Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens.

Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.

Celiac disease.

Celiac disease, with a prevalence around 1% of the general population, is the most common genetically-induced food intolerance in the world. Triggered by the ingestion of gluten in genetically predisposed individuals, this enteropathy may appear at any age, and is characterized by a wide variety of clinical signs and symptoms. Among them, gastrointestinal presentations include chronic diarrhea, abdominal pain, weight loss or failure to thrive in children; but extra-intestinal manifestations are also common, and actually appear to be on the rise. They include a large variety of ailments, such as dermatitis Herpetiformis, anemia, short stature, osteoporosis, arthritis, neurologic problems, unexplained elevation of transaminases, and even female infertility. For the clinician interested in oral diseases, celiac disease can lead to delayed tooth eruption, dental enamel hypoplasia, recurrent oral aphthae. Diagnosing celiac disease requires therefore a high degree of suspicion followed by a very sensitive screening test: serum levels of the autoantibody anti-tissue transglutaminase. A positive subject will then be confirmed by an intestinal biopsy, and will then be put on a strict gluten-free diet, that in most cases will bring a marked improvement of symptoms. Newer forms of treatment which in the future will probably be available to the non-responsive patients are currently being actively pursued.

Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro.

Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function.

Mode of action of heat-stable Escherichia coli enterotoxin. Tissue and subcellular specificities and role of cyclic GMP.

Some enteric strains of Escherichia coli release a heat-stable enterotoxin which, in contrast to cholera and heat-labile E. coli enterotoxins, stimulates guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2). We have examined the tissue spcificity of its action and the relation of its action to those of the 8-bromo analogues of cyclic GMP and cyclic AMP. Heat-stable enterotoxin stimulated guanylate cyclase activity and increased cyclic GMP concentration throughout the small and large intestine. It increased transepithelial electric potential difference and short-circuit current in the jejunum, ileum and caecum but not in the duodenum or distal colon. This pattern of electrical responses was mimicked by 8-bromo-cyclic GMP. However, 8-bromo-cyclic AMP produced an electrical response in all intestinal segments. The enterotoxin failed to stimulate guanylate cyclase inliver, lung, pancreas or gastric antral mucosa. In the intestines, it stimulated only the particulate and not the soluble form of the enzyme. Preincubation of the toxin with intestinal membranes did not render it capable of stimulating pancreatic guanylate cyclase. Cytosol factors did not enhance the toxin's stimulation of intestinal guanylate cyclase. This study supports the role of cyclic GMP as intracellular mediator for heat-stable enterotoxin and suggests that the toxin affects a membrane-mediated mechanism for guanylate cyclase activation that is unique to the intestines.

Prebiotics and probiotics in irritable bowel syndrome and inflammatory bowel disease in children.

Underlying pathophysiological mechanisms of irritable bowel syndrome (IBS), a common disorder characterized by abdominal pain associated to a change in stool consistency or frequency, include low-grade inflammation and intestinal microbiota changes. Few and disappointing data are available for prebiotics. A few controlled trials (RCTs) of probiotics are instead available with favourable effects, although most are limited by suboptimal design and small sample size. A recent report from the Rome foundation group included 32 RCTs of probiotics, most of which showed an overall modest improvement in symptoms, with the patients most benefitting from probiotics being those with predominant diarrhoea and those having a post-infectious IBS. A review focusing only on children with functional gastrointestinal disorders concluded that probiotics are more effective than placebo in the treatment of patients with abdominal pain-related functional gastrointestinal disorders, although no effect on constipation was evident. The role for probiotics in inflammatory bowel disease (IBD) appears logical: the endogenous intestinal microbiota plays a central role in their development, and various probiotics have been found effective in animal models of IBD. However, research in humans has been overall quite limited, and it would seem that after a phase of intense research in the first decade of this century, the pace has slowed down, with fewer clinical trials been published in the past 2-3 years. To summarize current evidence: no probiotic has proven successful in Crohn's disease. In ulcerative colitis, on the other hand, data are more promising, and a very recent meta-analysis, that included 23 randomized controlled trials, concluded that there is evidence of efficacy for the probiotic mixture VSL#3 in helping inducing and maintaining remission, as well as in maintaining remission in patients with pouchitis. It is fair to state that for both IBD and IBS, more well-designed, rigorous, randomized clinical trials must be performed.

Thyroxine effect on intestinal Cl-/HCO3- exchange in hypo- and hyperthyroid rats.

Changes in transepithelial water and electrolyte transport as causative or contributing factors of the diarrhoea and constipation found associated with changes in thyroid physiology were studied. Albino Wistar rats were pharmacologically made either hypothyroid or hyperthyroid. After sacrifice, the small intestine was mounted in Ussing chambers in order to measure in vitro ion net fluxes under short-circuit conditions. Hypothyroid animals showed an increase in intestinal transit time, Cl- absorption (mainly due to an increment in its mucosal to serosal component) and residual ion flux (which is believed to represent HCO3- secretion) when compared with euthyroid animals. The hyperthyroid animals showed a decrease in Cl- mucosal to serosal transport. Furthermore, a significant correlation was found between serum L-thyroxine (T4) levels and both net Cl- transport (r = -0.74, P < 0.00001) and residual ion flux (r = -0.55, P < 0.005). These results indicate that the effect of T4 is firstly to inhibit Cl-/HCO3- anion exchange thereby influencing transepithelial flux transport and secondly to affect intestinal motility. Such inhibition was not found when T4 was acutely added to rat ileum, suggesting that the effect on electrolyte transport probably requires protein synthesis. In conclusion, the phenomenon observed in vitro could explain the clinical manifestations of constipation and diarrhoea in hypo- and hyper-thyroidism respectively.

