonstrate a reduction in the incidence of acute diarrhea in a prospective randomized double-blind, placebo-controlled trial with a group of infants chronically hospitalized for management of long term conditions including, respiratory, orthopedic and neurologic problems, but no gastrointestinal disease or chronic use of antibiotics. The 55 enrolled infants, aged 5-24 months, were randomized to receive a standard infant formula or the same formula supplemented with Bifidobacterium bifidum (B. lactis) and S. thermophilus. During 17 months follow-up, 8 (31%) of the patients who received the control formula and 2 (7%) who received the supplemented formula developed diarrhea during the course of the study (p = 0.035). In this and other studies, B. lactis was supplemented to infant formula together with Streptococcus thermophilus. S. thermophilus was originally added to the formula to support viability of B. lactis. Specific probiotic effects of S. thermophilus have not been further documented. Additional results from this study are presented on the next slide. *P=0.035 Saavedra JM., et al. Lancet 1994;344: 35

Probiotics for Prevention of Acute Diarrhea
November 8, 2018 Probiotics for Prevention of Acute Diarrhea * The same first study showing a greater than 50% reduction in the incidence of acute diarrhea with B. lactis supplementation also showed a significant reduction in the incidence of shedding rotavirus. The excretion or shedding of rotavirus is a potential marker of the amount of viral replication (severity or duration of infection). The reduction in rotavirus shedding (percent of infants throughout the study that were regularly examined using ELYSSA) was noted independent of the occurrence of clinically evident acute diarrhea by the definition of the study. The reduction of a viral enteritis was a first clinical suggestion that the mechanism of action of probiotics is immune mediated rather than simply an increase in the number of potentially beneficial bifidobacteria versus other potential pathogenic bacteria in the intestinal lumen. Subsequent clinical and animal studies have suggested the improvement related to rotavirus enteritis with probiotics is associated with an increase in anti rotavirus secretory IgA. *P=0.025 Saavedra JM., et al. Lancet 1994;344: 36

Number of Scientific Publications on Probiotics
November 8, 2018 Number of Scientific Publications on Probiotics

Prevention of Atopic Dermatitis with Probiotics
November 8, 2018 Prevention of Atopic Dermatitis with Probiotics * * RR=0.57 CI ( ) RR=0.51 CI ( ) The occurrence of differences in intestinal flora in infants with allergic disease has led to the use of probiotic agents for the management of atopy. In a double-blind, randomized placebo-controlled trial, probiotics were given prenatally to mothers who had at least one first-degree relative (or partner) with atopic eczema, allergic rhinitis, or asthma, and postnatally for 6 months to their infants. Atopic eczema was subsequently diagnosed in 35% of the 132 children aged 2 years. Results from this study, which has now followed children for more than four years, showed that the consumption of probiotics, in this case Lactobacillus rhamnosus GG, in infants born to families with a history of allergy, significantly decreased atopy diagnosis at the two-year and four-year follow up period. *P<0.05 Kalliomäki M., et al. Lancet 2001; 357: Kalliomäki M., et al. Lancet 2003; 361: 38

*P=0.01 compared to EHF; SCORAD range in parenthesis.
November 8, 2018 Treatment of Atopic Disease with Probiotics ( ) * Probiotics have also been used for allergy treatment. In in a randomized double-blind placebo-controlled study, 27 infants who manifested atopic eczema during exclusive breast-feeding were weaned to an extensively hydrolyzed formula, or to the same formula supplemented with Lactobacillus rhamnosus GG or Bifidobacterium lactis. The severity of atopic dermatitis (SCORAD) after two months of intervention was significantly better in infants receiving the probiotic supplemented formula, than control. The formula containing B. lactis showed the most improvement in SCORAD. Results this study provided one of the first clinical demonstrations of specific probiotic strains modifying the changes related to allergic inflammation. * ( ) ( ) *P=0.01 compared to EHF; SCORAD range in parenthesis. Isolauri E., et al. Clin Exp Allergy 2000;30: 39

