Probiotics, Enteric and Diarrheal Diseases, and Global Health

Abstract

Enteric and diarrheal diseases are a major worldwide cause of death among children under the age of 5. In this age group, diarrhea occurs 2.5 billion times annually1The United Nations Children's Fund (UNICEF)/World Health Organization (WHO)Diarrhoea: why children are still dying and what can be done. UNICEF, New York2009Google Scholar and causes 15% of childhood deaths.2Black R.E. Cousens S. Johnson H.L. et al.Global, regional, and national causes of child mortality in 2008: a systematic analysis.Lancet. 2010; 375: 1969-1987Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar Diarrheal diseases claim 59 million disability-adjusted life years, nearly all from children in low- and middle-income countries.3Lopez A.D. Mathers C.D. Ezzati M. et al.Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data.Lancet. 2006; 367: 1747-1757Abstract Full Text Full Text PDF PubMed Scopus (2086) Google Scholar Despite this enormous burden, these numbers fail to capture the full impact of enteric and diarrheal diseases. Early and frequent exposure to intestinal pathogens begins a cycle (Figure 1A) that affects digestion, nutrient absorption, growth, and immunity.4Guerrant R.L. Oria R.B. Moore S.R. et al.Malnutrition as an enteric infectious disease with long-term effects on child development.Nutr Rev. 2008; 66: 487-505Crossref PubMed Scopus (88) Google Scholar Repeated infections, with either overt diarrhea or subclinical enteropathy, produce acute and chronic undernutrition,5Checkley W. Buckley G. Gilman R.H. et al.Multi-country analysis of the effects of diarrhoea on childhood stunting.Int J Epidemiol. 2008; 37: 816-830Crossref PubMed Scopus (78) Google Scholar which leads to more frequent and severe infections.6Schorling J.B. McAuliffe J.F. de Souza M.A. et al.Malnutrition is associated with increased diarrhoea incidence and duration among children in an urban Brazilian slum.Int J Epidemiol. 1990; 19: 728-735Crossref PubMed Google Scholar Undernutrition contributes to 53% of childhood deaths7Bryce J. Boschi-Pinto C. Shibuya K. et al.WHO estimates of the causes of death in children.Lancet. 2005; 365: 1147-1152Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar and is the leading risk factor for poor health outcomes in childhood8Black R.E. Allen L.H. Bhutta Z.A. et al.Maternal and child undernutrition: global and regional exposures and health consequences.Lancet. 2008; 371: 243-260Abstract Full Text Full Text PDF PubMed Scopus (1027) Google Scholar; survivors are at risk for developmental deficits in growth, fitness, and cognition that persist into adulthood with devastating consequences.4Guerrant R.L. Oria R.B. Moore S.R. et al.Malnutrition as an enteric infectious disease with long-term effects on child development.Nutr Rev. 2008; 66: 487-505Crossref PubMed Scopus (88) Google Scholar These consequences have a multiplicative effect on calculations of disability-adjusted life years from diarrheal disease.9Guerrant R.L. Kosek M. Lima A.A. et al.Updating the DALYs for diarrhoeal disease.Trends Parasitol. 2002; 18: 191-193Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Fortunately, there are strategies to break this cycle, although each approach has limitations. Sustainable access to potable water and improved sanitation reduces pathogen exposure; a $70 billion annual investment would only begin to reduce the 2.5 billion people without these necessities by 2015.10Hutton G. Bartram J. Global costs of attaining the Millennium Development Goal for water supply and sanitation.Bull World Health Organ. 2008; 86: 13-19Crossref PubMed Scopus (20) Google Scholar Antimicrobial agents are effective against specific pathogens but are expensive and can exacerbate toxin-mediated diseases, disrupt the human microbiome, and induce antibiotic-associated diarrhea as well as drug resistance. Immunization with enteric vaccines can reduce the burden of severe diarrhea, but vaccines must be kept in the cold, only protect against specific pathogens, and are less effective in regions of high mortality. For example, the efficacy of the live, attenuated rotavirus vaccine against severe disease is only 48.3% in southeast Asia11Zaman K. Anh D.D. Victor J.C. et al.Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-blind, placebo-controlled trial.Lancet. 2010; 376: 615-623Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar and 39.3% in sub-Saharan Africa.12Armah G.E. Sow S.O. Breiman R.F. et al.Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial.Lancet. 