Aetiology, risk factors and microbiota composition in children with prolonged diarrhoea: A prospective case-controlled cohort study.

AIM: Prolonged diarrhoea (ProD) refers to acute-onset diarrhoea that persists for longer than 1 week. As the aetiology, risk factors and management are poorly defined, we prospectively enrolled children hospitalised in a high-income setting to assess these outcomes and investigate the potential role of gut microbiota. METHODS: All children aged 30 days to 14 years admitted for acute-onset diarrhoea lasting 7-14 days were included. Children consecutively admitted in the same period for acute diarrhoea (AD) served as controls. High-throughput sequencing of 16S rRNA gene amplicons was used to analyse stool samples from a subset of patients and healthy controls. RESULTS: Sixty-eight with ProD and 104 with AD were enrolled. Intestinal infections were the main aetiology of diarrhoea in both groups (ProD 92.9% vs. AD 97.8%). ProD children showed a higher prevalence of bacterial infections compared to AD (30.8% vs. 8.9%, p = 0.024). Neither age, host-related factors, nor microbiome alterations were specifically linked to ProD. However, ProD children had a more severe initial clinical presentation than AD. CONCLUSION: ProD is often the result of an unusually severe intestinal infection that runs a course longer than expected but generally resolves without further problems. No specific management or therapies should be undertaken in most cases.

SARS-CoV-2 causes secretory diarrhea with an enterotoxin-like mechanism, which is reduced by diosmectite.

Background and aims: The pathophysiology of SARS-CoV-2-associated diarrhea is unknown. Using an experimental model validated for rotavirus-induced diarrhea, we investigated the effects of SARS-CoV-2 on transepithelial ion fluxes and epithelial integrity of human intestinal cells. The effect of the antidiarrheal agent diosmectite on secretion was also evaluated following its inclusion in COVID-19 management protocols. Methods: We evaluated electrical parameters (intensity of short-circuit current [Isc] and transepithelial electrical resistance [TEER]) in polarized Caco-2 cells and in colonic specimens mounted in Ussing chambers after exposure to heat-inactivated (hi) SARS-CoV-2 and spike protein. Spectrofluorometry was used to measure reactive oxygen species (ROS), a marker of oxidative stress. Experiments were repeated after pretreatment with diosmectite, an antidiarrheal drug used in COVID-19 patients. Results: hiSARS-CoV-2 induced an increase in Isc when added to the mucosal (but not serosal) side of Caco-2 cells. The effect was inhibited in the absence of chloride and calcium and by the mucosal addition of the Ca2+-activated Cl- channel inhibitor A01, suggesting calcium-dependent chloride secretion. Spike protein had a lower, but similar, effect on Isc. The findings were consistent when repeated in human colonic mucosa specimens. Neither hiSARS-CoV-2 nor spike protein affected TEER, indicating epithelial integrity; both increased ROS production. Pretreatment with diosmectite inhibited the secretory effect and significantly reduced ROS of both hiSARS-CoV-2 and spike protein. Conclusions: SARS-CoV-2 induces calcium-dependent chloride secretion and oxidative stress without damaging intestinal epithelial structure. The effects are largely induced by the spike protein and are significantly reduced by diosmectite. SARS-CoV-2 should be added to the list of human enteric pathogens.

Lacticaseibacillus rhamnosus GG Counteracts Rotavirus-Induced Ion Secretion and Enterocyte Damage by Inhibiting Oxidative Stress and Apoptosis Through Specific Effects of Living and Postbiotic Preparations.

Background: Administration of Lacticaseibacillus rhamnosus GG (LGG) to children with gastroenteritis is recommended by universal guidelines. Rotavirus (RV) causes diarrhea through combined cytotoxic and enterotoxic effects. Aim of this study was to evaluate the mechanisms of efficacy of LGG in an in-vitro model of RV diarrhea in its viable form (LGG) and conditioned medium (mLGG). Methods: Ion secretion corresponding to the NSP4 enterotoxic effect, was evaluated by short circuit current (Isc) and the cytotoxic effect by transepithelial electrical resistance (TEER) in Ussing chambers, upon exposure to RV in Caco-2 enterocyte monolayers treated or not with living probiotic or its culture supernatant. Mechanisms of enterotoxic and cytotoxic damage were evaluated including oxidative stress measured by reactive oxygen species, apoptosis evaluated by DAPI and nuclear staining, NFkß immunofluorescence. Results: RV induced Isc increase and TEER decrease, respectively indicating ion secretion and epithelial damage, the two established pathways of diarrhea. Both probiotic preparations reduced both diarrheal effects, but their potency was different. Live LGG was equally effective on both enterotoxic and cytotoxic effect whereas mLGG was highly effective on ion secretion and showed minimal protective effects on cytoskeleton, apoptosis and NFkß. Conclusions: LGG counteracts RV-induced diarrhea by inhibiting both cytotoxic and enterotoxic pathogenic mechanisms. Namely, LGG inhibits chloride secretion by specific moieties secreted in the medium with a direct pharmacologic-like action. This is considered a postbiotic effect. Subsequently, live bacteria exert a probiotic effect protecting the enterocyte structure.

Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion.

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

Potency of Oral Rehydration Solution in Inducing Fluid Absorption is Related to Glucose Concentration.

Oral rehydration solutions (ORSs) is the key treatment of acute diarrhea in children, as it restores the electrolyte balance by stimulating the intestinal sodium/glucose transporter SGLT1 to induce fluid absorption. The World Health Organization (WHO) and The European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) proposed ORSs with different chemical compositions. The main agent of childhood acute gastroenteritis is rotavirus (RV). We evaluate the effects of ORS with different concentration of glucose and sodium on RV induced secretion. Ussing chambers technique was used for electophysiology experiments to evaluate ion fluid flux. ESPGHAN ORS (sodium 60 mmol/L and glucose 111 mmol/L) induced a more potent proabsorptive effect in Caco-2 cells than WHO ORS, and this effect depended on the sodium/glucose ratio. Titration experiments showed that RV-induced fluid secretion can be reverted to a proabsorptive direction when sodium and glucose concentration fall in specific ranges, specifically 45-60 mEq/L and 80-110 mM respectively. The results were confirmed by testing commercial ORSs. These findings indicated that ORS proabsorptive potency depends on sodium and glucose concentrations. Optimal ORS composition should be tailored to reduce RV-induced ion secretion by also considering palatability. These in vitro data should be confirmed by clinical trials.

Differential effects of Clostridium difficile toxins on ion secretion and cell integrity in human intestinal cells.

BACKGROUND: Toxin A (TcdA), toxin B (TcdB), and binary toxin (CDT) produced by Clostridium difficile (CD) are thought to play a key role in inducing diarrhea. The aim of this study was to investigate the individual and combined roles of CD toxins in inducing enterotoxic and cytotoxic effect. METHODS: Ion secretion and epithelial damage were evaluated in the Ussing chambers as measure of enterotoxic or cytotoxic effect, respectively, in human-derived intestinal cells. RESULTS: When added to the mucosal side of Caco-2 cells, TcdB, but not TcdA, induced ion secretion and its effects increased in the presence of TcdA. CDT also induced ion secretion when added to either the mucosal or serosal compartment. Serosal addition of TcdB induced epithelial damage consistent with its cytotoxic effect. However, mucosal addition of TcdB had similar effects, but only in the presence of TcdA. CDT induced epithelial damage when added to the serosal side of cell monolayers, and this was associated with a late onset but prolonged effect. All data were replicated using human colon biopsies. CONCLUSIONS: These data indicate that CD, through the combined and direct activity of its three toxins, induces integrated and synergic enterotoxic and cytotoxic effects on the intestinal epithelium.

Acute Infectious Diarrhea.

Acute infectious diarrhea (AID) is one of the most common diseases in pediatric age with relevant burden both in high- and in low-income countries.Thanks to their direct action on enterocyte functions and indirect actions on mucosal and systemic immune system and intestinal microenvironment, probiotics are an ideal intervention to manage AID in childhood. However, their efficacy is strictly related to strains and indications, and practitioners should take this information into account in clinical practice.This chapter summarizes the main mechanisms of action of probiotics in AID, with a focus on proof of efficacy supporting their use in prevention and treatment of infant AID.The use of selected strains in appropriate doses is strongly recommended by guidelines of AID, based on large and consistent proofs of efficacy and safety. At present, therapy with probiotics of AID is arguably the strongest indication for probiotics in medicine. Future research should investigate probiotic efficacy in at-risk populations and settings where the evidence is missing.Their role in prevention of AID is however questionable in healthy population, whereas it should be considered in at-risk population. Evidence for prevention of diarrhea in day-care centers and communities is lacking, but consistent evidence supports efficacy in prevention of hospital acquired diarrhea.Overall, AID is the most convincing area for probiotic use in children, and effective strains should be used early after onset of symptoms.