November 8, 2018 Meta-analyses of Randomized Clinical Trials on Probiotic Treatment of Acute Diarrhea CI (19 – 42) CI (10 – 27) CI (14 – 26) CI (7 - 29) This slide depicts results from four meta analyses addressing the use of probiotics for acute diarrhea. As shown, the average reduction in duration in diarrhea varies from approximately fifteen to thirty hours of less duration with probiotics compared to placebo. Most of the clinical trials in these analyses are in pediatric patients; specifically, the meta analysis of Allen 200 is the only one including adult patients. These meta analyses include studies that used multiple probiotic bacteria in various forms. These include, among others, Lactobacillus rhamnosus GG, L. acidophilus, L. bulgaricus, L. reuteri, and various strains of bifidobacteria. Thus, this meta-analysis provides evidence of the efficacy of probiotic supplements in reducing the duration of acute nonbacterial diarrhea by approximately 1 day. . 8 RCTs 12 RCTs 9 RCTs 18 RCTs Adapted from: Szajewska H., et al. JPGN 2006;42: 40

Reduction in incidence (%)
Probiotics for Prevention of Acute Diarrhea * B. lactis LGG Other * * Reduction in incidence (%) * * This slide depicts individual randomized controlled clinical trials in infants and children using various probiotic bacteria in the prevention of acute enteritis. The specific bacteria which have been most commonly used in these trials include B. lactis, L. rhamnosus GG, and other lactobacilli. The reduction in incidence, similar to that seen for treatment of diarrhea, varies significantly among studies, from approximately 15-75%. In most of these studies, the greatest effect has been reported for an effect of probiotics on rotavirus enteritis. * *P<0.05 compared to incidence in control of each study.

Probiotics for Prevention of Antibiotic Associated Diarrhea
November 8, 2018 Probiotics for Prevention of Antibiotic Associated Diarrhea Controlled Clinical Trials * NS B. lactis LGG Other RR=0.29 CI ( ) RR=0.32 CI ( ) NS * % Reduction in incidence RR=0.47 CI ( ) RR=0.52 CI ( ) Several clinical trials have reported efficacy of probiotics for antibiotic-associated diarrhea. This slide summarizes various prospective controlled clinical trials using probiotic bacteria for prevention of antibiotic-associated diarrhea. Percent reduction in incidence of antibiotic-associated diarrhea ranged from 4% to 71%. Due to study design and other methodological variables, results have varying levels of statistical significance. Similar to those studies addressing treatment or prevention of acute viral diarrhea, no study to date has shown an increase (statistically significant or not) of any type of diarrhea with the use of a probiotic. NS RR=0.96 CI ( ) P<0.05 42

November 8, 2018 Premature Infants: Prime set-up for an Altered Microbiota and its potential consequences C-section birth Less chances of being breast fed NICU microbes Antibiotics Delayed establishment of microbiota Aberrant composition B. lactis has also been shown in various studies to help support gut barrier function and immunity, including decreasing gut permeability, increasing cytokines associated with immunologic tolerance and increasing secretory IgA and IgA secreting cells. To determine the effect of B. lactis on gut mucosal barrier function, a recent case-controlled, blinded trial by Stratiki and colleagues (2007) measured intestinal permeability using the sugar absorption test. Seventy-five premature infants were randomized to a preterm formula supplemented with B. lactis, or to unsupplemented formula. The Lactulose/mannitol ratio in the B. lactis supplemented infants was significantly lower after 30 days than in the control group (p=0.003). Improved intestinal permeability has important health implications, especially in these high risk infants. Another recent double-blind, placebo-controlled trial assessed the influence of probiotic supplementation on cytokines associated with immunologic tolerance. Rautava and colleagues (2006), randomized 81 full term infants that were approximately 1 month old to a probiotic (L. rhamnosus GG and B. lactis) supplemented formula, or to unsupplemented formula. At 12 months, serum concentrations of the soluble innate microbial receptor (sCD14) were significantly higher in the probiotic supplemented infants than the control. Stimulation of CD14 by microbial products has been previously demonstrated to induce TGF-B production in other studies. At 12 months an approximate 6 fold increase in serum TGF-B2 was observed in the supplemented infants. Although not statistically significant, microbial stimulation in the intestine by probiotics could lead to augmented TGF-B2 responses both systemically, and on the mucosa as a result of enhanced signalling through CD14. Another finding in this study was that the number of cow’s milk-specific IgA secreting cells were significantly higher in the probiotic supplemented infants, compared to control. Results from an additional study evaluating B. lactis on IgA levels in young children (Fukushima, 1998) is on the next slide. Inadequate GALT development and maturation Decreased gut barrier (mucin, permeability) Poor humoral and cellular immune response 43 43