2010; 376: 606-614Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Zinc reduces the propensity to develop recurrent diarrhea and oral rehydration solution attenuates overt symptoms of diarrhea and dehydration.13Walker C.L. Black R.E. Zinc for the treatment of diarrhoea: effect on diarrhoea morbidity, mortality and incidence of future episodes.Int J Epidemiol. 2010; 39: i63-i69Crossref PubMed Scopus (40) Google Scholar However, these approaches do not adequately address broader growth and developmental processes that could yield long-term benefits. Likewise, trials of therapeutics to reduce diarrhea severity, unplanned intravenous fluid administration, or duration of hospitalization fail to address the longer term, initially subclinical consequences of recurrent infections. Complementary outcome measures, including measures of growth and biomarkers of acute intestinal inflammation, barrier disruption, and impaired immunity, would provide greater insight into underlying pathology and therapeutic efficacy. Use of these measures could reduce acute, overt, as well as chronic, often unrecognized, intestinal diseases (Figure 1B). No single intervention is sufficient to eliminate the global burden of enteric and diarrheal diseases. Vaccines, for example, can protect against limited infectious agents, but immunization can be overwhelmed by heavily contaminated water. Multiple interventions could work synergistically, such as the combination of improved water and sanitation, vaccines, micro- and macronutrient provision, and selectively targeted antimicrobial therapy (eg, single-dose albendazole for intestinal helminths). Do current global health strategies use the best available interventions? One underexplored approach—probiotics—could combine favorable safety profiles with improved nutrition and microbiome function. Probiotics are live micro-organisms that confer a health benefit on the host,14The Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO)Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria. FAO/WHO, Basel, Switzerland2001Google Scholar and have been used to treat multiple gastrointestinal (GI) diseases.15Preidis G.A. Versalovic J. Targeting the human microbiome with antibiotics, probiotics, and prebiotics: gastroenterology enters the metagenomics era.Gastroenterology. 2009; 136: 2015-2031Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar Microbes are inexpensive to grow, and have the potential for rapid global scale-up. Is there compelling evidence to recommend developing probiotics-based strategies to complement current approaches against enteric and diarrheal diseases for children in developing countries? If so, what steps must be taken before these therapies are ready for clinical impact in the global health arena? In 1907, Russian Nobel laureate Elie Metchnikoff suggested that ingestion of microbes could benefit human health.16Metchnikoff E. The prolongation of life: optimistic studies. Springer, New York2004Google Scholar As we learned about multidrug-resistant pathogens and the role of the human microbiome in health and disease, numerous trials showed the safety of probiotics17Snydman D.R. The safety of probiotics.Clin Infect Dis. 2008; 46: S104-S111Crossref PubMed Scopus (91) Google Scholar and their beneficial outcomes in patients with various illnesses.18Shanahan F. Probiotics in perspective.Gastroenterology. 2010 Oct 19; ([Epub ahead of print])PubMed Google Scholar A systematic review that included 12 randomized, controlled trials (RCTs) in the Cochrane database (the majority from affluent countries) concluded that probiotics reduced the mean duration of acute diarrhea in children by 29.2 hours in a fixed-effects model and by 30.48 hours in a random-effects model.19Allen S.J. Okoko B. Martinez E. et al.Probiotics for treating infectious diarrhoea.Cochrane Database Syst Rev. 2004; (CD003048)Google Scholar Two meta-analyses that evaluated similar studies found statistically significant but modest reductions of diarrhea duration.20Huang J.S. Bousvaros A. Lee J.W. et al.Efficacy of probiotic use in acute diarrhea in children: a meta-analysis.Dig Dis Sci. 2002; 47: 2625-2634Crossref PubMed Scopus (204) Google Scholar, 21Szajewska H. Mrukowicz J.Z. Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children: a systematic review of published randomized, double-blind, placebo-controlled trials.J Pediatr Gastroenterol Nutr. 2001; 33: S17-S25Crossref PubMed Scopus (297) Google Scholar Although combination analyses of trials with microbes of different genera, species, strains, and doses provide limited information about specific therapeutic interventions, it is clear that many probiotics reduce the duration of acute diarrhea. There are studies of probiotics for enteric and diarrheal diseases targeting children in developing regions (Supplementary Table 1). The majority of RCTs studied acute gastroenteritis and reported modest reductions in diarrhea duration. However, the effects of probiotics were statistically equivalent to those of placebo in 25% of trials. Some of the negative results might be attributable to small sample size or administration of insufficient doses22Basu S. Chatterjee M. Ganguly S. et al.Efficacy of Lactobacillus rhamnosus GG in acute watery diarrhoea of Indian children: a randomised controlled trial.J Paediatr Child Health. 2007; 43: 837-842Crossref PubMed Scopus (30) Google Scholar, 23Basu S. Paul D.K. Ganguly S. et al.Efficacy of high-dose Lactobacillus rhamnosus GG in controlling acute watery diarrhea in Indian children: a randomized controlled trial.J Clin Gastroenterol. 2009; 43: 208-213Crossref PubMed Scopus (31) Google Scholar, 24Fang S.B. Lee H.C. Hu J.J. et al.Dose-dependent effect of Lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children.J Trop Pediatr. 2009; 55: 297-301Crossref PubMed Scopus (32) Google Scholar; each trial must be viewed in the context of the specific disease and probiotic strain analyzed. Two RCTs evaluated probiotics for children with persistent diarrhea and reported dramatic reductions in diarrhea duration—4.8 and 3.9 days in Argentina25Gaon D. Garcia H. Winter L. et al.Effect of Lactobacillus strains and Saccharomyces boulardii on persistent diarrhea in children.Medicina (B Aires). 2003; 63: 293-298PubMed Google Scholar and India,26Basu S. Chatterjee M. Ganguly S. et al.Effect of Lactobacillus rhamnosus GG in persistent diarrhea in Indian children: a randomized controlled trial.J Clin Gastroenterol. 2007; 41: 756-760Crossref PubMed Scopus (18) Google Scholar respectively. Two trials evaluated probiotics for diarrhea prevention; children in Peru had 13% fewer diarrheal episodes after 15 months of Lactobacillus rhamnosus,27Oberhelman R.A. Gilman R.H. Sheen P. et al.A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished Peruvian children.J Pediatr. 1999; 134: 15-20Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar whereas diarrhea frequency was reduced by 14% among children in India who received daily doses of Lactobacillus casei for 12 weeks, with a 12-week follow-up period.28Sur D. Manna B. Niyogi S.K. et al.Role of probiotic in preventing acute diarrhoea in children: a community-based, randomized, double-blind placebo-controlled field trial in an urban slum.Epidemiol Infect. 2010 Jul 30; ([Epub ahead of print])PubMed Google Scholar Few studies have examined markers of acute or chronic immunity or underlying intestinal function. Administration of Bifidobacterium bifidum and Streptococcus thermophilus increased numbers of CD4+ T cells in HIV-infected Brazilian children.29Trois L. Cardoso E.M. Miura E. Use of probiotics in HIV-infected children: a randomized double-blind controlled study.J Trop Pediatr. 2008; 54: 19-24Crossref PubMed Scopus (43) Google Scholar A similar effect was observed after administration of L rhamnosus to HIV-infected adults in Tanzania.30Irvine S.L. Hummelen R. Hekmat S. et al.Probiotic yogurt consumption is associated with an increase of CD4 count among people living with HIV/AIDS.J Clin Gastroenterol. 2010; 44: e201-e205Crossref PubMed Scopus (27) Google Scholar Tropical enteropathy was studied in Malawian children using urinary carbohydrate excretion; L rhamnosus failed to improve ratios of lactulose:mannitol excreted,31Galpin L. Manary M.J. Fleming K. et al.Effect of Lactobacillus GG on intestinal integrity in Malawian children at risk of tropical enteropathy.Am J Clin Nutr. 2005; 82: 1040-1045PubMed Google Scholar despite evidence that probiotics ameliorated GI permeability defects in children with atopic dermatitis in Germany.32Rosenfeldt V. Benfeldt E. Valerius N.H. et al.Effect of probiotics on gastrointestinal symptoms and small intestinal permeability in children with atopic dermatitis.J Pediatr. 2004; 145: 612-616Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar Probiotics improved growth among healthy children in Thailand33Nopchinda S. Varavithya W. Phuapradit P. et al.Effect of bifidobacterium Bb12 with or without Streptococcus thermophilus supplemented formula on nutritional status.J Med Assoc Thai. 2002; 85: S1225-S1231PubMed Google Scholar and Estonia,34Vendt N. Grunberg H. Tuure T. et al.Growth during the first 6 months of life in infants using formula enriched with Lactobacillus rhamnosus GG: double-blind, randomized trial.J Hum Nutr Diet. 2006; 19: 51-58Crossref PubMed Scopus (43) Google Scholar but not among HIV-exposed infants in South Africa.35Velaphi S.C. Cooper P.A. Bolton K.D. et al.Growth and metabolism of infants born to women infected with human immunodeficiency virus and fed acidified whey-adapted starter formulas.Nutrition. 