Differential expression of enteric neuroimmune-network in invasive and acute watery diarrhoea.

We aimed to evaluate the changes of nerve morphology and distribution of neurotransmitters and neuropeptides in the rectum of Shigella flexneri-infected patients and in the duodenum of Vibrio cholerae O1-infected patients. Nerve morphology was observed by transmission electron microscopy. Immunoreactivity of nerve growth factor (NGF), neurotransmitters and neuropeptides in tissues were studied by immunohistochemistry. Ultrastructural analysis of intestinal biopsy revealed persisting axons degeneration throughout the study period in all patients. Regeneration was already evident at the acute stage with marked increase at late convalescence. Both acute shigellosis and cholera were accompanied by increased expression of NGF and histamine and decreased expression of serotonin that was restored at convalescence. Immunoreactivity of vasoactive intestinal peptide (VIP) was increased during acute cholera, whereas in shigellosis VIP- and substance P-immunoreactive nerves appeared at early convalescence. Both shigellosis and cholera induced long-lasting degeneration of enteric neuronal axons, despite the presence of ongoing proliferation and regeneration processes. Neurotransmitters and neuropeptides may play differential roles in invasive and watery diarrhoea.

Acute dehydrating disease caused by Vibrio cholerae serogroups O1 and O139 induce increases in innate cells and inflammatory mediators at the mucosal surface of the gut.

BACKGROUND AND AIMS: The general concept is that as Vibrio cholerae is not invasive, it mediates a non-inflammatory type of infection. This is being re-evaluated based on available data that natural cholera infection or cholera toxin induces a Th2-type of immune profile and stimulates the humoral immune response, innate cells, and mediators in the host. METHODS: To perform a comprehensive analyses of the inflammatory components, we studied mucosal biopsies from patients, both adults and children with acute watery diarrhoea caused by V cholerae O1 and O139. Patients with cholera, adults (n = 30) and children (n = 18), as well as healthy controls (n = 24) were studied. Histochemical, immunohistochemical, and ultrastructural studies were carried out to elucidate the contribution of the different factors using paraffin and frozen duodenal and/or rectal sections as appropriate. Samples were collected during the acute stage and during early and/or late convalescence. RESULTS: Following natural cholera infection, patients responded with increases in neutrophil polymorphs during the acute stage (p<0.001) compared with healthy controls whereas mucosal mast cells (MMC) (p = 0.008) and eosinophils (p = 0.034) increased in the gut during convalescence. Electron microscopic analyses of duodenal biopsies from adult patients showed increased piecemeal degranulation in both MMC and eosinophils and accumulation of lipid bodies in MMC. Duodenal biopsies from V cholerae O1 infected patients showed upregulation of myeloperoxidase, lactoferrin, PGHS-1, SCF, tryptase, tumour necrosis factor alpha, alpha-defensin, and eotaxin during the acute stage and chymase, interleukin 3 and major basic protein during convalescence. CONCLUSION: We have shown that innate cells and their mediators are upregulated in acute watery diarrhoea. These cells and factors of the innate arm may be important in the host's defence against cholera. Such effects may need to be simulated in a vaccine to achieve long lasting protection from cholera.

Epidemiological and laboratory investigations of outbreaks of diarrhoea in rural South India: implications for control of disease.

Two epidemics of acute, watery diarrhoea in villages in North Arcot district, India, were investigated. The attack rates were 10.03 and 15.53 per 100 population, the median duration was 5 days and enteric pathogens were present in 56.8% and 60.3% of specimens from the two villages, but no predominant pathogen was identified. Examination of stools from a 20% age-stratified random sample of the population of one of the villages after the epidemic found 22.9% of asymptomatic subjects excreted bacterial enteric pathogens. Despite the high background of enteric pathogen carriage, the isolation rates for shigellae, enteropathogenic Escherichia coli and Shiga-toxin producing E. coli were significantly higher (P < 0.001, P < 0.02, P < 0.05) during the epidemic. The epidemics may have been caused by faecal contamination of well water following rain. Point-of-use techniques for water disinfection may be most effective for preventing such outbreaks, but further research into the development of appropriate technology is required.

Enteroaggregative Escherichia coli infection in a rabbit model.

