November 27, 2018 Diet, Environment, and Intestinal Flora: Determinants of Long term Health José M. Saavedra, MD Associate Professor of Pediatrics Johns.

November 27, 2018 Diet, Environment, and Intestinal Flora: Determinants of Long term Health José M. Saavedra, MD Associate Professor of Pediatrics Johns Hopkins University School of Medicine & Medical and Scientific Director, Nestlé Nutrition

The gut lumen is part of the environment
Diet GUT

Adult Microbiota: A Complex Ecosystem
November 27, 2018 Adult Microbiota: A Complex Ecosystem Esophagus No own microbiota Microbes from food and oral cavity Stomach 104 CFU/g Candida albicans Helicobacter pylori Lactobacillus Streptococcus species Duodenum CFU/g Bacteroides Candida albicans Lactobacillus Streptococcus Jejunum CFU/g Bacteroides Candida albicans Lactobacillus Streptococcus Colon CFU/g Bacteroides Bacillus Bifidobacterium Clostridium Enterococcus Eubacterium Fusobacterium Peptostreptococcus Ruminococcus Streptococcus Ileum CFU/g Bacteroides Clostridium Enterobacteriaceae Enterococcus Lactobacillus Veillonella

Surface Areas of Exposure to the Environment
November 27, 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 27, 2018 GALT Gut associate lymphoid tissue (GALT) comprises 70-80% of immunologic cells in the body GALT is the largest immune organ

Role of Intestinal Microflora:
November 27, 2018 Role of Intestinal Microflora: Compete with other bacteria Fermentation of substrates Metabolism of proteins, bile acids Vitamin synthesis Support gut barrier function (non-immune factors) Modulate gut immune response Innate immunity Adaptive immunity Optional Speaker Notes: Microflora supports the gut barrier and immune functions through various mechanisms. Diversity of the ecosystem in the microflora is necessary so that one species may compete with another to decrease the possibility of one or two species being predominate, particularly when there is the chance that a pathogenic species could prevail. Microflora ferment substrates resulting in the generation of short chain fatty acids which serve as important trophic factor for the epithelium, especially in the distal gut. Intestinal microflora play an important role in the metabolism of proteins and bile acids and allow for normal enterohepatic circulation of bile acids. Bacteria synthesize vitamins, including B vitamins and vitamin K. Both gut barrier function and modulation of immune response help develop innate immunity in growing infants and enhance their adaptive immunity response, particularly through the formation of antibodies. One of the more important physiologic roles of a healthy intestinal microflora is the adequate support of gut barrier function and modulation of the gut immune response of gut-associated lymphoid tissue (GALT). Microflora are critical in modulating both the innate and adaptive immune systems. 6 6

Germ-free vs. Colonized Gut

Neonatal Response to Colonization
November 27, 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.

Antigens Microflora Th0 Intestinal Lumen Epithelium Intestinal Mucosa
November 27, 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. 9

Antigens Microflora Th0 Th2 Th1 Intestinal Lumen Epithelium
November 27, 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. 10

Antigens Microflora Th0 Th2 Th1 TNF-α IL-4 Cytokines IFN-γ IL-5 IL-2
November 27, 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. An exaggerated response of Th1 type cells will lead to autoimmune disorders; examples are Crohn’s disease and type 1 diabetes. Exaggerated response of Th2 will lead to allergic disease. Therefore, a balanced response of the Th1 and Th2 subsets is critical to decrease the chances that the immune system overreacts and lead to one of the more common autoimmune or allergic conditions that we are facing today in modern society. Cytokines Cellular Immunity Pathogen protection IgM, IgG response Autoimmunity Humoral Immunity IgE, IgA response Allergy Response 11

Bacteria Support Gut Barrier and Immune Function
November 27, 2018 Bacteria Support Gut Barrier and Immune Function 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) Optional Speaker Notes: 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: 12 12