2008; 24: 203-211Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar In Malawi, probiotics failed to improve nutrition status in severely malnourished children who received inpatient nutritional rehabilitation. Despite the negative primary outcome, there was a trend toward decreased mortality among children treated with probiotics on an outpatient basis.36Kerac M. Bunn J. Seal A. et al.Probiotics and prebiotics for severe acute malnutrition (PRONUT study): a double-blind efficacy randomised controlled trial in Malawi.Lancet. 2009; 374: 136-144Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Probiotics also reduced the duration of rotavirus shedding in India.37Narayanappa D. Randomized double blinded controlled trial to evaluate the efficacy and safety of Bifilac in patients with acute viral diarrhea.Indian J Pediatr. 2008; 75: 709-713Crossref PubMed Scopus (5) Google Scholar Strategies to reduce fecal shedding of pathogens are important for the billions of people who live without adequate sanitation. Probiotics could also have a role in immunization programs. L rhamnosus increased the virus-specific antibody response in children with acute rotaviral gastroenteritis,38Kaila M. Isolauri E. Soppi E. et al.Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain.Pediatr Res. 1992; 32: 141-144Crossref PubMed Google Scholar so immunostimulatory probiotics might help children's immune systems to increase the memory responses to vaccines. Based on initial data from studies in industrialized regions, Bifidobacterium longum and L rhamnosus, administered during the first 6 months of life, increased vaccine-specific antibody production after vaccination against hepatitis B.39Soh S.E. Ong D.Q. Gerez I. et al.Effect of probiotic supplementation in the first 6 months of life on specific antibody responses to infant Hepatitis B vaccination.Vaccine. 2010; 28: 2577-2579Crossref PubMed Scopus (14) Google Scholar Infants that were given Lactobacillus paracasei from 4 to 13 months of age had increased titers of antibodies to Haemophilus influenzae type B capsular polysaccharide, diphtheria toxin, and tetanus toxoid.40West C.E. Gothefors L. Granstrom M. et al.Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines.Pediatr Allergy Immunol. 2008; 19: 53-60Crossref PubMed Scopus (39) Google Scholar Concentrations of antibodies against H influenzae type B increased among infants when women were given daily doses of probiotics during the final month of pregnancy; therapy continued for infants during their first 6 months of life.41Kukkonen K. Nieminen T. Poussa T. et al.Effect of probiotics on vaccine antibody responses in infancy—a randomized placebo-controlled double-blind trial.Pediatr Allergy Immunol. 2006; 17: 416-421Crossref PubMed Scopus (41) Google Scholar Taking probiotics during pregnancy and lactation seems to be safe42Dugoua J.J. Machado M. Zhu X. et al.Probiotic safety in pregnancy: a systematic review and meta-analysis of randomized controlled trials of Lactobacillus, Bifidobacterium, and Saccharomyces spp.J Obstet Gynaecol Can. 2009; 31: 542-552PubMed Google Scholar and may yield postnatal benefits. Intriguingly, maternal consumption of L rhamnosus or Bifidobacterium lactis increased the amount of immunoglobulin A detected in breast milk.43Prescott S.L. Wickens K. Westcott L. et al.Supplementation with Lactobacillus rhamnosus or Bifidobacterium lactis probiotics in pregnancy increases cord blood interferon-gamma and breast milk transforming growth factor-beta and immunoglobulin A detection.Clin Exp Allergy. 2008; 38: 1606-1614Crossref PubMed Scopus (59) Google Scholar Increased immunoglobulin A levels in breast milk might protect infants from enteric pathogens and serve as a biomarker for studies of probiosis in lactating women. It is a challenge to assimilate and analyze all the clinical evidence of the effects of probiotics. Study quality varies, randomization and blinding methods are rarely reported, and appropriate placebos are not always used. Exclusion criteria are numerous, limiting the generalization of findings to children who are very ill. Probiotic strain designations, bacterial growth phase, and variations in administration (in fermented dairy products, infant formula, solid food, oral rehydration