Altered intestinal microbiota composition, antibiotic therapy and intestinal inflammation in children and adolescents with cystic fibrosis.

The aim of the present study was to evaluate the effect of cystic fibrosis and antibiotic therapy on intestinal microbiota composition and intestinal inflammation in children and adolescents. A cross-sectional controlled study was conducted with 36 children and adolescents: 19 in the cystic fibrosis group (CFG) and 17 in the control group (CG) matched for age and sex. The CFG was subdivided based on the use of antibiotic therapy (CFAB group) and non-use of antibiotic therapy (CFnAB group). The following data were evaluated: colonization, antibiotic therapy, mutation, breastfeeding, use of infant formula, type of delivery, introduction of solid foods, body mass index, fecal calprotectin and intestinal microbiota composition (fluorescence in situ hybridization). Intestinal inflammation evaluated by fecal calprotectin was significantly higher in the CFG (median: 40.80 µg/g, IQR: 19.80-87.10, p = 0.040) and CFAB group (median: 62.95 µg/g, IQR: 21.80-136.62, p = 0.045) compared to the CG (median: 20.15 µg/g, IQR: 16.20-31.00), and the Bacteroides, Firmicutes, Eubacterium rectale and Faecalibacterium prausnitzii were significantly decreased (p < 0.05) in the CFG compared to the CG, whereas the bacteria Clostridium difficile, Escherichia coli and Pseudomonas aeruginosa were significantly increased in the CFG (p < 0.05). The main differences were found between the CG and CFAB group for Eubacterium rectale (p = 0.006), Bifidobacterium (p = 0.017), Escherichia coli (p = 0.030), Firmicutes (p = 0.002), Pseudomonas aeruginosa (p < 0.001) and Clostridium difficile (p = 0.006). The results of this study confirm intestinal inflammation in patients with CF, which may be related to changes in the composition of the intestinal microbiota.

Galacto-Oligosaccharide/Polidextrose Enriched Formula Protects against Respiratory Infections in Infants at High Risk of Atopy: A Randomized Clinical Trial.

BACKGROUND: Early nutrition affects the risk of atopy and infections through modifications of intestinal microbiota. The Prebiotics in the Prevention of Atopy (PIPA) study was a 24-month randomised, double-blind, placebo-controlled trial. It aimed to evaluate the effects of a galacto-oligosaccharide/polydextrose (GOS/PDX)-formula (PF) on atopic dermatitis (AD) and common infections in infants who were born to atopic parents and to investigate the relationship among early nutrition, gut microbiota and clinical outcomes. METHODS: A total of 201 and 199 infants were randomized to receive a PF and standard formula (SF), respectively; 140 infants remained on exclusive breastfeeding (BF). RESULTS: The cumulative incidence of AD and its intensity and duration were not statistically different among the three groups. The number of infants with at least one episode of respiratory infection (RI) and the mean number of episodes until 48 weeks of age were significantly lower in the PF group than in the SF group. The number of patients with recurrent RIs and incidence of wheezing lower RIs until 96 weeks were lower in the PF group than the SF group, but similar to the BF group. Bifidobacteria and Clostridium cluster I colonization increased over time in the PF group but decreased in the SF and BF groups. Bifidobacteria had a protective role in RIs, whereas Clostridium cluster I was associated with atopy protection. CONCLUSION: The early administration of PF protects against RIs and mediates a species-specific modulation of the intestinal microbiota. TRIAL REGISTRATION: clinicaltrial.gov Identifier: NCT02116452.

Effect of synbiotic supplementation in children and adolescents with cystic fibrosis: a randomized controlled clinical trial.

BACKGROUND/OBJECTIVES: Cystic fibrosis (CF) is characterized by excessive activation of immune processes. The aim of this study was to evaluate the effect of synbiotic supplementation on the inflammatory response in children/adolescents with CF. SUBJECTS/METHODS: A randomized, placebo-controlled, double-blind, clinical-trial was conducted with control group (CG, n = 17), placebo-CF-group (PCFG, n = 19), synbiotic CF-group (SCFG, n = 22), PCFG negative (n = 8) and positive (n = 11) bacteriology, and SCFG negative (n = 12) and positive (n = 10) bacteriology. Markers of lung function (FEV1), nutritional status [body mass index-for age (BMI/A), height-for-age (H/A), weight-for-age (W/A), upper-arm fat area (UFA), upper-arm muscle area (UMA), body fat (%BF)], and inflammation [interleukin (IL)-12, tumor necrosis factor-alpha (TNF-α), IL-10, IL-6, IL-1ß, IL-8, myeloperoxidase (MPO), nitric oxide metabolites (NOx)] were evaluated before and after 90-day of supplementation with a synbiotic. RESULTS: No significance difference was found between the baseline and end evaluations of FEV1 and nutricional status markers. A significant interaction (time vs. group) was found for IL-12 (p = 0.010) and myeloperoxidase (p = 0.036) between PCFG and SCFG, however, the difference was not maintained after assessing the groups individually. NOx diminished significantly after supplementation in the SCFG (p = 0.030). In the SCFG with positive bacteriology, reductions were found in IL-6 (p = 0.033) and IL-8 (p = 0.009) after supplementation. CONCLUSIONS: Synbiotic supplementation shown promise at diminishing the pro-inflammatory markers IL-6, IL-8 in the SCFG with positive bacteriology and NOx in the SCFG in children/adolescents with CF.

