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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 Ca 2+ -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., Competing Interests: The authors declare no conflict of interest., (© 2022 Published by Elsevier Ltd.)

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, NFkβ 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 NFkβ., 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., Competing Interests: The study was in part supported by Dicofarm S.p.A. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. All authors declare no other competing interests., (Copyright © 2022 Buccigrossi, Poeta, Cioffi, Terranova, Nunziata, Lo Vecchio and Guarino.)

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., (© 2021. The Author(s).)

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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 (FEV 1 ), 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-1β, 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 FEV 1 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.

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., Competing Interests: : A.G. has participated as a clinical investigator, and/or advisory board member, and/or consultant and/or speaker from Biocodex, Dicofarm, the Menarini Group, Malesci, Angelini, and Mead Johnson Nutrition. His institution received research grants from Mead Johnson USA, Dicofarm Italy, and Ippsen France. The study was supported by Mead Johnson, which produced the PF formula analysed in this study.

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.

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 H 2 -consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non-microbial routes., (© FEMS 2016.)

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.

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.

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.

The intestinal mucosa is an important target of human immunodeficiency virus (HIV) infection. HIV virus induces CD4+ T cell loss and epithelial damage which results in increased intestinal permeability. The mechanisms involved in nutrient malabsorption and alterations of intestinal mucosal architecture are unknown. We previously demonstrated that HIV-1 transactivator factor (Tat) induces an enterotoxic effect on intestinal epithelial cells that could be responsible for HIV-associated diarrhea. Since oxidative stress is implicated in the pathogenesis and morbidity of HIV infection, we evaluated whether Tat induces apoptosis of human enterocytes through oxidative stress, and whether the antioxidant N-acetylcysteine (NAC) could prevent it. Caco-2 and HT29 cells or human intestinal mucosa specimens were exposed to Tat alone or combined with NAC. In an in-vitro cell model, Tat increased the generation of reactive oxygen species and decreased antioxidant defenses as judged by a reduction in catalase activity and a reduced (GSH)/oxidized (GSSG) glutathione ratio. Tat also induced cytochrome c release from mitochondria to cytosol, and caspase-3 activation. Rectal dialysis samples from HIV-infected patients were positive for the oxidative stress marker 8-hydroxy-2'-deoxyguanosine. GSH/GSSG imbalance and apoptosis occurred in jejunal specimens from HIV-positive patients at baseline and from HIV-negative specimens exposed to Tat. Experiments with neutralizing anti-Tat antibodies showed that these effects were direct and specific. Pre-treatment with NAC prevented Tat-induced apoptosis and restored the glutathione balance in both the in-vitro and the ex-vivo model. These findings indicate that oxidative stress is one of the mechanism involved in HIV-intestinal disease., (© 2011 Buccigrossi et al.)

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.

The mechanisms of diarrhea due to rotavirus infection in humans are not fully understood; no specific therapy is available, but orally administered human serum immunoglobulins are effective in blocking stool output. We aimed to investigate the effect of rotavirus on ion transport and the role of NSP4 in human-derived enterocytes, and to test the efficacy of human serum immunoglobulin in a model of rotavirus infection. Soon after infection, rotavirus induces active chloride secretion in enterocytes. This effect is evident before viral replication leads to cell damage and correlates with NSP4 production. Inhibition of NSP4 prevents the early secretory phase but not cell damage. Incubation with human serum immunoglobulin blocks both ion secretion and cell damage. Rotavirus exerts an early NSP4-dependent ion secretion and subsequent tissue damage. The combined enterotoxic and cytotoxic effects may be responsible for the increased severity of diarrhea due to rotavirus infection, and both are counteracted by human serum immunoglobulin.

Human milk stimulates intestinal development through the effects of various moieties. Lactoferrin (LF) is a glycoprotein of human milk whose concentration is highest in colostrum decreasing in mature milk. LF promotes enterocyte growth in intestinal cell lines. We tested the hypothesis that LF induces a distinct effect on enterocyte proliferation and differentiation, depending on its concentration. We examined the dose-related effects by human-native LF (N-LF) in Caco-2 (human colon adenocarcinoma) cells. At high concentrations, N-LF stimulated cell proliferation in immature Caco-2 cells, as judged by 3H-thymidine incorporation. In contrast, sucrase and lactase activities were increased at low but not high LF concentrations and their mRNA were also increased, indicating a transcriptional effect. Because iron binds specific LF sites, we compared the potency of N-LF and iron-saturated LF (I-LF) and found the native form more potent. Finally, we tested the effects by bovine LF (bLF) in the same system and found the latter more potent than the human isoform in inducing cell growth and lactase expression. These results suggest that LF directly induces enterocyte growth and proliferation, depending on its concentration, thereby regulating the earlyx postnatal intestinal development. bLF could be added to infant formula as a growth factor in selected intestinal diseases.