Prevention of NEC with Probiotics in Premature Infants
November 8, 2018 Prevention of NEC with Probiotics in Premature Infants Reduction in Relative Risk * * RR=0.25 CI ( ) RR=0.20 CI ( ) RR=0.50 CI ( ) In humans, there are several published studies suggesting that probiotic supplementation can decrease the incidence of NEC. Three prospective studies are identified on this slide. The double-blind study by Dani and colleagues (2002) randomized 585 premature infants from12 NICUs, to receive Lactobacillus GG or placebo from the time of the first feed until discharge. The probiotic supplemented group was found to have a lower incidence of urinary tract infections and NEC than the control group; however, the difference was not statistically significant Bin-Nun and colleagues (2005) randomized 145 very low birth weight neonates (<1500g) to receive either a combination of bifidobacteria (Bifidobacteria infantis, Bifidobacteria bifidus, and Streptococcus thermophilus) or no probiotic supplement. The incidence of NEC was reduced from approximately16.0% in the control infants to 4% in the supplemented infants. Three of the fifteen babies who developed NEC died, and all of the deaths occurred in the non-supplemented group. Lin and others (2005) randomized 367 VLBW infants to either Lactobacillus acidophilus and Bifidobacterium infantis, or placebo from approximately day 7 of life until discharge. Results from these trials provided further support for the potential use of probiotics in reducing the risk of NEC. B. infantis, B. bifidus & S. thermophilus LGG L. acidophilus & B. infantis Bin-Nun A., et al. J Pediatr 2005;147: Dani C., et al. Biol Neonate 2002;82: Lin HC., et al. Pediatrics 2005;115:1-4. *P<0.05 44

Probiotics and mortality in prematurity

Safety of Bifidobacteria
November 8, 2018 Safety of Bifidobacteria No pathogenic characteristics Hundreds of thousands of metric tons of dairy products and supplements consumed annually Infant formula with B. lactis: 15 years, 30 countries Not a single documented episode of bifidobacteria infection More than 15 clinical trials involving more than 1,800 infants, half receiving B. lactis B. lactis is GRAS (Generally Regarded as Safe) and FDA has authorized for use in infant formula since birth Of probiotics currently being commercialized for infants, B. lactis has the longest safety track record. Because B. lactis is commercialized in food, including infant formula, it needs to have explicit authorization from FDA; and is considered GRAS. This is not the case for probiotics sold as supplements. Drugs – to be commercialized need to show a benefit in treating or preventing a condition and have side effects listed in the package. Food ingredients - to be used in infant formula can have no side effects at all to be considered GRAS. Thus, the safety bar is actually higher than that for drugs. Supplements - like probiotic capsules, do not require any claims oversight or GRAS status form FDA. 46 46

Birth Promote Promote Hinder Probiotic bifidobacteria
C- section Formula feeding Antibiotic use Environment (hospital & sterility) Vaginal delivery Breast feeding Probiotic bifidobacteria Promote Promote Hinder Establishment of “healthy” intestinal flora Predominance of bifidobacteria In review, an infant is born with a sterile gut lumen, which rapidly becomes colonized. A number of factors promote or hinder the establishment of a “healthy” intestinal flora in which bifidobacteria predominates. The probiotic bifidobacteria has been shown to benefit the host by promoting a “healthy” intestinal flora that is associated with gut barrier function and immune response. The establishment of a healthy intestinal flora is ultimately critical in the development of an adequate gut barrier function and adequate immune response. What Development of adequate gut barrier function Development of adequate immune response

Birth, Nutrition and Health
The development of a balanced intestinal flora is determined by the way we are born and how we feed infants the first few days of life- And has long term health consequences