Mechanisms of antidiarrhoeal effects by diosmectite in human intestinal cells.

BACKGROUND: Rotavirus (RV) induces diarrhoea through a sequence of enterotoxic and cytotoxic effects. The former are NSP4-dependent, induce calcium-dependent chloride secretion and involve oxidative stress. Diosmectite (DS) is a natural clay that has been recommended as an active therapy for diarrhoea, but the mechanism of its effect is not clear. Electrical parameters may be used to measure the direct enterotoxic and cytotoxic effects in polar epithelial intestinal cells. To investigate the effects of DS on RV-induced enterotoxic and cytotoxic damage. Caco-2 cells were used as a model of RV infection to evaluate chloride secretion, epithelial integrity, oxidative stress and viral infectivity in Ussing chambers. RESULTS: Diosmectite reduced the expression of NSP4 and oxidative stress, resulting in a strong inhibition of chloride secretion. Preincubating RV with DS reduced the cytotoxic effect. Finally, the viral load was reduced by DS but not by control clay. This result suggests that DS specifically affects the early events of RV infection protecting the enterocyte, whereas it does not restore already-established cell damage. CONCLUSION: These findings indicate that DS exerts an anti-diarrhoeal effect by inhibiting viral replication and the expression of NSP4. Both ion secretion and cell damage induced by RV are strongly inhibited consequent to the antiviral effect, which explains its clinical efficacy.

Impact of cystic fibrosis disease on archaea and bacteria composition of gut microbiota.

Cystic fibrosis is often associated with intestinal inflammation due to several factors, including altered gut microbiota composition. In this study, we analyzed the fecal microbiota among patients with cystic fibrosis of 10-22 years of age, and compared the findings with age-matched healthy subjects. The participating patients included 14 homozygotes and 14 heterozygotes with the delF508 mutation, and 2 heterozygotes presenting non-delF508 mutations. We used PCR-DGGE and qPCR to analyze the presence of bacteria, archaea and sulfate-reducing bacteria. Overall, our findings confirmed disruption of the cystic fibrosis gut microbiota. Principal component analysis of the qPCR data revealed no differences between homozygotes and heterozygotes, while both groups were distinct from healthy subjects who showed higher biodiversity. Archaea were under the detection limit in all homozygotes subjects, whereas methanogens were detected in 62% of both cystic fibrosis heterozygotes and healthy subjects. Our qPCR results revealed a low frequency of sulfate-reducing bacteria in the homozygote (13%) and heterozygote (13%) patients with cystic fibrosis compared with healthy subjects (87.5%). This is a pioneer study showing that patients with cystic fibrosis exhibit significant reduction of H2-consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non-microbial routes.

Early-Life Intestine Microbiota and Lung Health in Children.

The gastrointestinal microbiota plays a critical role in nutritional, metabolic, and immune functions in infants and young children and has implications for future lung health status. Understanding the role of intestinal dysbiosis in chronic lung disease progression will provide opportunities to design early interventions to improve the course of the disease. Gut microbiota is established within the first 1 to 3 years of life and remains relatively stable throughout the life span. In this review, we report the recent development in research in gut-lung axis, with focus on the effects of targeting microbiota of infants and children at risk of or with progressive lung diseases. The basic concept is to exploit this approach in critical window to achieve the best results in the control of future health.

Chloride secretion induced by rotavirus is oxidative stress-dependent and inhibited by Saccharomyces boulardii in human enterocytes.