Type strains of enteroaggregative Escherichia coli EAEC (17-2, serotype O3:H2; JM 221, serotype O92:H33), isolates from an adult and a child with diarrhoea and an asymptomatic colonised child were used to orally infect adult rabbits. The experimental animals were followed up and sacrificed at defined time periods. Colonisation of both small and large intestine was seen with all strains and isolates used. Isolates from an adult patient with diarrhoea (MP 27) and from an asymptomatic colonised child from the community (KM 1337) were recovered from the small intestine during the first week of infection and subsequently from the large intestine. A total of seven rabbits was infected with MP 27; while colonising the gastrointestinal tract of all seven rabbits, this isolate caused diarrhoea in only one. On ultrastructural examination, the rabbits infected with 17-2 showed invasion of lymphoid follicles. Bacteria were seen in intercellular spaces and within M cells, a finding that has not previously been described. It is clearly possible to produce gut colonisation by oral infection with EAEC in adult rabbits with normal flora.

A monkey model for enterohemorrhagic Escherichia coli infection.

Adult Macaca radiata (n=22) were infected intragastrically with 10(12) Escherichia coli O157:H7 strain 84-01, which produces Shiga toxins 1 and 2. Clinical symptoms and bacterial excretion were documented in each monkey for a specified time period before they were killed. At necropsy, samples were obtained for culture and histologic and ultrastructural examination. Seventeen monkeys had diarrhea: E. coli O157 was isolated from postinfection stool samples from all monkeys and from autopsy cultures for 14 of 22 monkeys. Histologic examination showed attaching-effacing lesions, which appeared at 12 h and persisted for 7 days, in 12 monkeys. Widening of the intercellular spaces, degeneration and vacuolization of the epithelial cells, epithelial tufting, extrusion of epithelial cells, and neutrophilic infiltration were characteristic features seen in 20 of the 22 infected monkeys but not in 4 control monkeys. This monkey model closely parallels the early stages of the disease produced by E. coli O157:H7 and would be useful in the further study of pathogenic mechanisms and prevention methods in enterohemorrhagic E. coli infections.

A prospective study of nosocomial enteric pathogen acquisition in hospitalized children in South India.

Screening for enteric pathogens in stool samples from 249 children under the age of 36 months, admitted to hospital for non-gastrointestinal disorders, was positive at admission in 41 (16.4 per cent) in a prospective study of enteric pathogen acquisition and diarrhoea in hospitalized children. Infection with multiple organisms was found in 31/41 (75.6 per cent) children who were positive when screened at admission. Of 194 children who had no enteric pathogens on admission and could be followed up for 3 days after discharge, clinical or laboratory data showed nosocomial enteric infections in 39 (20.1 per cent). Presumed nosocomial infection with more than one organism was seen in only two patients and no pathogens were isolated in 14 (35.8 per cent). Children presenting to hospital may asymptomatically carry enteric pathogens and potentially act as a source of nosocomial infections. One in five children admitted into hospital without an enteric infection is at risk of developing a nosocomial gastrointestinal infection, with rotavirus being the most common aetiological agent.

Glomerular injury induced in mice by intraperitoneal injection of Shiga-like toxins.

BACKGROUND AND OBJECTIVES: Shiga-like toxins I and II (Stx1 and Stx2) play an important role in the pathogenesis of renal disease by causing renal microvascular injury. A murine model was used to study glomerular lesions produced by Stx1 and Stx2. METHODS: Swiss albino mice of the Rockefeller strain were inoculated intraperitoneally with LD(50) doses of endotoxin-free Stx1 of Stx2 and observed for signs of disease. Samples of renal cortical tissue from mice were examined with the electron microscope. RESULTS: the mice developed systemic and neurological symptoms including hind limb paralysis and generalised convulsions. Renal arteriolar damage and glomerular endothelial cytoplasmic swelling, vacuolation, lysis and intravascular coagulation were present and resembled the microangiopathy seen in renal biopsies from patients. INTERPRETATION AND CONCLUSIONS: these experiments establish the role of Stx1 and Stx2 in glomerular vascular injury and provide a model for studying the pathogenesis of Shiga-like toxin related microangiopathy.

Innate immune responses in children and adults with Shigellosis.

