Barry N. Hudspith

Molecular characterization of rectal mucosa-associated bacterial flora in inflammatory bowel disease.

Background: Colorectal bacteria may play a role in the pathogenesis of inflammatory bowel disease (IBD). To test the hypothesis that, in affected patients, the numbers of potentially protective mucosal bacteria might be reduced and pathogenic species increased, we compared rectal mucosa‐associated flora in patients with IBD and normal controls. Methods: Snap‐frozen rectal biopsies taken at routine diagnostic colonoscopy from 33 patients with ulcerative colitis, 6 patients with Crohns disease, and 14 controls with normal colonoscopy were processed, and individual bacterial groups were counted using fluorescent in situ hybridization. Results: Bacteria were mostly found apposed to the epithelial surface and within crypts. Epithelium‐associated counts of bifidobacteria in active [median 15/mm of epithelial surface (range, 4‐56), n = 14] and quiescent ulcerative colitis [26/mm (range, 11‐140), n = 19] were lower than in controls [56/mm (range, 0‐144), n = 14; P = 0.006 and P = 0.03, respectively]. Conversely, epithelium‐associated Escherichia coli counts were higher in active [82/mm (range, 56‐136)] than inactive ulcerative colitis [6/mm (range, 0‐136), P = 0.0001] or controls [0/mm (range, 0‐16), P < 0.0001]. Epithelium‐associated clostridia counts were also higher in active [3/mm (range, 0‐9)] than inactive colitis [0/mm (range, 0‐9), P = 0.03] or controls [0/mm (range, 0‐1); P = 0.0007]. Epithelium‐associated E. coli counts were higher in Crohns disease [42/mm (range, 3‐90), n = 6] than controls (P = 0.0006). E. coli were also found as individual bacteria and in clusters in the lamina propria in ulcerative colitis and Crohns disease but in none of the controls (P < 0.01). Numbers of Lactobacillus and Bacteroides showed no differences between patient groups. Conclusions: The reduction in mucosa‐associated bifidobacteria and increase in E. coli and clostridia in patients with IBD supports the hypothesis that an imbalance between potentially beneficial and pathogenic bacteria may contribute to its pathogenesis.

Distinct microbial populations exist in the mucosa-associated microbiota of sub-groups of irritable bowel syndrome

Background  There is increasing evidence to support a role for the gastrointestinal microbiota in the etiology of irritable bowel syndrome (IBS). Given the evidence of an inflammatory component to IBS, the mucosa‐associated microbiota potentially play a key role in its pathogenesis. The objectives were to compare the mucosa‐associated microbiota between patients with diarrhea predominant IBS (IBS‐D), constipation predominant IBS (IBS‐C) and controls using fluorescent in situ hybridization and to correlate specific bacteria groups with individual IBS symptoms.

Independent and population-specific association of risk variants at the IRGM locus with Crohn's disease

DNA polymorphisms in a region on chromosome 5q33.1 which contains two genes, immunity related GTPase related family, M (IRGM) and zinc finger protein 300 (ZNF300), are associated with Crohns disease (CD). The deleted allele of a 20 kb copy number variation (CNV) upstream of IRGM was recently shown to be in strong linkage disequilibrium (LD) with the CD-associated single nucleotide polymorphisms and is itself associated with CD (P < 0.01). The deletion was correlated with increased or reduced expression of IRGM in transformed cells in a cell line-dependent manner, and has been proposed as a likely causal variant. We report here that small insertion/deletion polymorphisms in the promoter and 5′ untranslated region of IRGM are, together with the CNV, strongly associated with CD (P = 1.37 × 10−5 to 1.40 × 10−9), and that the CNV and the 5′-untranslated region variant −308(GTTT)5 contribute independently to CD susceptibility (P = 2.6 × 10−7 and P = 2 × 10−5, respectively). We also show that the CD risk haplotype is associated with a significant decrease in IRGM expression (P < 10−12) in untransformed lymphocytes from CD patients. Further analysis of these variants in a Japanese CD case–control sample and of IRGM expression in HapMap populations revealed that neither the IRGM insertion/deletion polymorphisms nor the CNV was associated with CD or with altered IRGM expression in the Asian population. This suggests that the involvement of the IRGM risk haplotype in the pathogenesis of CD requires gene–gene or gene–environment interactions which are absent in Asian populations, or that none of the variants analysed are causal, and that the true causal variants arose after the European–Asian split.

The role of prostaglandins in the modulation of histamine suppression of mitogen responses in atopic and normal subjects.

We have shown that lymphocytes from atopics are more sensitive to histamine-induced suppression of mitogen transformation than are cells from normal subjects. Recent reports have suggested that histamine suppression may act via prostaglandin synthesis. We have investigated this and found that indomethacin partially reversed the histamine suppression seen in atopics, but not that in normals. This effect was seen even when the direct enhancing effect of indomethacin was taken into account. The direct effect of indomethacin on mitogen-induced transformation was similar in both normal and atopic cultures, which suggests that these results are not due to the presence of PG producing cells in atopic cultures, but rather that histamine modulates the function of these cells. It was found that a specific H2 agonist, but not an H1 agonist could partially mimic the effect of histamine this system.

Quantification and characterization of mucosa-associated and intracellular Escherichia coli in inflammatory bowel disease.