Infant Feeding Options
November 8, 2018 Infant Feeding Options Human milk Well tolerated protein Non antigenic Not sterile “Modern formula” Less well tolerated intact cow proteins Antigenic Sterile Time to change the paradigm? 49 49

ADDITIONAL BACKUP SLIDES

Allergy Most common manifestation of immunologic (exaggerated) dysfunction Increasing incidence Role of food antigens Role of nutrition in primary prevention early in life

Role of Nutrition in Risk Reduction
Clinical trials show that specific extensive hydrolysates as well as specific partial hydrolysates can reduce the risk of developing allergies Most clinical trials (not all) have been done in infants at risk Current recommendations are directed at infants “at risk” How do we identify infants “at risk”

Family History as Allergy Predictor
Specificity of 86-91%* Sensitivity of 17-22%* Therefore, most infants at risk for atopic disease can not be identified * Bergmann et al., 1997 Clinical and Experimental allergy 27:

Prevalence of atopic disease in infants by age 2 according to the history of atopic disease (life-time prevalence) in their parents Prevalence of Parental Atopic History: % No Parental Hx % Uniparental 5% Biparental Prevalence within group: 10-15% 15-30% 30-40% Other refs: Halken S et al. Allergy 2000;55: Bousquet J. et al. J Allergy Clin Immunol 1986;78: ; Kjellman N. et al. Acta Paediatr Scan 1977;66: Exl BM, Nutr Res 2001;21: Bergman, et al. Clinical and Experimental Allergy 1998; 28:

Prevalence of atopic disease in infants by age 2 according to the history of atopic disease (life-time prevalence) in their parents Prevalence of Parental Atopic History: % No Parental Hx % Uniparental 5% Biparental (10-15%) (20-30%) (30-50%) Prevalence within group: 10-15% 15-30% 30-40% Other refs: Halken S et al. Allergy 2000;55: Bousquet J. et al. J Allergy Clin Immunol 1986;78: ; Kjellman N. et al. Acta Paediatr Scan 1977;66: Exl BM, Nutr Res 2001;21: Bergman, et al. Clinical and Experimental Allergy 1998; 28:

Prevalence by group: 9.6% 6.4% 1.6% Total Prevalence: 17.6%
Prevalence of atopic disease in infants by age 2 according to the history of atopic disease (life-time prevalence) in their parents Prevalence of Parental Atopic History: % No Parental Hx % Uniparental 5% Biparental (10-15%) (20-30%) (30-50%) Prevalence within group: 10-15% 15-30% 30-40% Prevalence by group: 9.6% % % Total Prevalence: 17.6% Other refs: Halken S et al. Allergy 2000;55: Bousquet J. et al. J Allergy Clin Immunol 1986;78: ; Kjellman N. et al. Acta Paediatr Scan 1977;66: Exl BM, Nutr Res 2001;21: Bergman, et al. Clinical and Experimental Allergy 1998; 28:

> 50% of children at risk for atopy do not have a family history
Prevalence of Parental Atopic History: % No Parental Hx % Uniparental 5% Biparental Prevalence within group: 10-15% 15-30% 30-40% Prevalence by group: 9.6% % % > 50% of children at risk for atopy do not have a family history

50% of children at risk for atopy do not have
Prevalence of Parental Atopic History: % No Parental Hx % Uniparental 5% Biparental Prevalence within group: 10-15% 15-30% 30-40% Prevalence by group: 9.6% % % 50% of children at risk for atopy do not have a family history For those who have a family history, there is no mandatory, standardized nor validated mechanism or tool to elicit parental history

Prevalence of Parental Atopic History:
Prevalence of Parental Atopic History: % No Parental Hx % Uniparental 5% Biparental Prevalence within group: 10-15% 15-30% 30-40% Prevalence by group: 9.6% % % One in every three infants is born to a family with a history of atopic disease

In summary The vast majority of infants at risk for developing allergy can not identified in standard clinical practice One in every three infants is at risk by family history Recommendations for primary prevention based on identifying “infants at risk” are impractical and ineffective