Microbiota Dynamics During Life
November 27, 2018 Microbiota Dynamics During Life First describe the slide!! This is how a normal colonisation occurs... By the time of weaning, the flora is basically acquired and stable. The first bacteria to colonise a previously sterile gut may establish a permanend niche by inducing specific and lasting glycosylation of the glycocalyx as well as by modulating enterocyte gene expression, thus putting later arrivers at competitive disadvantage. Since colostral and breast milk contain numerous factors that favor early colonisation by bifidobacteria and lactobacilli, feeding formulas may alter implantation and long-term colonisation of these organisms. Mitsuoka et al., 1996

Intestinal Flora and Feeding Type (%)
November 27, 2018 Breast fed Formula fed Bifidobacteria Bacteroides Coliforms Harmsen HJM et al.JPGN 2000;30:61

Bifidobacterial Counts in Breast Milk
Log 10 number of bifidobacteria per ml Optional Speaker Notes: 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 27, 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

Determinants of Intestinal Flora in Newborns
November 27, 2018 Determinants of Intestinal Flora in Newborns Early diet (Breast Milk vs Formula) Breast milk is not sterile Contains specific species (bifidobacteria) Contains bifidogenic factors (prebiotic oligosaccharides) Birth by C-section (Sterile procedure) Microbial environment, antimicrobials, etc

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 Optional Speaker Notes: 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

Microflora Antigens Th0 TReg Th2 Th1 Tolerance IBD Allergy TNF-α IL-4
November 27, 2018 Microflora Antigens 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 TGF-β Over Expression Over Expression Tolerance IBD Allergy

Chronic Disease Prevalence in the last 50 Years
November 27, 2018 Chronic Disease Prevalence in the last 50 Years Decrease in infections is associated with increase immune disorders (Th1) (Th1) (Th1) (Th2) Bach JF. N Engl J Med. 2002;347:

Increasing Prevalence of Asthma and Atopy
November 27, 2018 Increasing Prevalence of Asthma and Atopy Ninan et al., 1992; BMJ 304:

*P<0.05; bivariate analysis.
November 27, 2018 Atopic Sensitization and Allergy Symptoms Among Children Living on Farms and Children in Same Rural Community from Non-farming Families * * * * *P<0.05; bivariate analysis. Braun-Fahrlander CH. Clin Exp Allergy 1999;29:28-34.

Food Allergy to egg confirmed by testing at age 1 – 2.
November 27, 2018 Influence of Cesarean Delivery on Relative Risk of Childhood Food Allergy * CI = Adjusted Odds Ratio CI = *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:

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. In an attempt to prospectively relate microflora to allergy development, stool samples from 44 newborn infants were analyzed during the first week of life, and at various time periods through the first year. 18 infants had developed atopic dermatitis and/or positive skin prick test results by 2 years of age. 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:

Risk Factors for the Development of Allergies:
November 27, 2018 Risk Factors for the Development of Allergies: Infants C-section Not breastfeeding Exposure to intact cow milk protein 4-fold increase in allergies if given 1st week of life Family hx of allergy Children Urban living Antibiotics first 2 years of life Family hx of allergy Optional Speaker Notes: Changes in our microbial environment, including a lower exposure to bacteria, are considered to be one of the risk factors for immune disease, and specifically for allergy. Birth by sterile C-section procedure and not breastfeeding (feeding of sterile formula) are examples. Exposure to antigens, particularly intact cow milk proteins which can significantly increase risk (especially if given in the first few days of life), and family history are all risk factors for allergy development. In older children, urban verses rural living, the use of antibiotics in the first two years of life, which also drastically modifies intestinal microflora, as well as family history of allergy, have been associated with development of allergic disease. Marini A., et al. Acta Pediatrica Suppl 1996;414:1-22. Braun-Fahrlander CH., et al. Clin Exp Allergy 1999;29:28-34. Eggesbo M., et al. J Allergy Clin Immunol 2003;112: Farooqi IS and Hopkin JM. Thorax 1998;53: Wickens K., et al. Clin Exp Allergy 1999;29: von Mutius E., et al. Clin Exp Allergy 2000; 30: 25 25

Flora and Immune Response
November 27, 2018 Flora and Immune Response Determinants of Intestinal Flora in newborns are the same as the determinants of exaggerated immune response (allergic reactions).