Rotavirus (RV) infection causes watery diarrhea via multiple mechanisms, primarily chloride secretion in intestinal epithelial cell. The chloride secretion largely depends on non-structural protein 4 (NSP4) enterotoxic activity in human enterocytes through mechanisms that have not been defined. Redox imbalance is a common event in cells infected by viruses, but the role of oxidative stress in RV infection is unknown. RV SA11 induced chloride secretion in association with an increase in reactive oxygen species (ROS) in Caco-2 cells. The ratio between reduced (GSH) and oxidized (GSSG) glutathione was decreased by RV. The same effects were observed when purified NSP4 was added to Caco-2 cells. N-acetylcysteine (NAC), a potent antioxidant, strongly inhibited the increase in ROS and GSH imbalance. These results suggest a link between oxidative stress and RV-induced diarrhea. Because Saccharomyces boulardii (Sb) has been effectively used to treat RV diarrhea, we tested its effects on RV-infected cells. Sb supernatant prevented RV-induced oxidative stress and strongly inhibited chloride secretion in Caco-2 cells. These results were confirmed in an organ culture model using human intestinal biopsies, demonstrating that chloride secretion induced by RV-NSP4 is oxidative stress-dependent and is inhibited by Sb, which produces soluble metabolites that prevent oxidative stress. The results of this study provide novel insights into RV-induced diarrhea and the efficacy of probiotics.

Disrupted intestinal microbiota and intestinal inflammation in children with cystic fibrosis and its restoration with Lactobacillus GG: a randomised clinical trial.

BACKGROUND & AIMS: Intestinal inflammation is a hallmark of cystic fibrosis (CF). Administration of probiotics can reduce intestinal inflammation and the incidence of pulmonary exacerbations. We investigated the composition of intestinal microbiota in children with CF and analyzed its relationship with intestinal inflammation. We also investigated the microflora structure before and after Lactobacillus GG (LGG) administration in children with CF with and without antibiotic treatment. METHODS: The intestinal microbiota were analyzed by denaturing gradient gel electrophoresis (DGGE), real-time polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH). Intestinal inflammation was assessed by measuring fecal calprotectin (CLP) and rectal nitric oxide (rNO) production in children with CF as compared with healthy controls. We then carried out a small double-blind randomized clinical trial with LGG. RESULTS: Twenty-two children with CF children were enrolled in the study (median age, 7 years; range, 2-9 years). Fecal CLP and rNO levels were higher in children with CF than in healthy controls (184±146 µg/g vs. 52±46 µg/g; 18±15 vs. 2.6±1.2 µmol/L NO2 (-), respectively; P<0.01). Compared with healthy controls, children with CF had significantly different intestinal microbial core structures. The levels of Eubacterium rectale, Bacteroides uniformis, Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Faecalibacterium prausnitzii were reduced in children with CF. A similar but more extreme pattern was observed in children with CF who were taking antibiotics. LGG administration reduced fecal CLP and partially restored intestinal microbiota. There was a significant correlation between reduced microbial richness and intestinal inflammation. CONCLUSIONS: CF causes qualitative and quantitative changes in intestinal microbiota, which may represent a novel therapeutic target in the treatment of CF. Administration of probiotics restored gut microbiota, supporting the efficacy of probiotics in reducing intestinal inflammation and pulmonary exacerbations. TRIAL REGISTRATION: ClinicalTrials.gov NCT 01961661.

Time- and segment-related changes of postresected intestine: a 4-dimensional model of intestinal adaptation.

OBJECTIVES: The aim of the present study was to investigate the segment- and time-related changes in rat short bowel syndrome and construct a 4-dimensional (4D) geometrical model of intestinal adaptation. METHODS: Sprague-Dawley rats were divided into 3 groups: 2-day, 7-day, and 15-day postresection groups in which 75% of the jejunoileum was removed. Histological and morphometrical parameters in the remaining proximal to distal intestinal segments, from the jejunum to the distal colon, were comparatively evaluated in the groups. The data were used to construct a 4D geometric model in which villi were considered as cylinders, and their surface area was expressed as cylinder lateral area. RESULTS: Major adaptive changes were observed in the ileum consisting of an increase in both the diameter of base and the height of villi. A parallel reduction in their number/mm was observed. The resulting ileal architecture was characterized by a limited number of large villi. An opposite pattern was observed in the jejunum whose postresection structure consisted of an increased number of villi. No changes were observed in the colon. Postresection restructuring was early and faster in the ileum than in the jejunum resulting in an increase in absorptive area of 81.5% and 22.5% in the ileum and jejunum, respectively. CONCLUSIONS: Postresection adaptation is intestinal segment-specific because all of the major changes occur in the ileum rather than in the jejunum. Sparing ileal segments during resection may improve the outcome of patients undergoing extensive intestinal resection. Our 4D model can be used to test interventions aimed at optimizing postresection intestinal adaptation.

