Juan R. Malagelada

Diarrhoea-predominant IBS patients show mast cell activation and hyperplasia in the jejunum

Background: Increased numbers of mast cells and mast cell activation in distal gut segments are associated with symptom onset and severity in irritable bowel syndrome (IBS). Although upper gut symptoms are common, mast cells have not been thoroughly evaluated in proximal gut in IBS patients. Methods: Jejunal biopsies obtained by Watson’s capsule, aspiration of intestinal fluid and one blood sample were obtained in 20 diarrhoea-predominant patients with IBS (D-IBS) and 14 healthy volunteers (H). Psychological stress (Holmes-Rahe Scale) and depression (Beck’s Depression Inventory) were evaluated at baseline and food and respiratory allergy excluded. Biopsies were processed for H&E staining and microscopic inflammation assessed by counting intraepithelial lymphocytes. Mast cells in lamina propria were counted by immunohistochemistry with CD117 (c-kit). Tryptase concentration was measured in intestinal fluid and serum. Results: D-IBS patients showed higher psychological stress than healthy volunteers (D-IBS: 203 (SD 114) v H: 112 (SD 99); p = 0.019). Immunohistochemical staining of jejunal mucosa revealed mild increase in intraepithelial CD3+ cells in D-IBS patients (D-IBS: 15.3 (SD 5.5; 95% CI 12.7 to 17.9) v H: 10.3 (SD 3.9; 95% CI 8.0 to 12.5); p = 0.006). Moreover, D-IBS patients showed marked increase in mast cells numbers (D-IBS: 34 (SD 9.3); H: 15.3 (SD 4.4) mast cells/hpf; p<0.001) and higher tryptase concentration in jejunal fluid (D-IBS: 0.45 (SD 0.38); H: 0.09 (SD 0.10) µg/l; p = 0.005). Upper gut symptoms were not associated with gender, mast cell counts, jejunal tryptase or basal stress. Conclusion: This jejunal mucosal inflammatory profile may help identify diarrhoea-predominant IBS, a stress-related disorder.

Increased mucosal tumour necrosis factor alpha production in Crohn's disease can be downregulated ex vivo by probiotic bacteria.

Background and aims: Tumour necrosis factor α (TNF-α) plays a key role in the pathogenesis of intestinal inflammation in Crohn’s disease. The effect of bacteria on TNF-α release by intestinal mucosa was investigated. Methods: Ileal specimens were obtained at surgery from 10 patients with Crohn’s disease (ileal stricture) and five disease controls undergoing right hemicolectomy (caecal cancer). Mucosal explants from each specimen were cultured for 24 hours with either non-pathogenic Escherichia coli, Lactobacillus casei DN-114001, L bulgaricus LB10, or L crispatus (each study contained blank wells with no bacteria). Tissue and bacterial viability was confirmed by lactate dehydrogenase (LDH) release and culture. Concentrations of TNF-α were measured in supernatants and the phenotype of the intestinal lymphocytes was analysed by flow cytometry. Results: Coculture of mucosa with bacteria did not modify LDH release. Release of TNF-α by inflamed Crohn’s disease mucosa was significantly reduced by coculture with L casei or L bulgaricus; changes induced by L crispatus or E coli were not significant. The effect of L casei and L bulgaricus was not prevented by protease inhibitors. Coculture with L casei and L bulgaricus reduced the number of CD4 cells as well as TNF-α expression among intraepithelial lymphocytes from Crohn’s disease mucosa. None of the bacteria induced changes in non-inflamed mucosa. Conclusions: Probiotics interact with immunocompetent cells using the mucosal interface and modulate locally the production of proinflammatory cytokines.

Gastric wall tension determines perception of gastric distention

BACKGROUND & AIMS The primary mechanism that originates symptoms in response to gastric distention remains undefined. The aim of this study was to determine which factor, whether intragastric volume, pressure, or wall tension, determines perception of gastric distention. METHODS Healthy subjects underwent increasing gastric distentions (2-minute duration at 5-minute intervals) either at fixed pressure levels using a conventional barostat (n = 10) or at fixed tension levels using a newly developed computerized tensostat (n = 12); perception was scored by a 0-6 scale. Distentions were performed during basal conditions (intravenous saline) and during gastric relaxation by glucagon administration (4.8 microgram/kg intravenous bolus plus 9.6 microgram. kg-1. h-1 infusion). RESULTS Isobaric distentions with the conventional barostat produced more intense perception during glucagon (95% +/- 40% higher; P < 0.05). However, the factor that determined higher perception could not be ascertained, because at the same pressure levels both intragastric volume and wall tension were greater during glucagon administration (174% +/- 56% and 34% +/- 8% greater, respectively; P < 0.05 vs. saline for both). The tensostat evidenced that perception was selectively related to tension, not to elongation; during glucagon administration, intragastric volumes were significantly larger (80% +/- 28% larger increase; P < 0.05), but perception of isotonic distentions remained the same (27% +/- 22%; nonsignificant change). CONCLUSIONS Gastric wall tension, but not intragastric volume, determines perception of gastric distention, at least below nociception.