Diarrhoea-related morbidity is reduced by zinc supplementation in HIV-1-infected children. The mechanisms of this effect are largely undefined. We provide evidence for role for Tat (transactivating peptide produced by HIV-1) in the pathogenesis of diarrhoea in AIDS patients. In this study we showed that zinc, preventing Tat-induced fluid secretion, directly limits a specific mechanism of HIV-1-related diarrhoea. Our data support a 'zinc approach' in adjunct to specific antiretroviral therapy in HIV-1-infected children.

Aim: Growth hormone (GH) directly interacts with the enterocyte stimulating ion absorption and reducing ion secretion induced by agonists of cAMP. Since nitric oxide (NO) is involved in the regulation of transepithelial ion transport and acts as a second messenger for GH hemodynamic effects, we tested the hypothesis that NO may be involved in the resulting effects of GH on intestinal ion transport., Methods: Electrical parameters reflecting trans-epithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers and exposed to GH and cholera toxin (CT) alone or in combination, in the presence or absence of the NO synthase (NOS) inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME). Similar experiments were conducted to determine cAMP and nitrite/nitrate concentrations. NOS expression was assayed by Western blot analysis., Results: L-NAME causes total abrogation of absorptive and anti-secretory effects by GH on intestinal ion transport. In addition, L-NAME was able to inhibit the GH-effects on intracellular cAMP concentration under basal conditions and in response to CT. GH induced a Ca(2+)-dependent increase of nitrites/nitrates production, indicating the involvement of the constitutive rather than the inducible NOS isoform, which was directly confirmed by Western blot analysis., Conclusion: These results suggest that the GH effects on intestinal ion transport, either under basal conditions or in the presence of cAMP-stimulated ion secretion, are mediated at an intracellular level by the activity of cNOS.

Objective: The pathophysiology of HIV-1-related intestinal dysfunction is largely unknown. We previously found that the transactivator factor peptide (Tat) produced by HIV-1 induces ion secretion and inhibits cell proliferation in human enterocytes. Because sugar malabsorption is a frequent feature in AIDS patients, we evaluated whether Tat inhibits intestinal glucose absorption., Design and Methods: We measured Na-D-glucose symporter (SGLT-1) activity and determined its phenotypic expression in Caco-2 cells, in the presence and absence of Tat, in uptake experiments using a non-metabolized radiolabelled glucose analogue, and by western blot analysis, respectively. alpha-Tubulin staining was used to study the effects exerted by Tat on cell structure., Results: Tat dose dependently inhibited glucose uptake by human enterocytes. This effect was prevented by anti-Tat polyclonal antibodies and by L-type Ca channels agonist Bay K8644. Western blot analysis of cellular lysates and brush-border membrane preparations showed that Tat induced SGLT-1 missorting. Tat also caused a dramatic decrease in alpha-tubulin staining, which indicates dysruption of the cytoskeleton organization., Conclusions: Tat acutely impairs intestinal glucose absorption through SGLT-1 missorting. This result indicates that Tat is directly involved in AIDS-associated intestinal dysfunction.

We previously detected specific binding activity of Escherichia coli heat-stable enterotoxin (ST), the guanylin exogenous ligand, in rat colonic basolateral membranes. Because guanylin circulates in the bloodstream, we tested the hypothesis that it modulates intestinal ion transport by acting on the serosal side of intestinal cells. The effects of the mucosal and serosal addition of ST and guanylin on ion transport were investigated in the rat proximal colon and in Caco-2 cells in Ussing chambers, by monitoring short-circuit current (Isc). cGMP concentration was measured in Caco-2 cells by RIA. Mucosal ST addition induced an increase in Isc in rat proximal colon consistent with anion secretion. Serosal addition induced the same effects but to a lesser extent. The electrical effects observed in Caco-2 cells paralleled those observed in rat proximal colon. A pattern similar to the electrical response was observed with cGMP concentration. Guanylin addition to either side of Caco-2 cells induced the same effects as ST, although to a lesser extent. In all conditions, the electrical effect disappeared in the absence of chloride. ST directly interacts with basolateral receptors in the large intestine inducing chloride secretion through an increase of cGMP. However, the serosal effects are less pronounced compared with those observed with mucosal addition. Guanylin shows the same pattern, suggesting that it plays a role in the regulation of ion transport in the colon, but the relative importance of serosally mediated secretion remains to be determined.