An array of pro- and anti-inflammatory mediators of the innate immune system was analyzed in stool, urine, and rectal mucosa samples from adults and children with shigellosis to better understand their role in recovery from and in the immunopathogenesis of the disease. Increased concentrations of lactoferrin (Lf), myeloperoxidase (MPO), prostaglandin E(2), and leukotriene B(4) (LTB(4)) in stool during acute shigellosis in both children and adults indicated that activated cells of the innate defense system at the mucosal site were secreting the mediators. Increased concentration of MPO and 8-iso-prostaglandin F(2alpha) and lower levels of superoxide dismutase (SOD) activity in stool during acute Shigella infection suggested increased formation of reactive oxygen species, free radical-catalyzed peroxidation of membrane lipids, and decreased scavenging of the reactive oxygen radicals. In children, lower expression of SOD in tissue with severe inflammation and lower levels of SOD activity in stool for longer periods compared to adults may further worsen the tissue damage and predispose the children to a lowered defense. Both adult and pediatric patients had significantly higher expression of inducible nitric oxide synthase (iNOS) in the rectum with severe inflammation, compared to that seen with mild inflammation, accompanied by persistently up-regulated iNOS mRNA, reflecting increased production of nitric oxide at the local site. However, in contrast to adults, reduced urinary nitrate levels in pediatric patients during acute shigellosis suggested lower production of nitric oxide in the renal compartment. Persistent production of Lf in pediatric patients may contribute to chronic inflammation in the rectum. In addition, increased production of proinflammatory mediators in the rectum of patients with severe histology suggested contribution of these molecules to the immunopathogenesis of severe colitis caused by shigellae.

Butyrate and glucose metabolism by colonocytes in experimental colitis in mice.

BACKGROUND/AIMS: Impaired colonocyte metabolism of butyrate has been implicated in the aetiopathogenesis of ulcerative colitis. Colonocyte butyrate metabolism was investigated in experimental colitis in mice. METHODS: Colitis was induced in Swiss outbred white mice by oral administration of 4% dextran sulphate sodium (DSS). Colonocytes isolated from colitic and normal control mice were incubated with [(14)C]butyrate or glucose, and production of (14)CO(2), as well as of intermediate metabolites (acetoacetate, beta-hydroxybutyrate and lactate), was measured. The effect of different substrate concentrations on oxidation was also examined. RESULTS: Butyrate oxidation (micromol/h per mg protein; mean (SEM)) was significantly reduced in DSS colitis, values on day 7 of DSS administration being 0.177 (0.007) compared with 0.406 (0.035) for control animals (p<0.001). Glucose oxidation (micromol/h per mg protein; mean (SEM)) on day 7 of DSS administration was significantly higher than in controls (0.06 (0.006) v 0.027 (0.004), p<0.001). Production of beta-hydroxybutyrate was decreased and production of lactate increased in DSS colitis compared with controls. Increasing butyrate concentration from 10 to 80 mM enhanced oxidation in DSS colitis (0.036 (0.002) to 0.285 (0.040), p<0.001), although it continued to remain lower than in controls. Surface and crypt epithelial cells showed similar ratios of butyrate to glucose oxidation. When 1 mM DSS was added to normal colonocytes in vitro, it did not alter butyrate oxidation. The initial histological lesion of DSS administration was very patchy and involved crypt cells. Abnormal butyrate oxidation became apparent only after six days of DSS administration, at which time histological abnormalities were more widespread. CONCLUSIONS: Colonocyte metabolism of butyrate, but not of glucose, is impaired in DSS colitis, and may be important in pathophysiology. Histological abnormalities preceded measurable defects in butyrate oxidation.

Prevalence of enteroaggregative and other HEp-2 cell adherent Escherichia coli in asymptomatic rural south Indians by longitudinal sampling.

Enteroaggregative and other HEp-2 cell adherent Escherichia coli can produce acute and persistent diarrhoea in children and adults, but their prevalence in asymptomatic individuals in the community is not known. In this study, faecal specimens were obtained at 3-4 monthly intervals from 349 subjects constituting a 20% age-stratified sample of a rural community for a period of two years. HEp-2 cell adherent E. coli were found in 210 subjects, and repeat isolations of enteroaggregative E. coli belonging to the same serogroup were found in 12.6% of children less than 12 years of age, indicating that this organism can asymptomatically colonise the intestinal tract. These children may act as a reservoir of infection for the community.