Release of mast cell mediators into the jejunum by cold pain stress in humans.

BACKGROUND & AIMS The central nervous system regulates gut functions via complex interactions between the enteric nervous and immune systems. The aim of this study was to investigate whether mast cell mediators are released into the human jejunal lumen during stress. METHODS A closed-segment perfusion technique was used to investigate jejunal release of tryptase, histamine, prostaglandin D2, and water flux in response to the cold pressor test in 8 healthy subjects and 9 patients with food allergy. In 6 food-allergic patients, jejunal biochemical responses to cold pain stress were compared with those induced by food intraluminal challenge. RESULTS Cold pain stress elevated heart rate and blood pressure and increased luminal release of mast cell mediators and jejunal water secretion in both groups. Stress-induced release of tryptase and histamine, but not of prostaglandin D2 and water flux, was greater in food-allergic patients than in healthy volunteers. In food-allergic patients, jejunal biochemical responses induced by cold pain stress were similar to those induced by antigen challenge. CONCLUSIONS These results show the ability of the central nervous system to modulate intestinal mast cell activity and suggest that mast cells have a role in stress-related gut dysfunction.

Dietary inulin improves distal colitis induced by dextran sodium sulfate in the rat

OBJECTIVES:Inulin stimulates intracolonic generation of butyrate and growth of lactic acid bacteria. This study investigated whether inulin protects against colitis.METHODS:Rats with dextran sodium sulfate colitis received inulin either orally (1% in drinking water, or 400 mg/day) or by enema. Matched groups received vehicle. In addition, fecal water obtained from inulin-fed rats was administered by enema to rats with colitis and compared with fecal water from control rats. Finally, rats with colitis received daily enemas of either butyrate (at 40 or 80 mmol/L) or vehicle. Inflammation was assessed by eicosanoid asssay in rectal dialysates and MPO activity in colonic tissue. Mucosal lesions were blindly scored by microscopic examination. Luminal pH was measured from cecum to rectum by a surface microelectrode.RESULTS:Oral inulin prevented inflammation, as evidenced by lower lesion scores (p < 0.05), decreased release of mediators (p < 0.05), and lower tissue MPO (p < 0.05) as compared with controls. Inulin induced acidic environment (pH <7.0) from cecum to left colon and increased counts of lactobacilli. Fecal water from inulin-fed rats also reduced scores (p < 0.05) and inflammation (p < 0.05). However, inulin or butyrate enemas had no effect.CONCLUSIONS:Oral inulin reduces the severity of dextran sodium sulfate colitis. The effect seems to be mediated by modification of the intracolonic milieu.

Intestinal gas dynamics and tolerance in humans

BACKGROUND & AIMS Abdominal symptoms are often attributed to intestinal gas. In humans, gas production and composition have been previously investigated, but intestinal gas dynamics and tolerance remain virtually unknown. The aim of this study was to establish the relationship between intestinal gas loads, evacuation, perception of symptoms, and objective abdominal distention in healthy humans. METHODS A dose-response study was performed in 46 healthy subjects; intestinal gas was infused for 3 hours (0, 1, 4, 12, and 30 mL/min), and anal gas evacuation, symptom perception, and abdominal distention were measured. A mixture of gases was infused in venous proportions to minimize diffusion. Anal gas recovery and calculations of gas retention were validated using sulfur hexafluoride as a nonabsorbable gaseous marker. RESULTS At all of the infusion rates, gas evacuation rapidly equilibrated and paralleled gas infusion without significant differences in perception. Only 6 subjects retained >400 mL gas, and 5 of them developed abdominal distention and symptoms. By contrast, all but 4 of the 41 subjects without retention tolerated the gas loads without discomfort. CONCLUSIONS Intestinal gas tolerance is normally high, because expeditious gas transit and evacuation prevent gas pooling and symptoms. When this protective mechanism is insufficient, gas retention occurs, and it causes abdominal symptoms and distention.

Effects of Nonpathogenic Bacteria on Cytokine Secretion by Human Intestinal Mucosa

OBJECTIVE:The human intestine harbors a complex microbial ecosystem, and the mucosa is the interface between the immune system and the luminal environment. The aim of this study was to elucidate whether host–bacteria interactions influence mucosal cytokine production.METHODS:Macroscopically normal colonic specimens were obtained at surgery from eight patients with neoplasm, and inflamed ileal specimens were obtained from two patients with Crohns disease. Mucosal explants were cultured for 24 h with either nonpathogenic Escherichia coli ECOR-26, Lactobacillus casei DN-114 001, L. casei DN-114 056, L. casei ATCC-334, or Lactobacillus bulgaricus LB-10. Each study included blank wells with no bacteria. Tissue and bacteria viability were confirmed by LDH release and culture. Concentration of tumor necrosis factor (TNF)α, transforming growth factor β1, interleukin (IL)-8, and IL-10 was measured in supernatants. In parallel experiments, neutralizing anti-TNFα antibody was added to the culture.RESULTS:Co-culture of mucosa with bacteria did not modify LDH release. Co-culture with L. casei strains significantly reduced TNFα release, whereas E. coli increased it. These effects were observed both in normal and inflamed mucosa. In combination studies, L. casei DN-114 001 prevented TNFα stimulation by E. coli. L. casei DN-114 001 also reduced IL-8 release via a TNFα-independent pathway. L. casei DN-114 056 or E. coli increased IL-10 release in the presence of neutralizing anti-TNFα.CONCLUSIONS:Nonpathogenic bacteria interact with human intestinal mucosa and can induce changes in cytokine production that are strain specific.