Background: Because zinc deficiency in malnourished children is associated with severe diarrhea, use of zinc supplementation has been proposed as an adjunct to oral rehydration. However, the effects of zinc on enterocyte ion transport are largely unknown. The objective of the present study was to investigate the effects of zinc on transepithelial ion transport under basal conditions and under conditions of enterotoxin-induced ion secretion., Methods: Ion transport was investigated by monitoring electrical parameters in human intestinal Caco-2 cells that were mounted in Ussing chambers and exposed to increasing concentrations of zinc, both in the absence and presence of either cholera toxin (CT) or Escherichia coli heat-stable enterotoxin (ST). Intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) concentrations were also determined., Results: The addition of zinc to the luminal or basolateral side of enterocytes induced a chloride-dependent, dose-related decrease in short-circuit current, indicating ion absorption. It also resulted in a substantial reduction in CT-induced ion secretion and in cAMP concentration. E. coli ST-induced ion secretion and cGMP concentration were not affected. Ion absorption peaked at 35 mu mol/L zinc, whereas excess zinc load induced active ion secretion., Conclusions: By causing a decrease in cAMP concentration, zinc directly promotes ion absorption and substantially reduces CT-induced, but not E. coli ST-induced, ion secretion.

The role of nitric oxide (NO) in the intestinal basal ion transport and under conditions of enterotoxin-induced ion secretion is controversial. Namely it is not clear whether NO enhances or counteracts intestinal ion secretion and whether the effects on transport result from a direct interaction with the enterocyte. The cell origin of NO is also unclear. We have tested the hypothesis that NO produced by the enterocyte directly regulates ion transport processes either in basal condition or in response to cholera toxin-induced secretion. Electrical variables reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers exposed to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester, in the presence or absence of cholera toxin. cAMP concentrations were also measured. NO release was determined by nitrite-nitrate concentration. NO synthase activities were assayed by Western blot analysis. Nomega-nitro-l-arginine methyl ester had a secretory effect, as judged by increased basal short-circuit current and cAMP concentration. It also increased cholera toxin-induced electrical response and cAMP production. Either cholera toxin or the cAMP analog 8-bromo-cAMP induced a rapidly progressive and Ca2+-dependent increase in NO concentration, suggesting a homeostatic up-regulation of the constitutive form of NO synthase. Western blot analysis showed an increase in constitutive NO synthase enzyme isoform. These results indicate that the enterocyte regulates its own ion transport processes, either in basal condition or in the presence of active secretion, through the activation of a constitutive NO synthase-NO pathway, functioning as a braking force of cAMP-induced ion secretion.

Background & Aims: Severe diarrhea and enteropathy of unknown origin are frequent in patients infected with human immunodeficiency type 1 virus (HIV-1). The HIV-1 transactivating factor protein (Tat) is a key factor in the pathogenesis of acquired immunodeficiency syndrome. We investigated whether Tat could directly induce ion secretion and cell damage in enterocytes., Methods: Electrical parameters (ion transport studies) were measured in Caco-2 cell monolayers and in human colonic mucosa specimens mounted in Ussing chambers. The effect of Tat on intestinal mucosa integrity was determined by monitoring the transepithelial electrical resistance of Caco-2 cell monolayers. (3)H-thymidine incorporation and cell count were used to evaluate the effect of Tat on cell growth. Intracellular calcium concentrations were measured at the single-cell level using microfluorometry technique., Results: Tat protein induced ion secretion in Caco-2 cells and in human colonic mucosa similar to that induced by bacterial enterotoxins. It also significantly prevented enterocyte proliferation. In both instances, the effect of Tat was maximum at concentrations within the range detected in the sera of HIV-1-infected patients. Anti-Tat antibodies inhibited both effects. Ion secretion and the antiproliferative effects were mediated by L-type Ca(2+) channels. An increase in intracellular calcium concentration in Caco-2 cells was found after addition of Tat., Conclusions: These results indicate that Tat may be involved in HIV-1-related intestinal disease through direct interaction with enterocytes.