solution, water, juice, capsules) are often unreported. It is important that studies report these parameters so that findings can be reproduced—proteins and metabolites synthesized by live microorganisms are strain specific and vary with growth conditions. Many probiotics have shown beneficial effects, but improving our knowledge of the mechanisms that mediate these effects would facilitate identification of more potent probiotics for specific applications. There are 3 general classes of probiotic antipathogenic mechanisms: direct antagonism, immunomodulation, and exclusion (Figure 2). Many probiotics secrete small molecules or bioactive peptides that have antimicrobial activities. Lactobacillus salivarius UCC118 protects mice against infection with Listeria monocytogenes; UCC118 produces a broad-spectrum bacteriocin (antimicrobial peptide) that kills Listeria in the lumen of the GI tract, preventing translocation and systemic spread of infection.44Corr S.C. Li Y. Riedel C.U. et al.Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118.Proc Natl Acad Sci U S A. 2007; 104: 7617-7621Crossref PubMed Scopus (229) Google Scholar Mice were not protected when they were fed a derivative of UCC118 that no longer produced the bacteriocin or when they were infected with an engineered strain of Listeria that was resistant to the bacteriocin. Listeria pathogens were eliminated within 30 minutes after oral administration of UCC118, indicating a direct effect of the probiotic on the pathogen. UCC118 also protects mice against Salmonella infection, but the bacteriocin is not involved. Thus, a single probiotic can protect mammals against different pathogens via multiple mechanisms. Several in vitro studies have reported down-regulation of virulence factors in pathogens exposed to probiotics or their cell-free supernatants.45Medellin-Pena M.J. Wang H. Johnson R. et al.Probiotics affect virulence-related gene expression in Escherichia coli O157:H7.Appl Environ Microbiol. 2007; 73: 4259-4267Crossref PubMed Scopus (54) Google Scholar, 46Carey C.M. Kostrzynska M. Ojha S. et al.The effect of probiotics and organic acids on Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157:H7.J Microbiol Meth. 2008; 73: 125-132Crossref PubMed Scopus (37) Google Scholar, 47de Sablet T. Chassard C. Bernalier-Donadille A. et al.Human microbiota-secreted factors inhibit Shiga toxin synthesis by enterohemorrhagic Escherichia coli O157:H7.Infect Immun. 2009; 77: 783-790Crossref PubMed Scopus (29) Google Scholar Lactobacillus acidophilus LA-5 suppresses transcription of Escherichia coli O157:H7 genes involved in adherence; this corresponds with reduced colonization in mice.48Medellin-Pena M.J. Griffiths M.W. Effect of molecules secreted by Lactobacillus acidophilus strain La-5 on Escherichia coli O157:H7 colonization.Appl Environ Microbiol. 2009; 75: 1165-1172Crossref PubMed Scopus (17) Google Scholar Probiotics can also interfere with toxin production or directly antagonize enterotoxins. Saccharomyces cerevisiae var. boulardii (S boulardii), which reduces Clostridium difficile-associated diarrhea,49McFarland L.V. Systematic review and meta-analysis of Saccharomyces boulardii in adult patients.World J Gastroenterol. 2010; 16: 2202-2222Crossref PubMed Scopus (83) Google Scholar secretes a 54-kDa serine protease that hydrolyzes toxin A (a C difficile virulence factor) and its receptor, which is present in the intestinal brush border.50Pothoulakis C. Kelly C.P. Joshi M.A. et al.Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum.Gastroenterology. 1993; 104: 1108-1115Abstract PubMed Google Scholar, 51Castagliuolo I. LaMont J.T. Nikulasson S.T. et al.Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.Infect Immun. 1996; 64: 5225-5232Crossref PubMed Google Scholar Probiotics elicit a variety of responses from immune cells in vitro and in vivo, through mostly unknown mechanisms.52Forsythe P. Bienenstock J. Immunomodulation by commensal and probiotic bacteria.Immunol Invest. 2010; 39: 429-448Crossref PubMed Scopus (38) Google Scholar The responses of specific immune cells to particular microbes result from complex interactions between surface-bound and secreted ligands (eg, pathogen-associated molecular patterns) and host Toll-like receptors (TLRs).53Kawai T. Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.Nat Immunol. 