| 1. |
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| 2. |
- Bas, A, et al.
(författare)
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Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.
- 2004
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 59:6, s. 566-73
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Tidskriftsartikel (refereegranskat)abstract
- The accuracy of 18S rRNA, beta-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, beta-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 beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) 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.
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| 3. |
- Fahlgren, A, et al.
(författare)
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Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.
- 2003
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Ingår i: Clinical and Experimental Immunology. - 0009-9104 .- 1365-2249. ; 131:1
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Tidskriftsartikel (refereegranskat)abstract
- The impact of chronic inflammation on the expression of human alpha-defensins 5 and 6 (HD-5, HD-6), beta-defensins 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 alpha-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.
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| 4. |
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| 6. |
- Huber, M., et al.
(författare)
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Phase memory relaxation times of spin labels in human carbonic anhydrase II : Pulsed EPR to determine spin label location
- 2001
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Ingår i: Biophysical Chemistry. - 0301-4622 .- 1873-4200. ; 94:3, s. 245-256
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Tidskriftsartikel (refereegranskat)abstract
- Phase memory relaxation times (TM or T2) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters TM and x. TM values of seven positions are between 1.6 ╡s for the most buried residue (L79C) and 4.7 ╡s for a residue at the protein surface (W245C). In deuteriated buffer, longer TM are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose TM as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism. ⌐ 2001 Elsevier Science B.V. All rights reserved.
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| 7. |
- Melgar, S, et al.
(författare)
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Cytolytic capabilities of lamina propria and intraepithelial lymphocytes in normal and chronically inflamed human intestine
- 2004
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 0300-9475 .- 1365-3083. ; 60:1-2, s. 167-177
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Tidskriftsartikel (refereegranskat)abstract
- Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. 'Classical' cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.
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| 8. |
- Melgar, S, et al.
(författare)
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Over-expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis.
- 2003
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Ingår i: Clinical and Experimental Immunology. - 0009-9104 .- 1365-2249. ; 134:1, s. 127-37
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Tidskriftsartikel (refereegranskat)abstract
- Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohn's disease and control patients were analysed for cytokine mRNA levels by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)-2, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, but not IL-4, IL-5 or IL-10. In UC, a highly significant increase in IL-10 mRNA levels in T lymphocytes and an increased frequency of IL-10 positive cells was seen in colon. IL-10 mRNA levels were also elevated in T lymphocytes of the non-inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL-10 mRNA. IL-2, IFN-gamma and TNF-alpha mRNA levels were decreased in colonic T lymphocytes, and virtually no IL-2, IFN-gamma, TNF-alpha or TGF-beta positive cells were detected in basal lymphoid aggregates. However, scattered IL-10 positive cells were found here. Lamina propria outside the aggregates contained IL-10-, IFN-gamma, TNF-alpha and TGF-beta but not IL-2 positive cells. T cells of UC patients did not express IL-4 or IL-5. Taken, together the data suggest a generalized activation of IL-10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL-10.
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| 9. |
- Yeung, M M, et al.
(författare)
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Characterisation of mucosal lymphoid aggregates in ulcerative colitis : immune cell phenotype and TcR-gammadelta expression.
- 2000
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Ingår i: Gut. - 0017-5749 .- 1468-3288. ; 47:2, s. 215-27
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND AIMS: A histopathological feature considered indicative of ulcerative colitis (UC) is the so-called basal lymphoid aggregates. Their relevance in the pathogenesis of UC is, however, unknown. We have performed a comprehensive analysis of the immune cells in these aggregates most likely corresponding to the lymphoid follicular hyperplasia also described in other colitides.METHODS: Resection specimens of UC and normal colon were analysed by immunomorphometry, immunoflow cytometry, and immunoelectron microscopy, using a large panel of monoclonal antibodies.RESULTS: (1) In all cases of UC, colonic lamina propria contained numerous basal aggregates composed of lymphocytes, follicular dendritic cells, and CD80/B7.1 positive dendritic cells. (2) CD4(+)CD28(-) alphabeta T cells and B cells were the dominant cell types in the aggregates. (3) The aggregates contained a large fraction of cells that are normally associated with the epithelium: that is, gammadelta T cells (11 (7)%) and alpha(E)beta(7)(+) cells (26 (13)%). The gammadelta T cells used Vdelta1 and were CD4(-)CD8(-). Immunoelectron microscopy analysis demonstrated TcR-gammadelta internalisation and surface downregulation, indicating that the gammadelta T cells were activated and engaged in the disease process. (4) One third of cells in the aggregates expressed the antiapoptotic protein bcl-2.CONCLUSIONS: Basal lymphoid aggregates in UC colon are a consequence of anomalous lymphoid follicular hyperplasia, characterised by abnormal follicular architecture and unusual cell immunophenotypes. The aggregates increase in size with severity of disease, and contain large numbers of apoptosis resistant cells and activated mucosal gammadelta T cells. The latter probably colonise the aggregates as an immunoregulatory response to stressed lymphocytes or as a substitute for defective T helper cells in B cell activation. gammadelta T cells in the aggregates may be characteristic of UC.
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| 10. |
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| 11. |
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| 12. |
- Frängsmyr, L., et al.
