Maria Antolin

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.

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.

Reshaping the gut microbiome with bacterial transplantation and antibiotic intake

The intestinal microbiota consists of over 1000 species, which play key roles in gut physiology and homeostasis. Imbalances in the composition of this bacterial community can lead to transient intestinal dysfunctions and chronic disease states. Understanding how to manipulate this ecosystem is thus essential for treating many disorders. In this study, we took advantage of recently developed tools for deep sequencing and phylogenetic clustering to examine the long-term effects of exogenous microbiota transplantation combined with and without an antibiotic pretreatment. In our rat model, deep sequencing revealed an intestinal bacterial diversity exceeding that of the human gut by a factor of two to three. The transplantation produced a marked increase in the microbial diversity of the recipients, which stemmed from both capture of new phylotypes and increase in abundance of others. However, when transplantation was performed after antibiotic intake, the resulting state simply combined the reshaping effects of the individual treatments (including the reduced diversity from antibiotic treatment alone). Therefore, lowering the recipient bacterial load by antibiotic intake prior to transplantation did not increase establishment of the donor phylotypes, although some dominant lineages still transferred successfully. Remarkably, all of these effects were observed after 1 mo of treatment and persisted after 3 mo. Overall, our results indicate that the indigenous gut microbial composition is more plastic that previously anticipated. However, since antibiotic pretreatment counterintuitively interferes with the establishment of an exogenous community, such plasticity is likely conditioned more by the altered microbiome gut homeostasis caused by antibiotics than by the primary bacterial loss.

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.

Unstable Composition of the Fecal Microbiota in Ulcerative Colitis During Clinical Remission

BACKGROUND AND AIM:Studies have identified abnormal characteristics of the gut microbiota in patients with active IBD, but whether the changes are causal or secondary to inflammation remains uncertain. We investigated dynamics of fecal microbiota in ulcerative colitis (UC) during remission by genomic technology.PATIENTS AND METHODS:Patients in clinical remission and on stable maintenance mesalazine therapy were recruited (N = 33). Fecal samples were collected at regular intervals over a period of 1 yr. Sixteen patients who remained in remission and eight healthy controls were included in the analysis. Variable V6 to V8 regions of the 16S rRNA gene in DNA extracts from fecal samples were amplified by polymerase chain reaction. Amplicons were separated by denaturant gradient gel electrophoresis, band profiles were compared by software, and similarity indices were calculated from densitometric curves.RESULTS:Band profiles showed unique patterns with low similarity index between individuals, suggesting host specificity in the predominant microbiota. Within the same individual, profiles were stable in controls but varied notably over time in patients. In controls, the similarity index was remarkably stable (78 ± 8% mean ± SD) over a period of 24 months. However, patients showed a steady decline in similarity index versus the initial profile, dropping down to 42 ± 24% at month 3 of follow-up and to 23 ± 19% at month 12 (P < 0.001). Biodiversity of the dominant microbiota, as estimated by number of bands, was lower in patients (17 ± 4) than controls (23 ± 4, P < 0.01).CONCLUSION:Molecular analysis of fecal bacteria in patients with inactive UC shows low biodiversity and temporal instability.

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.

Oral oligofructose-enriched inulin supplementation in acute ulcerative colitis is well tolerated and associated with lowered faecal calprotectin

Background Inulin and oligofructose promote selective growth of saccharolytic bacteria with low inflammatory potential.

Role of intestinal microflora in chronic inflammation and ulceration of the rat colon.

Bacteria and their products stimulate inflammatory responses. The effects of different antimicrobial regimens (amoxicillin/clavulanic acid, tobramycin, imipenem, vancomycin, metronidazole) were investigated on the course of experimental colitis induced by trinitrobenzenesulphonic acid (TNB) in the rat. On day 7 and 21 after the induction of colitis, matched groups of control and antibiotic treated rats were subjected to colonic dialysis to measure eicosanoid release, and killed for morphological assessment of the colonic lesions (macro and microscopic scores). Stool samples were cultured. Selective antibiotic treatment against Gram positive, Gram negative or anaerobic bacteria had no effect on colonic lesion scores. By contrast, certain broad spectrum antibiotics (amoxicillin/clavulanic acid or the association of imipenem plus vancomycin) significantly reduced macro and microscopic scores. Rats receiving these antibiotics did not develop chronic colitis as shown by the virtual absence of colonic strictures, adhesions, fibrosis, and granulomas. On day 21 after TNB, the intracolonic release of prostaglandin E2, thromboxane B2, and leukotriene B4 was significantly higher in control than in antibiotic treated rats. Control stool cultures showed abundant colony forming units of both aerobic and anaerobic bacteria. Amoxicillin/clavulanic acid and imipenem plus vancomycin induced appreciable reductions in luminal bacteria. In conclusion, certain broad spectrum antibiotics prevent chronic colitis. The normal colonic flora seems to play an important pathogenetic part in the progression of inflammatory colonic lesions to chronicity.

Colonisation by Faecalibacterium prausnitzii and maintenance of clinical remission in patients with ulcerative colitis

Although incrimination of the intestinal microbiota in the pathogenesis of IBD is widely accepted, few data are available about the role of specific bacteria. Potentially, Faecalibacterium prausnitzii, bacteria with anti‐inflammatory properties, might be deficient in ulcerative colitis (UC).

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.