“Modern” Lifestyle Has Decreased Exposure to Bacteria
November 27, 2018 “Modern” Lifestyle Has Decreased Exposure to Bacteria Lower microbial exposure Sterile processed food Decrease in naturally fermented foods Increased hygiene measures Urban life Cesarean sections Antibiotics Altered Intestinal microbiota Inadequate immune response

Today’s “modern formula” for Non-breastfed Infants
November 27, 2018 Today’s “modern formula” for Non-breastfed Infants Sterile, intact cow milk protein formula as sole source of feeding Any alternatives?

Risk Factors for Allergy at 1 year of Age: Diet
November 27, 2018 Marini et al Acta Pediatrica 1996, 414:1-22

Cumulative Incidence of Atopic Manifestations Partially Hydrolyzed Whey Formula vs Cow Milk
80 Partially Hydrolyzed Whey Intact Cow Milk p<0.05 60 p=0.021 p<0.05 p<0.05 p=NS p<0.001 Cumulative Incidence of AM (%) 40 p=0.063 p<0.05 20 p=0.109 Becker 2004 Von Berg Exl 2000 Chandra Marini 1996 Vandenplas de Seta 1994 Willems Vandenplas 2003 1997 1995 1993 1988 * Graph depicts only published, peer-reviewed, prospective trials with data collection at timepoints ≤12 months. ** For all studies except Becker 2004, AM includes AD as one of the allergic outcomes assessed; for Becker 2004, AM refers to asthma alone. *** 4 months: Vandenplas 1988; 6 months: Exl 2000, De Seta 1994; 12 months: Becker 2004, Von Berg 2003, Chandra 1997, Marini 1996, Vandenplas 1995, Willems 1993 **** p-values in italics indicate that no p-value is reported in publication; p-value is based on calculated OR and CI

Cumulative Incidence of Atopic Dermatitis Partially Hydrolyzed Whey Formula vs Cow Milk Formula in Prevention Studies 60.0 Partially Hydrolyzed Whey Intact Cow Milk p<0.05 40.0 p=NS p=0.004 p<0.02 p<0.05 Cumulative Incidence of AD (%) p>0.05 20.0 p=0.048 p>0.05 0.0 Von Berg Chan 2002 Exl 2000 Chandra 1997 Marini 1996 Vandenplas Tsai 1991 Vandenplas 2003 1995 1988 * Graph depicts only published, peer-reviewed, prospective trials with data collection at timepoints ≤12 months. ** 4 months: Vandenplas 1988; 6 months: Exl 2000; 12 months: Von Berg 2003, Chandra 1997, Marini 1996, Vandenplas 1995, Tsai 1991 **** p-values in italics indicate that no p-value is reported in publication; p-value is based on calculated OR and CI

Cumulative Incidence of Atopic Dermatitis
November 27, 2018 Cumulative Incidence of Atopic Dermatitis 5 10 15 20 25 Birth 12mo 24mo 36mo Intact Cow Milk Partially Hydrolyzed Whey Extensively Hydrolyzed Whey Extensively Hydrolyzed Casein * 3 Yr. GINI study : von Berg et al JACI, 2007

November 27, 2018 Conclusions “The data confirm a long-term allergy preventive effect of hydrolyzed infant formula on atopic manifestations and atopic eczema until 6 years of age” J Aller Clin Immunol 2008; 121:

Intestinal Flora (A Balanced Ecosystem)
November 27, 2018 Intestinal Flora (A Balanced Ecosystem) Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Pseudomonas Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli As mentioned these bacteria in intestine for a complex ecosystem Represnted here major genus of bact Streptococci Eubacteria Bifidobacteria Bacteroides From: Gibson GR. J Nutrition 1995; 125:

Intestinal Flora (A Balanced Ecosystem) Potential Probiotic Bacteria
November 27, 2018 Intestinal Flora (A Balanced Ecosystem) Potentially Harmful Bacteria Diarrhea/constipation Infections Production of Toxins Pseudomonas Potentially Helpful Bacteria Inhibition of exogeneous and/or harmful bacteria Stimulation of immune functions Aid in digestion and/or absorption Synthesis of vitamins Proteus Staphylococci Clostridia Enterococci E. coli Lactobacilli As mentioned these bacteria in intestine for a complex ecosystem Represnted here major genus of bact Streptococci Potential Probiotic Bacteria (When Ingested) Eubacteria Bifidobacteria Bacteroides From: Gibson GR. J Nutrition 1995; 125:

Ingestion of Bacteria 3500 BC 2000 BC AD 2000
November 27, 2018 Ingestion of Bacteria Sumerians (Cheese) Celts & Huns (Khefir) Pasteurization Abraham (Milk & curds) Monks (Refine Fermentation) 3500 BC 2000 BC AD 2000 Optional Speaker Notes: Humans have ingested bacteria and have been exposed to a heavy microbial environment for thousands of years. The purposeful use of bacteria for food preservation more than 5000 years ago led to the large consumption of fermented foods. Fermentation of milk for manufacturing of cheese dates back to the earliest times of recorded history. Biblical references refer to the use of milk and fermented curds for human consumption. During the middle ages, the fermentation of fruits and milk led to a refinement in the production of these fermented foods. With the introduction of pasteurization in the last 100 years ingestion of bacteria as part of our food supply has dramatically changed. In the past 50 years, antibiotics became readily available which has also changed not only our microbial environment, but has significantly altered the intestinal flora of individuals, particularly children who consume antibiotics in the first two years of life. In the past 20 years, the reintroduction of potentially beneficial bacteria (the concept of probiotics) has gained attraction as a way to address, at least in part, the microbial imbalance of our current modern society. Antibiotics Probiotics In the last 100 years, we drastically changed our ingestion of microbes and our microbial environment. 39 39

November 27, 2018 PROBIOTICS Live microbial feed supplement which beneficially affects the host animal by improving its microbial balance. Bifidobacteria Lactobacilli

Promoted yogurt and fermented foods as healthy
November 27, 2018 Suggested that ingested bacteria could have positive influence on normal microbial flora in intestinal tract Hypothesized that lactobacilli were important for human health and longevity Promoted yogurt and fermented foods as healthy Elie Metchnikoff ( )

Probiotic Characteristics:
November 27, 2018 Probiotic Characteristics: Be nonpathogenic in nature 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 Optional Speaker Notes: 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: 42 42

Bifidobacterium lactis
November 27, 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 Optional Speaker Notes: 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 43 43

Bifidobacteria Supplementation Can Increase Enteric Bifidobacteria
November 27, 2018 Bifidobacteria Supplementation Can Increase Enteric Bifidobacteria * Optional Speaker Notes: 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: 44

Incidence of Diarrhea (%)
November 27, 2018 (p=0.035) Saavedra et al., 1994; The Lancet 344:

Rotaviral Shedding (%)
November 27, 2018 (p=0.025) Saavedra et al., 1994; The Lancet 344:

Published Scientific Papers on Probiotics
November 27, 2018 Published Scientific Papers on Probiotics 50 100 150 200 250 300 350 400 450 500 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Refer to slide. The number of publications on the topic of probiotics have risen exponentially…

November 27, 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 Optional Speaker Notes: 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. 48 48

November 27, 2018 Meta-analyses of Randomized Clinical Trials on Probiotic Treatment of Acute Diarrhea 95% CI = 95% CI = 95% CI = 95% CI = 8 RCTs 12 RCTs 9 RCTs 18 RCTs Adapted from: Szajewska H., et al. JPGN 2006;42:

RCTs on Prevention of Probiotics and Diarrheal Disease in Infants
November 27, 2018 RCTs on Prevention of Probiotics and Diarrheal Disease in Infants * B. lactis L GG Other * * * Reduction in incidence (%) * * *P<0.05 compared to incidence in control of each study.

RCTs on Prevention of Antibiotic Associated Diarrhea
November 27, 2018 RCTs on Prevention of Antibiotic Associated Diarrhea Reduction in Relative Risk * NS B. lactis L. GG Other CI ( ) CI ( ) * NS CI ( ) % r CI ( ) % Reduction in incidence NS CI ( ) P< p.0.05.