Use of serology, the urease test & histology in diagnosis of Helicobacter pylori infection in symptomatic & asymptomatic Indians.

Age-specific prevalence of IgA and IgG antibodies in 714 subjects without gastrointestinal complaints aged 6 months to 90 yr was measured by an enzyme linked immunoassay using an acid-glycine extract of H. pylori as the antigen. The urease test and histology were used for the diagnosis of H. pylori infection in 83 subjects with a clinical diagnosis of dyspepsia, and these results were compared with measurement of IgG, IgA and IgM antibodies. The age specific prevalence of IgG and IgA antibodies respectively was 57 and 43 per cent for subjects aged 6 months to 4 yr and showed an increase with age to a maximum of 90 per cent for IgG in subjects > 60 yr of age and to 87 per cent for IgA in subjects between 51 and 60 yr. In symptomatic patients, there was a high degree of correlation between severity of H. pylori infection on histopathological examination and IgG (P < 0.02) levels. The use of IgG and IgA estimation could have identified H. pylori infection without endoscopy in 50 of the 83 patients. Serology for IgG and IgA antibodies against H. pylori may play a major role in decreasing the need for endoscopy, but cut-off values must be determined for each assay based on the prevalence of antibodies in the population.

Cytomegalovirus infection of the human gastrointestinal tract.

BACKGROUND: Current interest in cytomegalovirus (CMV) is largely due to an increase in the number of cases of acquired immunodeficiency syndrome and organ transplantation in recent years. The proper recognition of CMV-infected cells in gastrointestinal mucosal biopsies is critical for effective treatment of this condition. METHODS: A total of 6580 endoscopic mucosal biopsies from 6323 patients in the 8-year period (1989-1996) were examined for CMV inclusion bodies. The endoscopic appearance and particularly the presence of ulcers were also analysed. RESULTS AND CONCLUSIONS: The prevalence of cytomegalovirus (CMV) inclusions was 9 per thousand in the gastrointestinal mucosal biopsies from an unselected group of patients. Of the 54 patients with CMV infection, 37 were immunocompromised and 17 apparently immunocompetent. Typical Cowdry inclusions and atypical inclusions were present, the latter more frequently in immunocompromised patients. The maximum prevalence of inclusions was in the oesophageal mucosa in immunocompromised individuals.

Enteric pathogens in southern Indian HIV-infected patients with & without diarrhoea.

This study was undertaken to determine the carriage rate of various enteric pathogens in southern Indian patients with HIV infection, both with and without diarrhoea. Stool from 111 consecutive HIV-positive patients (50 without and 61 with diarrhoea) was examined by microscopy and culture. Jejunal biopsy and fluid examination were carried out if diarrhoea persisted, with negative stool examination. Enteric pathogens were detected from stool in 57.4 per cent of diarrhoeal patients compared to 40 per cent of those without diarrhoea (P > 0.05). Jejunal biopsy and fluid examination provided 11 additional diagnoses. Protozoa accounted for 71.8 per cent of all pathogens isolated. Isospora was significantly more common in patients with (11/61) than in those without (2/50) diarrhoea (P < 0.05). Bacterial pathogens were isolated more commonly from patients with diarrhoea (12/61 compared to 2/50, P < 0.05). Isolation rate of pathogens was higher from patients with diarrhoea for more than 2 wk, compared to those with less than 2 wk duration. Remission of diarrhoea either spontaneously or with symptomatic therapy was observed in 22 patients with acute diarrhoea. A high enteric carriage of a number of pathogens was noted in HIV patients without diarrhoea, but I. belli and bacterial enteropathogens were more likely to be associated with diarrhoea.

Effect of shiga toxin on NaCl transport in rat distal colon.

