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Background and Aim E scherichia coli can be isolated from lamina propria macrophages in Crohn's 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. Methods Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E . coli strains ( CD-derived adherent-invasive [ AI] and non- AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [ TNFα], interleukin [ IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E . coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles ( NOD2, IL-23R, ATG16L1 and IRGM). Results Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E . coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non- AIEC and E . coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. Conclusions CD macrophage responses to infection with E . coli are deficient, regardless of clinical phenotype, CD genotype or E . coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E . coli persistence in CD. [ABSTRACT FROM AUTHOR]

Background: Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage., Methods: Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR., Results: E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls., Conclusion: In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

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 Crohn's 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.

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., Methods: Forty-seven patients with IBS (27 IBS-D and 20 IBS-C) and 26 healthy controls were recruited to the study. Snap-frozen rectal biopsies were taken at colonoscopy and bacterial quantification performed by hybridizing frozen sections with bacterial-group specific oligonucleotide probes., Key Results: Patients with IBS had significantly greater numbers of total mucosa-associated bacteria per mm of rectal epithelium than controls [median 218 (IQR - 209) vs 128 (121) P = 0.007], and this was chiefly comprised of bacteroides IBS [69 (67) vs 14 (41) P = 0.001] and Eubacterium rectale-Clostridium coccoides [52 (58) vs 25 (35) P = 0.03]. Analysis of IBS sub-groups demonstrated that bifidobacteria were lower in the IBS-D group than in the IBS-C group and controls [24 (32) vs 54 (88) vs 32 (35) P = 0.011]. Finally, amongst patients with IBS, the maximum number of stools per day negatively correlated with the number of mucosa-associated bifidobacteria (P < 0.001) and lactobacilli (P = 0.002)., Conclusions & Inferences: The mucosa-associated microbiota in patients with IBS is significantly different from healthy controls with increases in bacteroides and clostridia and a reduction in bifidobacteria in patients with IBS-D., (© 2011 Blackwell Publishing Ltd.)

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 Crohn's 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 Crohn's 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 Crohn's 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.

Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.

This study examined whether or not there is progressive loss of individual myocytes in established heart failure, accounting for the progressive left ventricular dysfunction; whether such loss is by necrosis or apoptosis; and whether such loss is more pronounced in ischaemic heart disease or idiopathic dilated cardiomyopathy. Tissue for patients undergoing cardiac transplantation for clinical end-stage heart disease was used. The clinical diagnosis was not known to the observer at the time of analysis. Indices of potential myocyte loss were: detection of apoptotic nuclei in situ, using the TUNEL method, immunohistochemistry for CD120a, CD120b, CD95, perforin and granzyme B; binding of C9 complex; and lipofuscin deposition within macrophages. Interstitial macrophages and T cells and their relationship to myocyte loss were also examined. There is indeed low grade myocyte loss in chronic heart failure, but there was no difference between the disease groups; rather, there was marked patient-to-patient variation within each category. Thus in chronic heart failure myocyte loss does occur, and both necrosis and apoptosis contribute to this loss, irrespective of the underlying nature of the disease. Any mechanism which accounts for myocyte loss must be common to both conditions, rather than specific for a pre-operative diagnosis., (Copyright 1999 John Wiley & Sons, Ltd.)

Keratinocytes in normal ectocervix did not express major histocompatibility complex class II molecules. In low-grade intraepithelial lesions expression was confined to HLA-DR, while in high-grade disease there was expression of HLA-DR and occasional expression of HLA-DQ. HLA-DR was expressed constitutively on the majority of Langerhans cells. In contrast, few Langerhans cells expressed HLA-DQ in normal cervix, but there was a steady upregulation of the proportion expressing HLA-DQ which paralleled the severity of disease. There was no direct correlation between human papillomavirus 16 and the expression of major histocompatibility complex class II by keratinocytes and Langerhans cells. Significant upregulation of HLA-DQ by Langerhans cells is observed in high-grade intraepithelial cervical lesions, suggesting antigen-presenting cell activation in papillomavirus-related premalignant disease.

Objective: To examine localisation of tumour necrosis factor (TNF alpha) and transforming growth factor beta (TGF beta) mRNA synthesis in human coronary artery atheromatous plaques, to explore how synthesis of these cytokines relates to distribution of macrophages and smooth muscle cells, and to correlate this with plaque micro-environments., Method: In situ hybridisation with digoxigenin-labelled sense and anti-sense riboprobes was used, combined with immunohistochemistry to detect TNF alpha protein, macrophage, lymphocyte and smooth muscle cell markers., Results: In the intimal plaque TNF alpha mRNA is synthesised by monocytes/macrophages as well as by smooth muscle cells. Both TNF alpha and TGF beta mRNAs were present at the margins of the lesions and in reactive areas, where there was little lipid and fibrosis. Focal aggregates of macrophages in the adventitia expressed both TNF alpha mRNA and protein and TGF beta mRNA., Conclusion: Synthesis of these two cytokines by macrophages as well as smooth muscle cells contributes to the pathobiology of the plaque and that this is part of the 'reaction to injury', rather than a feature of a specific cell, or a specific layer, within the vessel wall.