With Extensively Hydrolyzed Formula (EHF) and Probiotics
November 27, 2018 Treatment of Atopic Disease With Extensively Hydrolyzed Formula (EHF) and Probiotics ( ) * * Population: Eczema 4-6 mo on exclusive breast feeding N= 27, results at 2 months of intervention ( ) ( ) *P=0.01 compared to EHF; SCORAD range in parenthesis. Isolauri E., et al. Clin Exp Allergy 2000; 30:

November 27, 2018 Probiotics for the Prevention of Antibiotic Associated Diarrhea (AAD) in Infants * Optional Speaker Notes: The results of the Correa study, one of the studies shown on the previous slide, are highlighted here. In this double-bind, formula controlled study, 80 infants, 6 to 36 months of age were randomized to B. lactis supplemented formula, or placebo at the initiation of antibiotic therapy for 15 days. S. thermophilus, not thought to have a probiotic effect was added to the formula to help maintain viability of the B. lactis. Results indicated a near 2-fold reduction in the incidence of diarrhea in infants receiving the probiotic supplemented formula, compared to control. In most cases, probiotics have been concomitantly used with the prescription of an antibiotic and these have shown a reduction in incidence or duration of loose stools while on antibiotic therapy for various populations of infants and children. *P<0.05 Correa NBO., et al. J Clin Gastroenterol 2005;39: 53

B. lactis Fed to Infants Can Modify Intestinal Microflora:
November 27, 2018 B. lactis Fed to Infants Can Modify Intestinal Microflora: Increase counts of bifidobacteria in infants fed B. lactis Increase short chain fatty acids, and lower stool pH Decrease Clostridia and other Enterobacteriaceae Optional Speaker Notes: Due to its use and viability in the food supply, B. lactis has been shown to increase the counts of bifidobacteria when fed to infants. In a recent double-blind, placebo-controlled clinical trial, Mohan and colleagues (2006) randomized 69 perterm infants to a B. lactis supplemented formula, or unsupplemented formula for their first 21 days of life. Results from analysis of stool samples, collected weekly for 3 weeks, indicated that cell numbers of Bifidobacterium were significantly increased and Enterobacteriaceae and Clostridium significantly decreased in B. lactis supplemented infants, compared to control. In another report of the same cohort of infants (Mohan 2006), fecal pH was significantly lower in the B. lactis supplemented infants compared to control. Low pH facilitates the proliferation of lactic acid bacteria and bifidobacteria and inhibits growth of some pathogenic microorganisms. Reduced pH can result from increased short chain fatty acids, as seen by the increased fecal acetate in the Mohan (2006a) report. An earlier trial by Fukushima and colleagues (1997) evaluated the effect of a follow-up formula containing B. lactis on microflora and fecal metabolites in healthy children age months old. In this prospective trial, 7 toddlers consumed the supplemented formula for 21 days; stool was analyzed prior to supplementation, three times during the intervention, and again one week after cessation of the supplement. Stool analysis results indicated that Clostridia was significantly decreased during intake, and Bifidobacterium, strain Bb 12, was significantly increased, but returned to undetectable levels within one week after the supplementation period. In addition, changes in fecal metabolic products such as significantly decreased ammonia, and significantly increased short chain fatty acids (acetic acid) were found in stool samples during intake, but also returned to levels similar to before intake one week after supplementation. Fukushima Y., et al. Bioscience Microflora 1997;16:65-72. Mohan R., et al. J Clin Microbio 2006;44: Mohan R., et al. JPGN 2006;45:E36-E37(abstr). 54 54

November 27, 2018 Effects of documented with Probiotic Supplementation on Protective Gut Barrier Function and Immune Function Decreased gut permeability Increase d mucin production Increased IgA secreting cells and secretory IgA Increased natural killer cell tumor-killing activity Increased production of macrophages and activated phagocytosis Immune modulation towards antigen tolerancetolerance Optional Speaker Notes: 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: 55 55