Functions of intestinal microflora in children.

PURPOSE OF REVIEW: This review discusses the structural composition of intestinal microbiota, the functional relationship between the latter and the host, and the role of abnormal microflora in chronic diseases. RECENT FINDINGS: A more complete view of the gut microbiota is being developed following the Human Microbiome Project. The microflora in children is plastic, susceptible to changes in response to diet modifications, antibiotic treatment and other events, providing the opportunity to study its functional role. Increasing evidence highlights the role of nutrition in the age-related development of microflora. Eubiosis, that is, a normal microflora structure, provides protection against infections, educates the immune system, ensures tolerance to foods, and contributes to nutrient digestion and energy harvest. Changes in microflora, consisting in the overpresence of harmful species or underpresence of commensal species, or dysbiosis produce dysfunctions, such as intestinal inflammation or dysmotility. Moreover abnormal pattern of microflora have been consistently detected in specific diseases. SUMMARY: A relationship exists between eubiosis and functions and conversely between dysbiosis and dysfunctions or even diseases. Abnormalities in microflora composition may trigger or contribute to specific diseases. This raises the hypothesis to target microflora in order to restore eubiosis through the use of antibiotics, probiotics or nutrients.

Composition and roles of intestinal microbiota in children.

OBJECTIVE: To provide an update of the advantages of new-generation molecular diagnostics to study the diversity of intestinal microflora and to evaluate its alteration in human diseases. METHODS: We review recent advances in understanding the complex ecosystem of gut microbiota based on a dynamic and mutual interaction with the host. RESULTS: In vaginal delivery, the contact with the mother's vaginal and intestinal flora is an important source of Lactobacillus, Prevotella and other Bifidobacterium. On the opposite, in cesarean delivery, direct contact of the mouth of the newborn with vaginal and intestinal microbiota is replaced by exogenous non-maternally derived bacteria colonizing the infants' intestine producing a less diverse flora. The original microbiome settings evolve during the growth converging to three main clusters defined "enterotypes" in the adult age. CONCLUSIONS: The key role in human health could depend on the balance between beneficial and harmful microbial species populating the gut, therefore the intestinal microflora can been considered as a potential biomarker and/or therapeutic target in intestinal and extra-intestinal diseases.

Polar Effects on Ion Transport and Cell Proliferation Induced by GC-C Ligands in Intestinal Epithelial Cells.

Guanylin receptor guanylate cyclase (GC-C) peaks in neonatal intestine and is involved in either enterocyte proliferation or chloride secretion. The latter is more potent when GC-C activator guanylin, or its analog Escherichia coli heat-stable enterotoxin (ST), is added to the mucosal rather than serosal side of intestinal monolayers. By using Ussing chambers, we investigated transepithelial ion transport and enterocyte proliferation and their mechanisms in response to the addition of guanylin or ST to the mucosal or serosal side of Caco-2 monolayers and in ileal specimens from neonates. GC-C activation showed a polar pattern of the effects. GC-C mucosal activation resulted in a potent cGMP-chloride secretion activation and in a marginal enterocyte proliferation. Conversely, serosal GC-C activation induced a potent enterocyte proliferation, through MAP kinase ERK 1/2. Finally, the inhibition of ERK1/2 enhanced the Isc increase in response to serosal but not to mucosal ST stimulation, indicating that ERK1/2 also acts as a brake of chloride secretion. These data suggest that the guanylin/GC-C system plays a key role in early postnatal intestinal adaptation exploiting the polar structure of enterocyte.

Mechanisms of action of zinc in acute diarrhea.

PURPOSE OF REVIEW: For over a decade, the importance of zinc in the treatment of acute diarrhea has been recognized. More recently, the mechanisms of action of zinc are becoming clearer. This review is focused on the new evidence on the mechanisms of action of zinc in acute diarrhea. RECENT FINDINGS: The vast majority of data derive from in-vitro studies using intestinal cell lines or from animal model. The positive action by zinc in acute diarrhea derives from a regulation of intestinal fluid transport, mucosal integrity, immunity, gene expression, and oxidative stress. A complex homeostatic network is also able to regulate zinc status at cellular and extracellular level. SUMMARY: All these data support the use of zinc in the treatment of acute diarrhea, but further clinical studies are needed to explore the selective effects of zinc against specific pathogens responsible for diarrhea.