BACKGROUND: Shiga toxin causes net fluid secretion in rabbit jejunum by selectively targeting, and inhibiting protein synthesis in, absorptive villous cells. The effect of Shiga toxin on the colon, where it is presumably produced, is not known. This study was undertaken to investigate the effect of Shiga toxin on the rat distal colon. METHODS: Net absorption of water and Na was determined by in vivo perfusion of closed loops of rat colon pre-exposed to Shiga toxin or saline. Unidirectional and net fluxes of 22Na and 36Cl were measured in vitro under voltage-clamp conditions across rat distal colon mucosa pre-exposed to Shiga toxin. Shiga toxin binding to sections of rat distal colon was localized by immunohistochemistry. Protein synthesis was measured in surface and crypt colonocytes with 3H-leucine incorporation. RESULTS: In the in vivo perfusion studies net absorption of Na and water was increased in Shiga toxin-treated colon compared with controls (P < 0.01). In the studies carried out in vitro, J(net)Na and J9net)Cl across Shiga toxin-treated mucosa were found to be significantly higher than in control tissue (P < 0.001 and P < 0.01, respectively). Net absorption of Na or Cl did not increase further in the presence of 25 mM butyrate, indicating the absence of short-chain fatty acids (SCFA)-linked NaCl absorption in Shiga toxin-treated colon. Moreover, Shiga toxin-treated colon failed to respond to theophylline, which induced secretion in the normal colon. Immunohistochemistry showed Shiga toxin binding to crypt cells but not to surface cells in the distal colon. Shiga toxin inhibited protein synthesis (by 27.3%) in crypt cells but not in surface cells (P < 0.05). CONCLUSIONS: An unexpected increase in water and NaCl absorption was noted in Shiga toxin-treated rat distal colon, which appears to result from selective effects of the toxin on secretory crypt cells.

Endoscopic mucosal biopsies are useful in distinguishing granulomatous colitis due to Crohn's disease from tuberculosis.

BACKGROUND: Intestinal tuberculosis and Crohn's disease are chronic granulomatous disorders that are difficult to differentiate histologically. AIMS: To characterise distinctive diagnostic features of tuberculosis and Crohn's disease in mucosal biopsy specimens obtained at colonoscopy. METHODS: Selected histological parameters were evaluated retrospectively in a total of 61 biopsy sites from 20 patients with tuberculosis and 112 biopsy sites from 20 patients with Crohn's disease. The patients were chosen on the basis of clinical history, colonoscopic findings, diagnostic histology, and response to treatment. RESULTS: The histological parameters characteristic of tuberculosis were multiple (mean number of granulomas per section: 5.35), large (mean widest diameter: 193 microm), confluent granulomas often with caseating necrosis. Other features were ulcers lined by conglomerate epithelioid histiocytes and disproportionate submucosal inflammation. The features characteristic of Crohn's disease were infrequent (mean number of granulomas per section: 0.75), small (mean widest diameter: 95 microm) granulomas, microgranulomas (defined as poorly organised collections of epithelioid histiocytes), focally enhanced colitis, and a high prevalence of chronic inflammation, even in endoscopically normal appearing areas. CONCLUSIONS: The type and frequency of granulomas, presence or absence of ulcers lined by epithelioid histiocytes and microgranulomas, and the distribution of chronic inflammation have been identified as histological parameters that can be used to differentiate tuberculosis and Crohn's disease in mucosal biopsy specimens obtained at colonoscopy.

Collagenous gastritis and collagenous colitis: a report with sequential histological and ultrastructural findings.

The case is reported of a young adult man with collagenous gastritis, an extremely rare disorder with only three case reports in the English literature, who subsequently presented with collagenous colitis. Sequential gastric biopsies showed a notable increase in thickness of the subepithelial collagen band. Ultrastructural study of gastric and rectal mucosa showed the characteristic subepithelial band composed of haphazardly arranged collagen fibres, prominent degranulating eosinophils, and activated pericryptal fibroblasts.

Salicylate inhibits fimbriae mediated HEp-2 cell adherence of and haemagglutination by enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAggEC) are associated with both acute and persistent diarrhoea in children. Bowel colonisation due to fimbrial adherence factors appears to play a major role in the disease process. In this study, we investigated the effect of sodium salicylate and 5-aminosalicylic acid on adherence of a type strain and 40 clinical isolates of EAggEC to HEp-2 cells and erythrocytes from different species. Growth in the presence of 10 mM salicylate resulted in markedly decreased adherence to tissue culture cells with 33/40 (82.5%) isolates, and was also associated with inhibition of haemagglutination in 20/33 (60.6%) isolates. Complete or partial inhibition of adherence was also seen in two of five isolates showing localised adherence and three of five isolates with diffuse adherence. Decrease in adherence was associated with decreased or absent expression of fimbriae in 28/40 (70%) of the EAggEC isolates, although production of outer membrane proteins was not affected. Salicylates appear to inhibit adherence mediated by fimbrial adhesins.