Increased bacterial degradation of bile acids in cholecystectomized patients.

Bile acid metabolism was studied in 10 patients previously cholecystectomized for cholesterol cholelithiasis, in 12 healthy subjects, and in 13 patients with cholesterol cholelithiasis (disease control subjects). The rate of bacterial deconjugation and 7-dehydroxylation was measured by using [2, 4- 3 H ]cholyl- [1- 14 C]glycine; measurements of synthesis and pool size were made simultaneously. Total bile acid pool size and biliary bile acid composition were determined by gas-liquid chromatography. Compared with healthy subjects, patients with cholecystectomy showed significantly increased deconjugation and dehydroxylation of the labeled cholylglycine. However, the pool of cholylglycine was considerably smaller, so that the absolute rate of deconjugation was similar. The pool of deoxycholylglycine was twice as large as that in the healthy control and the disease control subjects. In addition, ketohydroxy bile acids (resulting from bacterial dehydrogenation) composed 7 to 15% of the bile acid pool. The 7-keto and 12-keto derivatives of cholic acid were identified by mass spectrometry. The total bile acid pool in cholecystectomized patients did not differ from that in healthy control subjects but was larger than that in the disease control subjects. The size of the total bile acid pool in cholecystectomized patients correlated highly with the size of the pool of secondary bile acids, suggesting that its apparent return to a normal size in these patients after cholecystectomy could be explained in part by the increased input of secondary bile acids from the intestine.

Lactobacillus casei downregulates commensals' inflammatory signals in Crohn's disease mucosa

Background: The interaction of commensal bacteria with the intestinal immune system is an essential factor in the development of inflammatory bowel disease (IBD). The study of isolated commensal bacterias effects on the mucosal immune response might be relevant for a better understanding of pathophysiological mechanisms in IBD. Methods: We investigated the immune responses to signals from the commensal Escherichia coli ATCC 35345 and the probiotic Lactobacillus casei DN‐114 001 in Crohns disease (CD) mucosa. Ileal specimens were obtained during surgery from CD patients. Mucosal explants were incubated with L. casei or its genomic DNA; TNF‐&agr;, IFN‐&ggr;, IL‐2, IL‐6, IL‐8, and CXCL1 were measured in the supernatant. Second, tissue expression of key proinflammatory cytokines (IL‐6, TGF‐&bgr;, IL‐23p19, IL‐12p35, IL‐17F), and chemokines (IL‐8, CXCL1, CXCL2) was evaluated after incubation with L. casei or E. coli. Finally, combination experiments were carried out by incubating both strains with mucosal explants at different timepoints. Results: Live L. casei significantly decreased secretion of TNF‐&agr;, IFN‐&ggr;, IL‐2, IL‐6, IL‐8, and CXCL1 by CD mucosa, but the effect was not reproduced by L. casei DNA. Second, live L. casei downregulated expression of IL‐8, IL‐6, and CXCL1 and did not modify expression of IL‐23p19, IL‐12p35, and IL‐17F. In contrast, E. coli significantly upregulated expression of all these cytokines. Interestingly, combination experiments revealed the ability of L. casei to prevent and counteract the proinflammatory effects of E. coli. Conclusions: Live L. casei can counteract the proinflammatory effects of E. coli on CD inflamed mucosa by specific downregulation of key proinflammatory mediators.

Altered Pancreatic and Biliary Function after Vagotomy and Pyloroplasty

Changes in pancreatic enzyme secretion and gallbladder contraction occurring after vagotomy and pyloroplasty were investigated and compared with findings from healthy volunteers and patients with duodenal ulcer. Total lipase and bile acid outputs were quantified, using duodenal perfusion, under basal conditions and in response to both endogenous cholecystokinin-pancreozymin (CCK-PZ) (released by intraduodenal perfusion of essential amino acids) and to exogenous (porcine) CCK-PZ given by vein. After vagotomy and pyloroplasty, basal pancreatic enzyme outputs were reduced, as were responses to both intraduodenal essential amino acid perfusion and lower doses of intravenous CCK-PZ, but the response to maximal doses of intravenous CCK-PZ was normal. By contrast, gallbladder contraction occurred at a lower dose of intravenous CCK-PZ than in healthy subjects or in patients with duodenal ulcer, although the response to intraduodenal essential amino acids was normal. Our results suggest that (a) vagal impulses contribute to pancreatic enzyme secretion in the interdigestive periods; and (b) after vagotomy and pyloroplasty, sensitivity of the pancreas to CCK-PZ decreases and that of the gallbladder increases.