Background:Mucosa-associated Escherichia coli are abundant in inflammatory bowel disease (IBD), but whether these bacteria gain intracellular access within the mucosa is uncertain. If E. coli does gain intracellular access, the contribution of bacterial pathogenicity to this requires further elucidation. This study aimed to quantify and characterize mucosa-associated and intracellular E. coli in patients with IBD and in healthy control subjects (HC). Methods:Mucosal biopsies from 30 patients with Crohns disease (CD), 15 with ulcerative colitis (UC), and 14 HC were cultured with or without gentamicin protection to recover intracellular or mucosa-associated E. coli, respectively. Overall, 40 strains (CD: n = 24, UC: n = 9, and HC: n = 7) were characterized by phylogenetic typing, adhesion and invasion assays, detection of virulence factors, antimicrobial resistance genes, and proteomic analysis. Results:Mucosa-associated E. coli were more abundant in CD and UC than in HC (2750 versus 1350 versus 230 median colony-forming units per biopsy; P = 0.01). Intracellular E. coli were more prevalent in CD (90%) than in UC (47%) or HC mucosal biopsies (0%) (P < 0.001). Of 24 CD strains, 2 were adherent and invasive, but there were no unifying pathogenicity determinants that could distinguish most CD strains from UC or HC strains, or intracellular isolates from mucosa-associated isolates. Conclusions:Intracellular E. coli are more common in CD than in UC and not identified in HC. Most intracellular E. coli did not have characterizing pathogenic features, suggesting a significant role for defects in mucosal immunity or barrier dysfunction in their ability to gain intracellular access.

Prostaglandin E2-mediated enhancement of human plasma cell differentiation

Addition of prostaglandin E2 (PGE2) to blood mononuclear cell cultures containing pokeweed mitogen (PWM) enhances plasma cell (PC) differentiation measured by intracytoplasmic immunoglobulin 7 days later. T-cell mitogenesis to concanavalin A is inhibited using the same concentrations of PGE2. PGE2 failed to enhance the PC differentiation of lymphocytes from patients with systemic lupus erythematosus (SLE). Indomethacin, on the other hand, either had no effect or suppressed PC differentiation. The data is discussed in terms of the effect of PGE2 on human suppressor T-cell function.

Effect of Breadmaking Process on In Vitro Gut Microbiota Parameters in Irritable Bowel Syndrome

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria–mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small -molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.

Defective macrophage handling of Escherichia coli in Crohn's disease

 Escherichia coli can be isolated from lamina propria macrophages in Crohns disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD‐derived adherent‐invasive E. coli (AIEC) and less pathogenic E. coli strains.

The effect of the major mediators of type I hypersensitivity on lymphocytes from normal and atopic subjects

The effects of some of the major mediators of the Type I reaction, (histamine, SRS-A, PGE2 and PGF2α) were investigated in vitro on lymphocytes from normal and atopic individuals. Specific H1 and H2 agonists were also examined. Both PGE2 and histamine suppressed lymphocyte transformation, induced by concanavalin A, atopic being more sensitive to histamine induced suppression of lymphocyte transformation than normals. This differences was obvious at low levels of mitogen stimulation. The effect of histamine appeared to be mediated via the receptor characterised specific H2 agonists although specific antagonists did not significantly reverse the effect. PGF2α and high concentrations of the specific H1 agonist 2-methyl histamine enhanced lymphocyte transformation in atopics but either had no effect or slightly suppressed function in normals.

Defective Macrophage Function in Crohn's Disease: Role of Alternatively Activated Macrophages in Inflammation

Introduction The aetiology of Crohns disease (CD) involves a genetically determined dysregulated immune response to commensal intestinal microflora. In CD, viable E coli are found within lamina propria macrophages (MΦs) and E coli intracellular survival is prolonged in CD-derived MΦs in vitro. Different MΦ subpopulations exist, M1 cells are inflammatory cells, M2a cells are involved in tissue repair and M2c are regulatory cells that limit inflammation. The role these different MΦs play in this abnormal handling of bacteria in CD is unclear. Methods The aim of this study was to examine in vitro M1 and M2 MΦ maturation in CD patients and healthy individuals and how these cells respond to challenge with CD-derived E coli. To do this we monitored MΦ morphology, surface markers and cytokine production and intracellular bacterial survival. Peripheral blood monocytes were isolated from CD patients and healthy controls and treated with cytokines to generate distinct MΦ subpopulations: IFNγ for M1, IL4/IL13 for M2a and IL10 for M2c. MΦ morphology was assessed by H&E staining and surface marker expression of CD14, CD16 and CD33, chemokine receptor CCR2, scavenger receptor CD163, co stimulatory molecule CD40 and mannose receptor CD206 using flow cytometry. IL10 and TNFα production were measured by ELISA and intracellular E coli survival was measured using the gentamicin protection assay. Results In contrast to M1 MΦs, both CD-derived M2 populations failed to develop the characteristic MΦ dendrite morphology seen in control macrophages. Surface expression of CD40 in M2 CD-derived MΦs was 3.4-fold lower for M2a and 4.4-fold lower for M2c compared to controls after E coli challenge. CD163 was higher in M1 CD cells but reduced by 50% in M2 cells compared to healthy cells. After E coli challenge, TNFα production by M2 but not M1 MΦs was significantly lower in CD than in controls (M2a 38%, M2c 27% respectively less than controls) but there were no differences in IL10 production. Prolonged intracellular survival of E coli was demonstrated in CD M2 cells but effective killing occurred in all M1 CD MΦs and all control MΦs. Conclusion In CD, M2 (but not M1) MΦs display abnormal morphological maturation, lower TNFα levels after E coli challenge, prolonged intracellular bacterial survival and differences in surface marker expression. The results are consistent with an innate MΦ defect in CD relating particularly to a failure of the normal role of M2 MΦs to limit and control inflammation.