Intestinal ion and nutrient transport in health and infectious diarrhoeal diseases.

Absorption of water from the intestine occurs in response to the osmotic gradient as a passive consequence of the active transfer of solutes (nutrients and electrolytes, with Na absorption playing a key role) from the intestinal lumen to the serosal side. During intestinal infections, several possible derangements of such a situation may occur, ultimately leading to the shift of net water absorption to secretion and, thus, to diarrhoea. In rotaviral diarrhoea, the mature enterocytes are invaded by the virus and exfoliate, thus inducing villous atrophy and crypt hyperplasia. Consequently, undigested and unabsorbed nutrients cause an osmolar diarrhoea, while the ongoing process of crypt secretion contributes by adding active anion and water secretion. In bacterial intestinal infections, the pathogenetic mechanisms are essentially mucosal invasion, adherence, cytotoxicity or release of enterotoxins. The pathophysiology of bacterial diarrhoea is best known for the latter mechanism; heat-labile and heat-stable families of enterotoxins have been described and characterised that act by inducing, respectively, an increase in the enterocyte's cyclic AMP or cyclic GMP content. Such alteration leads, in a morphologically intact mucosa, to changes in the major electrolyte transport processes that reverse net absorption of ions and water to net secretion and thus to secretory diarrhoea. As for nutrient absorption, although experimental evidence indicates an impairment of glucose and amino acid absorption in rotaviral diarrhoea, many clinical trials have shown the successful use of oral rehydration solutions in such circumstances. The same applies to bacterial-induced diarrhoeas; the well-established observation that, in enterotoxic diarrhoea of all kinds, the coupled transport of Na and nutrients such as glucose or amino acids is intact has proved to be the cornerstone of the highly successful, widespread use of oral rehydration solutions.

Perturbed bioelectrical properties of the mouse cecum following hepatectomy and starvation: the role of bacterial adherence.

Previous work in our laboratory has demonstrated that bacterial adherence alone to the intestinal epithelium, as occurs following catabolic stress, significantly perturbs the normal electrophysiology of the cecal mucosa. The aim of this study was to further characterize these effects in the mouse cecum following hepatectomy and short-term starvation, and to define the role of bacterial adherence in this process. Groups of mice underwent a surgical hepatectomy and were either fed or starved during the postoperative period. Groups of controls underwent sham operations and were either fed or starved postoperatively. Electrophysiologic studies in Ussing chambers at 48 hours were performed. Bacterial adherence to the mucosa was assessed by culture and histologic staining. To determine the role of bacteria in the altered electrophysiologic response, ciprofloxacin decontamination studies were performed. Only mice subjected to both hepatectomy and starvation developed bacterial adherence of sufficient magnitude (>10(5) cfu/gm) to alter mucosal electrophysiology (short-circuit current and basal potential difference). Ciprofloxacin decontamination completely abrogated this effect. Ion replacement studies suggested that active sodium transport was primarily responsible for the observed changes in mucosal electrophysiology. Bacterial-epithelial cell interactions may be responsible for altered mucosal ion transport observed following operative catabolic stress and short-term starvation.

Asymptomatic carriage of intestinal Cryptosporidium in immunocompetent and immunodeficient children: a prospective study.

Little information is available on asymptomatic carriage of Cryptosporidium in immunocompetent and immunodeficient children. We prospectively studied a group of asymptomatic children, 78 immunocompetent and 50 immunodeficient, to document the incidence of asymptomatic carriage of cryptosporidiosis in such a population. We also investigated whether the treatment of children who carried asymptomatic cryptosporidiosis could help in reducing their risk of gastrointestinal symptoms as well as the shedding of infectious oocysts. The occurrence of multiple infections with common intestinal pathogens including Giardia lamblia was also investigated. Asymptomatic cryptosporidiosis was documented in 6.4% of immunocompetent and 22% of immunodeficient children. In a control symptomatic population Cryptosporidium was found in 4.4% of immunocompetent and 4.8% of immunodeficient children. Asymptomatic carriage of Cryptosporidium was documented in 2 human immunodeficiency virus-infected children, one of whom also carried Giardia asymptomatically. Treatment with spiramycin (100 mg/kg daily for 14 days) reduced significantly the duration of the shedding of potentially infectious oocysts. Finally no gastrointestinal symptoms developed in children treated for asymptomatic infection with Cryptosporidium, whereas children who were not treated developed gastrointestinal symptoms.

Current controversies in oral rehydration solution formulation.