B. Lactis safety Facts The safety of B. lactis is documented based on multiple sources of evidence Thorough understanding of the organism itself, its bacterial properties, including its full genome: No invasive or identifiable pathogenic potential. No undesirable metabolic characteristics (no D- lactate , no toxin production, etc.). Bifidobacteria are the species most commonly found in the GI tract of breast fed infants Bifidobacteria are regularly found in human breast milk in significant amounts

B. Lactis safety Facts B) Not a single documented bacteremia or infection associated with ingestion of bifidobacteria in general or B. lactis specifically, despite extensive history of use C) History of safe use Hundreds of thousands of metric tons of B. lactis containing products are consumed globally each year Infant formula with B. lactis has been commercialized for more than 15 years, in more than 30 countries No adverse effects documented with free use in the food supply nor in infant formula

B. Lactis safety Facts D) Safety in clinical trials
Not a single adverse effect reported in more than 60 controlled human clinical trials with B. animalis and specifically B. animalis sub-species lactis in humans, including premature infants B. lactis is currently the only bacteria with GRAS status for routine use in infants from birth After through evaluation and assessment , B. lactis has been approved by the FDA, for use in routine infant formula from birth.

Probiotic Quality Facts
Quality assurance criteria are different for supplements, foods, and drugs Foods are strictly regulated, and infant formula much more so. They contain the species stated in the label in the amounts stated by the manufacturer In the US, a drug is a product specifically commercialized to prevent or treat disease. A supplement is a dietary component that does not replace part of a normal diet

Probiotic Quality Facts
In the US, a product commercialized to prevent or treat disease is a drug; and can be commercialized even if it produces side effects (as long as the side effects outweigh the benefits) In the US a product commercialized as a food must have NO side effects Thus, in practice, the “safety” bar is in fact higher for a food than for a drug

Summary Although much remains to be learned, the safety and efficacy documentation to date support the statement that B. lactis, incorporated into the diet, and at the levels found in infant formula in the US, “helps support a healthy immune system” Use of certain probiotic bacteria, including B. lactis, have been shown to have specific prophylactic and therapeutic effects. However, in the US they are not commercialized as drugs (for treatment or prevention of disease )

Infant Feeding and Microflora
Breast feeding: Promotes a Bifidobacterium rich microflora Presence of bifidobacteria in milk Presence of oligosaccharides that encourage bifidobacteria growth Formula feeding: Less bifidobacteria present in microflora Not the predominant organism Optional Speaker Notes: Breastfeeding and formula feeding profoundly affect the type of flora which colonizes the newborn gut in the first several days of life. Breastfed babies, in general, have a significantly higher percentage of bifidobacteria compared to bacteroides, coliforms and other species. Formula fed babies will have variable amounts of these species and bifidobacteria will not tend to be the predominant species. Factors likely responsible for a higher bifidobacteria level in breastfed infants include: - the presence of specific bacteria in maternal milk, including bifidobacteria. - the presence of specific oligosaccharides (galacto-oligosaccharides) that favor the growth of bifidobacteria in the intestinal lumen. - up to 30% of the carbohydrate in mature maternal milk is composed of non-digestible carbohydrates, including galacto-oligosaccharides. The selective effect on the growth of bifidobacteria by galacto-oligosaccharides present in maternal milk is considered to be a prebiotic effect (discussed in more detail in a later slide). Breast milk contains bifidobacteria and specific Bifidobacterium species that may promote healthy microbiota development. The next 2 slides show results from studies in which the microflora among infants that were breast and formula fed were analyzed. Swanson D. Indigenous Flora. In Feigin RD et al., eds. Textbook of Pediatric Infectious Diseases. 5th ed. Philadelphia: WB Saunders;2004: Gueimonde M., et al. Neonatology 2007;92:64-66.

Postnatal Morbidity as a Result of Delivery Mode
November 8, 2018 Postnatal Morbidity as a Result of Delivery Mode P = 0.01 P = 0.02 P<0.0001 P<0.0001 P<0.0001 P<0.0001 n = 4463 n = 676 n = 1349 n = 1149 n = 311 n = 411 n=26975 n = 747 n = 6271 n = 7370 n = 323 n = 1783 1 month after birth 1 year after birth Chang JH et al. Acta Paediatrica 2006; 95:

Neonatal Response to Colonization
November 8, 2018 Neonatal Response to Colonization 10 20 30 40 50 60 70 80 90 100 110 2 4 6 8 12 14 16 18 22 24 26 28 32 34 36 38 42 44 Age in days Percent of infants with detectable salivary A Later Colonization Initial Colonization J. Pediatr. 1968; 72:685.

