No abstract

The accuracy of 18S rRNA, b-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, b-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of b-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gdTCR þ , CD4 þ and CD8 þ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

SUMMARYThe impact of chronic inflammation on the expression of human a -defensins 5 and 6 (HD-5, HD-6), bdefensins 1 and 2 (hBD-1, hBD-2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real-time quantitative RT-PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD-5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD-2, HD-5, HD-6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD-5, HD-6, hBD-1 and hBD-2 mRNA. HD-5 and lysozyme protein was demonstrated in metaplastic Paneth-like cells in UC colon. There was no correlation between hBD-2 mRNA levels and HD-5 or HD-6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohn's colitis patients showed increased mRNA levels of HD-5 and lysozyme mRNA whereas ileal epithelial cells of Crohn's patients with ileo-caecal inflammation did not. Chronic inflammation in colon results in induction of hBD-2 and a -defensins and increased lysozyme expression. hBD-1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

No abstract

The human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and unusual subpopulations of IEL are present at different levels of human intestine. IEL phenotypes in normal jejunum, ileum and colon were compared using immunoflow cytometry and immunohistochemistry. The expression of mRNA for recombination-activating gene-1 (RAG-1) in IEL from all three levels was compared using reverse-transcription polymerase chain reaction, and the morphology of IEL in situ was determined using immunoelectron microscopy. Surface marker profiles of isolated intestinal epithelial cells at all three levels were also investigated. On average the proportion of TCR gamma delta IEL was comparable in jejunum than ileum and colon and varied in phenotype with gut level. CD4-CD8-TCR alpha beta IEL dominated in colon but were absent in jejunum. CD8+ TCR alpha beta IEL were present at all levels but only in jejunum did they constitute the majority of all IEL. CD4+ TCR alpha beta IEL were present in similar frequencies at all levels of the gut. In general, the majority of IEL had an activated phenotype (CD45RO+, alpha E beta 7+). Furthermore, IEL exhibited phenotypes which are rare in peripheral blood. The thymocyte markers CD1a and CD1c as well as the NK cell marker CD56 were expressed on a fraction of TCR alpha beta and TCR gamma delta IEL. A small population of 'null' cells (CD45+ TCR/CD#-CD20-CD14-CD15- cells) was also present at equal proportions along the gut. Jejunal but not colonic IEL expressed RAG-1 mRNA suggesting that extrathymic T cell maturation occurs in the epithelium of small intestine. RAG-1 was expressed in CD2+TCR/CD3- and CD3+/TCR-IEL. Ultrastructurally, IEL often formed small clusters and intimate contacts with epithelial cells, suggesting cell cooperation within the epithelium. Some IEL had pseudopodium-like extensions penetrating the epithelial basement membrane suggesting transmigration. Epithelial cells in small intestine but not colon expressed heat shock protein 60 and HLA-DR. CD1a, CD1b and CD1c were not expressed on intestinal epithelial cells at any level. The distinct surface marker profiles of IEL and epithelial cells along small and large intestine suggest functional regional specialization and are compatible with the hypothesis that TCR alpha beta IEL participate in immune reactions to lumenal antigens while TCR gamma delta IEL perform surveillance of the epithelium.

No abstract

Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.

Rod-shaped bacteria, probably of the indicated species, constituted a significant fraction of the proximal small intestine microbiota in children born during the Swedish CD epidemic and may have been an important risk factor for CD contributing to the fourfold increase in disease incidence in children below 2 years of age during that time.