2010; 11: 373-384Crossref PubMed Scopus (1423) Google Scholar Reductionist approaches to studying these interactions, such as analyses of knockout or transgenic mice, have provided limited information about probiotic immunomodulation. In one successful example, the presence of d-alanines in teichoic acids in the cell wall of Lactobacillus plantarum elicited production of proinflammatory cytokines by peripheral blood mononuclear cells.54Grangette C. Nutten S. Palumbo E. et al.Enhanced antiinflammatory capacity of a Lactobacillus plantarum mutant synthesizing modified teichoic acids.Proc Natl Acad Sci U S A. 2005; 102: 10321-10326Crossref PubMed Scopus (205) Google Scholar Co-culture of dendritic cells with polysaccharide A derived from Bacteroides fragilis induced naïve T cells to generate an interleukin-10–producing regulatory T-cell population.55Mazmanian S.K. Liu C.H. Tzianabos A.O. et al.An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.Cell. 2005; 122: 107-118Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar Purified polysaccharide A prevents intestinal inflammation in multiple mouse models.56Mazmanian S.K. Round J.L. Kasper D.L. A microbial symbiosis factor prevents intestinal inflammatory disease.Nature. 2008; 453: 620-625Crossref PubMed Scopus (533) Google Scholar The immunomodulatory effects of probiotics can be species-57Christensen H.R. Frokiaer H. Pestka J.J. Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells.J Immunol. 2002; 168: 171-178Crossref PubMed Google Scholar and strain-specific,58Lin Y.P. Thibodeaux C.H. Pena J.A. et al.Probiotic Lactobacillus reuteri suppress proinflammatory cytokines via c-Jun.Inflamm Bowel Dis. 2008; 14: 1068-1083Crossref PubMed Scopus (58) Google Scholar, 59Thomas C.M. Versalovic J. Probiotics-host communication: modulation of signaling pathways in the intestine.Gut Microbes. 2010; 1: 1-16Crossref Google Scholar and involve multiple mammalian signaling pathways that affect immune cell phenotypes. Some probiotic Lactobacillus strains increase production of tumor necrosis factor through activation of the transcription factors nuclear factor-κB and STAT,60Miettinen M. Lehtonen A. Julkunen I. et al.Lactobacilli and Streptococci activate NF-kappa B and STAT signaling pathways in human macrophages.J Immunol. 2000; 164: 3733-3740PubMed Google Scholar whereas others suppress tumor necrosis factor production by inactivating nuclear factor-κB61Petrof E.O. Kojima K. Ropeleski M.J. et al.Probiotics inhibit nuclear factor-kappaB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition.Gastroenterology. 2004; 127: 1474-1487Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 62Iyer C. Kosters A. Sethi G. et al.Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid leukemia-derived cells by modulation of NF-kappaB and MAPK signalling.Cell Microbiol. 2008; 10: 1442-1452Crossref PubMed Scopus (49) Google Scholar or mitogen-activated protein kinase and c-Jun signaling.58Lin Y.P. Thibodeaux C.H. Pena J.A. et al.Probiotic Lactobacillus reuteri suppress proinflammatory cytokines via c-Jun.Inflamm Bowel Dis. 2008; 14: 1068-1083Crossref PubMed Scopus (58) Google Scholar Differential immune regulation might prime the immune system to limit infections, inflammation, and pathogen-mediated damage. Probiotic signaling molecules that regulate immunity have not been identified. Exclusion is used as a “catch-all” term for probiotic mechanisms that make the GI environment less hospitable for pathogens. These mechanisms include altering the resident microbiota, decreasing luminal pH, improving epithelial barrier function, interfering with pathogen binding by down-regulating specific host receptors, and stimulating production of defense-associated factors, including mucins and defensins. Multiple probiotics have been implicated in each of these functions, but clear links between individual bacterial compounds and specific responses have been difficult to establish.63Ohland C.L. Macnaughton W.K. Probiotic bacteria and intestinal epithelial barrier function.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G807-G819Crossref PubMed Scopus (92) Google Scholar Rats that consumed the probiotic mixture VSL#3 increased their luminal mucin content by 60% through an unidentified, heat-resistant, secreted, soluble compound.64Caballero-Franco C. Keller K. De Simone C. et al.The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G315-G322Crossref PubMed Scopus (135) Google Scholar Some bacterial products, including short-chain fatty acids produced by fermentation, can stimulate epithelial cell differentiation and improve barrier function65Cook S.I. Sellin J.H. Review article: short chain fatty acids in health and disease.Aliment Pharmacol Ther. 1998; 12: 499-507Crossref PubMed Scopus (135) Google Scholar, 66Johnson-Henry K.C. Donato K.A. 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