Birth C-section Formula feeding Vaginal delivery Antibiotics
November 27, 2018 Birth C-section Formula feeding Antibiotics Environment Vaginal delivery Breast feeding Probiotic Bifidobacteria Positively promote Positively promote Hinder Establishment of Healthy Balanced Microflora Predominance of Bifidobacteria Adequate gut barrier function Adequate immune response

Intrauterine vs Postnatal Mature Gut

November 27, 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 Optional Speaker Notes: 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 58 58

Effect of B. lactis in Preterm Infants: Fecal Bacterial Groups
November 27, 2018 Effect of B. lactis in Preterm Infants: Fecal Bacterial Groups * * * * *P<0.05. Mohan R., et. al., 2006 J Clin Microbio 2006;44:

Effect of B. lactis in Preterm Infants: Improved Gut Maturation
November 27, 2018 Effect of B. lactis in Preterm Infants: Improved Gut Maturation Placebo = 32 Probiotic = 37 * ** Significant decrease in calprotectin, a marker of inflamation, in probiotic treated group. The difference in levels between infants also receiving the antibiotic was NS. *P<0.05; **P<0.01 Mohan R. et., al. EUROBIO 2006; abstract.

Effect of B. lactis in Preterm Infants:
November 27, 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 27, 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:

Prevention of NEC with Probiotics in Premature Infants
November 27, 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 ( ) Optional Speaker Notes: 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 this blinded trial 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 63

Probiotics in Stage 2 NEC

Probiotics in Sepsis

Probiotics in mortality

Probiotics and Time to Full Feeds

Specific Bifidobacteria and Lactobacilli
Mechanism of action Probiotics Specific Bifidobacteria and Lactobacilli Support Gut Barrier function throughout the GI tract Support GI and systemic immune function Extensive clinical research, including treatment of acute diarrhea, antibiotic associated diarrhea and NEC

Mechanism of action Prebiotics Probiotics
Specific Bifidobacteria and Lactobacilli Stimulate growth of multiple species of bacteria including some bifidobacteria which are already present in the colon Support Gut Barrier function throughout the GI tract Support GI and systemic immune function Support Gut Barrier function in the colon May supports and modulates immune function Extensive clinical research, including treatment of acute diarrhea, antibiotic associated diarrhea and NEC Limited clinical research Limited areas of benefit

Pre- and Probiotics Documented Clinical Evidence
Randomized Controlled Clinical Trials Simple search “Probiotic”, “Prebiotic” RCTs PubMed NLM,

November 27, 2018 Conclusions Probiotics are a concept that has clearly evolved to proof of purpose There is a theoretical rationale for introduction of safe bacteria to infant nutrition There are specific benefits consistently demonstrated by specific bacteria There are mechanisms described which explain the benefits Specific agents have excellent safety record

November 27, 2018 Summary Modulation of antigenic environment early in life appears to be key in the prevention and management of most chronic inflammatory diseases

Probiotics: Reported Mechanisms and Clinical Benefits in Pediatrics
November 27, 2018 Probiotics: Reported Mechanisms and Clinical Benefits in Pediatrics Mechanisms Clinical Benefits ↑Ratio of Bifidobacteria & Lactobacilli to pathogens Increased mucin production Enhanced gut permeability Modulation of gut immune response - ↑ humoral immunity (IgA and other antibodies) - Modulation of Th1/Th response towards antigen tolerance Balanced intestinal microflora ↓ duration of acute diarrhea ↓ incidence of acute diarrhea ↓ antibiotic associated diarrhea ↓ in severity & incidence of atopic disease ↓ NEC Gut Mucosa Gut Lumen GALT PROBIOTICS Saavedra JM NCP, 2007

November 27, 2018 Diet, Environment, and Intestinal Flora: Determinants of Long term Health José M. Saavedra, MD Associate Professor of Pediatrics Johns.

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November 27, 2018 Diet, Environment, and Intestinal Flora: Determinants of Long term Health José M. Saavedra, MD Associate Professor of Pediatrics Johns.

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Presentation on theme: "November 27, 2018 Diet, Environment, and Intestinal Flora: Determinants of Long term Health José M. Saavedra, MD Associate Professor of Pediatrics Johns."— Presentation transcript:

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