Although the importance of oral rehydration therapy for acute diarrhea is unquestioned, controversy remains about the preparation and formulation of the oral rehydration solutions (ORS). There is disagreement about whether the ORS should be homemade or commercially prepared, what constitutes the optimal sodium content, what base (if any) should be present in the ORS, and whether other nutrients can be substituted entirely or in part for glucose. These issues are discussed and recommendations for the ideal ORS are presented.

Evidence favouring the gastro-oral route in the transmission of Helicobacter pylori infection in children.

OBJECTIVE: Several studies support the view that Helicobacter pylori is acquired in early life and within families. However, the exact route of transmission remains unknown. Given that H. pylori colonizes only the human gastric mucosa, the hypothesis that history of vomiting in siblings may be a relevant risk factor was tested in a paediatric setting. METHODS: One hundred urban children (age range 0.8-16.6 years, median 9), 44% with evidence of active H. pylori infection, were recruited. A structured questionnaire dealing with socio-economic issues was completed. Vomiting siblings and siblings of vomiting index children were screened for H. pylori by means of (13)C-urea breath test. Serum samples from index children were assayed for immunoglobulin G to hepatitis A (HAV) and Epstein-Barr virus (EBV) in order to check for faecal-oral and oral-oral exposure, respectively. RESULTS: Vomiting siblings of H. pylori-infected index children and siblings of H. pylori-infected vomiting index children had a high rate of active H. pylori infection (60 and 67%, respectively). History of vomiting in siblings was positively associated with active H. pylori infection in the index children (multivariate odds ratio 2.4, 95% confidence interval 1.3-4.3). Seropositivity for HAV and EBV was found in 1 and 68 index children, respectively. The agreement between active H. pylori infection and EBV seropositivity was not significant (kappa = 0.26). CONCLUSIONS: History of vomiting in siblings is an independent risk factor for H. pylori. Nowadays, transmission of H. pylori in urban children may involve the gastro-oral route more than the faecal-oral or oral-oral pathways.

Use of Lactobacillus-GG in paediatric Crohn's disease.

The potential role of luminal bacteria in initiating the abnormal immune response seen in inflammatory bowel disease is stressed by many observations. A defect in mucosal barrier function could allow luminal bacterial antigens to initiate the chronic relapsing inflammation in Crohn's disease. The potential role of luminal bacteria in initiating the abnormal immune response seen in inflammatory bowel disease is stressed by many observations. A pilot study to investigate the possible effect of Lactobacillus GG in children with active Crohn's disease was conducted. Four male patients were enrolled, median age 14.5 years (range 10-18). In terms of clinical outcome, the patients showed significant improvement. In three patients on Lactobacillus GG, it was possible to taper the dose of steroids. Thus, although our data are obviously very preliminary, Lactobacillus GG appears to be effective in improving the clinical status of children with Crohn's disease. A multicentre study is currently being carried out in 7 US University centres in a randomized, double-blind, placebo-controlled fashion to establish the efficacy of this probiotic in children with Crohn's disease.

In vitro effects of conalbumin on iron transport by rat intestinal mucosa.

The possible effects of conalbumin (a glycoprotein closely resembling lactoferrin) on iron transport across the brush border of intestinal cells of the rat were evaluated in vitro. Iron influx was measured in proximal and distal tracts of small intestine from either normal or iron-deprived rats, both in the presence of 1 or 10 mg/ml conalbumin and in its absence. Conalbumin did not affect iron uptake at any concentration and in any intestinal segment, either in normal or in iron-deprived animals. It was concluded that the luminal presence of conalbumin, even at a relatively high concentration, does not interfere with iron intestinal transport.

[Oral rehydration in infants with acute diarrhea: using a new preparation of reduced osmolarity]. / Reidratazione orale in bambini con diarrea acuta: utilizzo di una preparazione a ridotta osmolarità.

Oral rehydration solutions (ORS) are widely employed in the treatment of acute diarrhea in children. Although several studies in the recent literature prove the efficacy and safety of newly formulated, reduced-osmolarity solutions, and a specific working group of the European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) has made similar recommendations, such evidence in our Country is lacking. To this aim, we enrolled 38 children (age from 3 to 24 months, average: 8.2) with acute diarrhea lasting from no more than 5 days (average: 2.5). At observation, dehydration was found to be: absent in 12 (31.6%), mild in 19 (50%), moderate in 5 (13.2%), severe in 2 (5.2%). The last 2 patients were admitted and treated i.v., thus excluded from the study. In the 36 remaining children, a pathogen was identified in 25 (69.4%): Rotavirus in 18, Salmonella in 4, EPEC in 2, Cryptosporidium in 1. Stool Clinitest was positive in 7/31 children, all with Rotavirus infection. All the patients received ad libitum for the first 4-6 hours exclusively a reduced-osmolarity ORS formulated according to the ESPGAN criteria. Afterwards, they were fully re-fed and were also given the same ORS until diarrhea stopped. Most of the children accepted the ORS; those who refused it were either not dehydrated and/or older than 15 months. Thirty-three out of 36 children were fully rehydrated after 12 hours, without any side effects. We conclude that such an ORS is safe and effective in the treatment of children with acute diarrhea in our settings.