Eosinophilic gastroenteritis--an Indian experience.

Eosinophilic gastroenteritis is an uncommon disorder with varied presentation. Records of 7 patients (6 men) with eosinophilic gastroenteritis, presenting to a large referral hospital over a 10-year period, were analyzed. The patients were young adults (mean age 32 years) with short duration (median 2 months) of illness. Symptoms included abdominal pain and vomiting (100%), weight loss (57%), diarrhea (43%) and abdominal distension (43%). Peripheral eosinophilia was present in all the patients. The diagnosis was made at duodenal (2 patients) or cecal (1) biopsy or surgical full-thickness jejunal biopsy (4). Three patients had predominantly mucosal disease (Klein type I), whereas two patients each had predominantly muscular (type II) and submucosal (type III) disease. Surgical resection was curative in one patient with type II disease. The other six responded to prednisolone, with complete resolution in one patient.

Mucosal mitochondrial function and antioxidant defences in patients with gastric carcinoma.

BACKGROUND: Cancer cells have alterations in energy metabolism due to defective mitochondrial function. This may be due to generation of excessive free radicals and/or defective antioxidant enzyme systems. The aim of the present study was to assess mitochondrial function and antioxidant defences in the gastric mucosa of patients with gastric carcinoma (CA). METHODS: Gastric mucosal mitochondrial function was assessed by means of the reduction of tetrazolium dye (MTT), and levels of antioxidants such as glutathione S-transferase (GST), catalase, superoxide dismutase (SOD), and thiols were measured in biopsy specimens taken from the tumour mucosa (TM) and tumour-free (TF) mucosa, 2 cm away from the tumour, in 49 patients with gastric CA and compared with that in 54 controls. In a further 10 patients with gastric CA, these studies were done on TM and TF mucosa 2 cm and > or = 5 cm away from the tumour. In 10 patients and 5 controls, specimens were obtained for electron microscopy as well. Helicobacter pylori infection was diagnosed by means of histology. RESULTS: MTT reduction and GST and SOD activities were significantly decreased in TM and TF mucosa in patients with CA compared with controls (P < 0.01). The levels of thiols and catalase activity were significantly increased in CA as compared with controls (P < 0.01). H. pylori positivity did not influence most of these variables but did give significant decrease in MTT reduction in CA (TF) mucosa (P=0.01) and significant increase in thiol levels in CA (TM) mucosa (P=0.04). Electron microscopy showed mitochondrial alterations in tumour cells in all patients and in adjacent mucosa of 10%-50% of the cells. CONCLUSIONS: 1) In gastric CA the cancer mucosal cells and the non-involved cells adjacent to the tumour have defective mitochondrial function, which may be due to altered antioxidant defences and possibly altered free radical formation. 2) Ultrastructural mitochondrial abnormalities are shown to parallel these biochemical abnormalities.

Quantitative and ultrastructural analysis of rectal mucosal mast cells in acute infectious diarrhea.

The role of mast cells, potential mediators of mucosal immunity and inflammation, was studied morphologically in the rectal mucosa in two acute diarrheal diseases, cholera and shigellosis. Quantitation of mucosal mast cells showed that they were significantly higher in the deeper lamina propria where blood vessels and nerves were more abundant. There was no difference in mast cell counts or degranulation in the mucosa in both groups of patients and controls. Intraepithelial mast cells were decreased in the patients. The prevalence of lipid bodies was significantly higher in mast cells from patients with cholera and shigellosis (P < 0.01). These findings suggest that mast cell populations are more dense around blood vessels and nerves and that inflammatory mediators derived from arachidonic acid metabolites, as indicated by the lipid bodies, are the response of mast cells to the alterations in diarrhea, despite differences in the etiology of diarrhea.