Bifidobacteria and Total Bacterial Cells at 1 Month of Age (95% CI)
November 8, 2018 Bifidobacteria and Total Bacterial Cells at 1 Month of Age (95% CI) * 120 x 109 Vaginal delivery Cesarean delivery 100 x 109 80 x 109 * 60 x 109 40 x 109 20 x 109 2 x 109 Intestinal infections are often first mediated by adhesion of pathogenic bacteria to mucosal surfaces in the GIT. The purpose of this in vitro study was to determine if probiotics could inhibit pathogen adhesion, and/or displace pre-adhered pathogens from various parts of the intestine of young (9-10 month old), healthy, piglets. Probiotic incubation of piglet mucus included B. lactis and LGG, due to their ability to inhibit pathogen adhesion to human intestinal mucus. Pathogenic bacteria, including Salmonella, Clostridium, and E. coli were tested due to their relevance in GIT infections and mortality in piglets. Results indicated that treatment of intestinal mucus with B. lactis and LGG, alone or in combination, significantly reduced the adhesion of the tested pathogens. Results for B. lactis and E. coli are shown here. Results presented on the left side of the chart indicated that treatment of intestinal mucus with B. lactis significantly reduced the adhesion of E. coli in all intestinal mucus tested. Transcending colon mucus inhibited the adhesion of E. coli by 38%.. Results from the right side of the chart show that pre-adhered E. coli was displaced significantly when all mucus was incubated with B. lactis. B. lactis was able to displace 49% of E. coli pre-adhered in the small intestine (ileum) mucus. Results indicate that B. lactis has the ability to inhibit the adhesion and displace pre-adhered pig pathogens. 1 x 109 0 x 109 Bifidobacterium Total bacterial cells *P<0.001 Huurre A et al. Poster at ESPGHAN Congress, Dresden 2006.

Number of IgA-secreting Cells
November 8, 2018 Number of IgA-secreting Cells * * * Age (days) *P<0.05 Huurre A et al. Poster at ESPGHAN Congress, Dresden 2006.

Development of Atopic Disease During the First 2 Years of Life
November 8, 2018 Development of Atopic Disease During the First 2 Years of Life P = 0.04 P = 0.09 (n.s.) P = 0.04 P = 0.03 n = 1940 n = 401 n = 2002 n = 414 n = 1935 n = 397 n = 1681 n = 348 pig pathogens. Negele K et al. Pediatr Allergy Immunol 2004; 15:

Incidence of Allergic Manifestations
November 8, 2018 Incidence of Allergic Manifestations P = 0.007 P = P = 0.04 Renz-Polster H et al. Clin Exp Allergy 2005; 35:

Prevalence of bifidobacteria in Stools of Atopic and Healthy Infants
*** ** * Optional Speaker Notes: The increased occurrence of allergy in infants has also been associated with differences in intestinal flora. The prevalence of colonization with bifidobacteria in infants who developed allergy during the first 2 years of life was significantly less than for those that did not develop atopy. As shown, less than 30% of the atopic infants were colonized with bifidobacteria soon after birth, at 3 months, or at 1 year. Differences in intestinal flora in infants developing allergy suggest that factors in early life (such as sterile formula feeding and birth by C-section) may be a determinant in the later development of immunologic disease, including allergy. *P=0.02; **P=0.03; ***P=0.05 comparing prevalence at a given age Björksten B., et al. J Allergy Clin Immunol 2001;108:

Bifidobacteria Supplementation Can Increase Enteric Bifidobacteria
November 8, 2018 Bifidobacteria Supplementation Can Increase Enteric Bifidobacteria * Results from another double-blind, randomized controlled study, in which a number of formula fed infants provided B. lactis realized an increase in stool bifidobacteria, are presented here. In this study, 20 full-term infants were provided formula supplemented with B. lactis, and an equal number provided non-supplemented formula. 14 Breastfed infants served as controls. Results indicated a similar percentage of infants fed B. lactis formula were colonized with bifidobacteria (having > 10 (6) CFU/ml stool) as were the breast milk cohort of infants. A significantly lower percentage of infants were colonized with bifidobacteria after receiving standard formula with no probiotics. In this and other studies, B. lactis was supplemented to infant formula together with Streptococcus thermophilus. S. thermophilus has not typically been found to have a probiotic effect and in some of these formulations it was incorporated for the purposes of helping probiotic viability in the product. *P<0.05 compared to standard formula. Adapted from: Langhendries JP., et al. JPGN 1995;21: 76

Effect of B. lactis in Preterm Infants:
November 8, 2018 Effect of B. lactis in Preterm Infants: Improved Immune Secretory Function Placebo = 16 Probiotic = 19 * * *P<0.05. Mohan R. et., al. EUROBIO 2006; abstract.

Effect of B. lactis in Preterm Infants:
November 8, 2018 Effect of B. lactis in Preterm Infants: Improved Weight Gain Placebo = 32 Probiotic = 35 * * Singificantly increased weight gain with B. lactis compared to placebo; the effect is dependent on abx. *P<0.01. Mohan R., et. al., 2006 J Clin Microbio 2006;44:

Probiotics in Stage 2 NEC

Proposed as Beneficial
November 8, 2018 Ingestion of Bacteria Proposed as Beneficial Suggested that ingested bacteria could have positive influence on microflora in the intestinal tract Hypothesized that lactobacilli were important for human health and longevity Promoted yogurt and fermented foods as healthy The purposeful ingestion of bacteria for providing a benefit to the host was originally proposed by Elie Metchnikoff, an immunologist of whom won the Nobel Prize for describing phagocytosis. Metchnikoff’s treatise, The Prolongation of Life, is one of the first publications suggesting that the regular ingestion of fermented foods containing live microbes may be beneficial to the host. His writings were based on epidemiologic observations of Bulgarian peasants who consumed large amounts of yogurt and lived long lives. Metchnikoff postulated that the ingestion of lactic acid-producing bacteria would have a benefit to the host and support longevity. As seen on the next slide, consumption of bacteria in the food supply that bring a health benefit to the host is part of the definition of a probiotic. Elie Metchnikoff ( ) 80 80

Intestinal Microflora
November 8, 2018 Components of Gut Barrier and Gut Immune System Intestinal Microflora Complex “ balanced” flora Bacterial competition “balances” species Mucus (mucin) Part of barrier function Tight Junctions (between cells) Decrease gut permeability Immune Cells Gut associated lymphoid tissue (GALT) 70% of immune cells are located in the GI tract Innate: macrophages, white cells, mast cells Adaptive: T and B lymphocytes The gut barrier and gut immune function are fundamental for maintaining health of the host. Numerous layers of the gut immune system are involved. Bacteria diversity allows the possibility for certain beneficial species to compete with those considered less beneficial to help maintain a “healthy” bacterial balance. Secretion of mucus at the level of the epithelium, containing mucin, forms a critical barrier by trapping and decreasing the chances for pathogenic contact and translocation through the gut epithelium. Tight junctions, governed by the cellular activity of the epithelium, are a primary component of maintaining gut permeability. Looseness in tight junctions will increase gut permeability and allow the passage of macromolecules and other intestinal contents of the lumen. A vital component of gut barrier function relates to gut-associated lymphoid tissue (GALT). Lymphocytes in the villi, lamina propria, Peyer’s patches, and mesenteric lymph nodes are essential in preventing pathogens from entering the circulation (innate immunity) and GALT plays a major role in developing adequate defense mechanisms including antibodies (adaptive immunity) to potential pathogens. Intestinal associated lymphoid cells can form 70-80% of all immune cells in the body. The GI tract is, in that way, the largest immune organ of the body. 81 81

Training of the GALT has systemic immune effects
Bacteria “train” the GALT Immune effects are manifested systemicaly Antigens activate GALT 82

Diet and Environment: Early Determinants of Immune Response

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Diet and Environment: Early Determinants of Immune Response

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