(författare)
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Evolution of the carcinoembryonic antigen family. Structures of CGM9, CGM11 and pregnancy-specific glycoprotein promoters
- 2000
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Ingår i: Tumor Biology. - 1010-4283 .- 1423-0380. ; 21:2, s. 63-81
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Tidskriftsartikel (refereegranskat)abstract
- Earlier studies have demonstrated that the genes of the human carcinoembryonic antigen (CEA) family can be divided into three subgroups, the CEA subgroup (n = 12), the pregnancy-specific glycoprotein (PSG) subgroup (n = 11), and a third subgroup (n = 6). To further characterize the CEA gene family, we have determined the genomic structures of CGM9 and CGM11, analyzed the promoter regions of all eleven PSGs, studied the CGM15-PSG13 intergenic region and the evolutionary relationships between the CEA family genes. CGM9, a typical CEA subgroup member, was a pseudogene with the exon structure [5'UTR-L-L/N-TM-Cyt-3'UTR]. CGM11 contained a mixture of exons derived from CEA and PSG subgroup genes. The formula of the CGM11 pseudogene was [5'UTRL- L/N-C-3'UTR]. Thus both genes lacked the IgC2-like domains typically found in CEA subfamily members. The upstream promoter regions of all eleven PSGs were characterized. All PSG promoters lacked the classical TATA and CCAAT elements, but had putative PEA3 box(es), CACCC box(es), a RARE box, and poly (dG-dT) repeats of different lengths. Five PSGs also had an SP1 site. The complete 10-kb intergenic region between CGM15 and PSG13 was sequenced. Clusters of different types of repetitive sequences were seen. The time of divergence of the CEA and PSG subfamilies was estimated to be 107.7 ± 17.1 million years, or at about the time of human-rodent divergence. Models for the evolution of CEA and PSG and the third family subgroup genes are proposed.
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| 13. |
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| 14. |
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| 15. |
- Michaëlsson, Gerd, et al.
(författare)
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Gluten-free diet in psoriasis patients with antibodies to gliadin results in decreased expression of tissue transglutaminase and fewer Ki67+ cells in the dermis
- 2003
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Ingår i: Acta Dermato-Venereologica. - : Medical Journals Sweden AB. - 0001-5555 .- 1651-2057. ; 83:6, s. 425-429
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies have shown that 16% of patients with psoriasis vulgaris have IgA and/or IgG antibodies to gliadin, but few have antibodies to endomysium. The increase in duodenal intraepithelial lymphocytes was mild. Still, highly significant clinical improvement was observed after 3 months on a gluten-free diet. This study surveys certain immunohistological aspects of involved and non-involved skin in 28 AGA-positive psoriasis patients before and after 3 months of a gluten-free diet. Staining was performed for CD4+ T lymphocytes, Langerhans' cells, endothelium, proliferating (Ki67) cells and tissue transglutaminase. In the entire group of patients, as well as in those on a gluten-free diet as the only treatment, Ki67 + cells in involved dermis were highly significantly decreased after the diet. There was a significant decrease in Ki67 + cells even in patients without increased intraepithelial lymphocytes. Tissue transglutaminase was highly overexpressed in involved skin in the papillary endothelium, and decreased by 50% after gluten-free diet. The possible role of tissue transglutaminase in the pathogenesis of psoriasis needs further investigation.
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| 16. |
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| 17. |
- Olofsson, Katarina, et al.
(författare)
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Human uvula : characterization of resident leukocytes and local cytokine production
- 2000
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Ingår i: Annals of Otology, Rhinology and Laryngology. - 0003-4894 .- 1943-572X. ; 109:5, s. 488-496
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Tidskriftsartikel (refereegranskat)abstract
- Upper airway infections often lead to macroscopic changes in the architecture of the uvula. Using immunomorphometric analysis, we investigated the frequency and distribution of immune cells and of cytokine-producing cells in uvular samples. Tissue macrophages, alphabeta T cells, gammadelta T cells, and B cells were, in declining order, the main cell populations. Gammadelta T cells and B cells exhibited reciprocal localization, with almost all gammadelta T cells residing in the vicinity of the epithelium, and all B cells in the glandular area. The presence of cells expressing the suppressor phenotype CD8+CD28- alphabeta T cells is suggested. Fifteen to twenty-five percent of the immune cells expressed the down-regulatory cytokine tumor growth factor beta. Most macrophages were located subepithelially, in the vicinity of the basal lamina. The composition and cytokine profile of leukocytes in the tissue suggest that the uvula may be a site, additional to the jejunal mucosa, for induction of mucosal tolerance to inhaled and ingested antigens. Concomitantly, the uvula appears to be protected from invasion of microbial pathogens by a subepithelial barrier of macrophages and gammadelta T cells.
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| 18. |
- Qian, Bi-Feng, et al.
(författare)
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Neuroendocrine changes in colon of mice with a disrupted IL-2 gene
- 2000
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 120:3, s. 424-433
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Tidskriftsartikel (refereegranskat)abstract
- Neuroendocrine peptides have a variety of physiological functions in the gastrointestinal tract. This study was carried out to investigate the impact of IL-2 deficiency on the neuroendocrine system in normal colon, and the neuroendocrine changes during colonic inflammation. Mice with homozygous disrupted IL-2 gene (IL-2-/-) spontaneously developed a bowel disease with similarities to human ulcerative colitis. Different types of colonic endocrine cells and myenteric nerves were analysed in the IL-2-/- mice using immunomorphometry. The neuropeptide contents in the colonic tissues were determined by radioimmunoassay. Age-matched healthy IL-2+/- and IL-2+/+ mice served as controls and the colonic IL-2 levels were compared between these two groups of mice by ELISA. Our data showed that less than half the amount of IL-2 was synthesized in the colon of IL-2+/- mice compared with the IL-2+/+ wild-type mice. Two major differences in the neuroendocrine colon were found between the mice with an intact and disrupted IL-2 gene. One was age-related. The frequencies of various endocrine cells and myenteric nerves increased with age in the IL-2+/+ mice. However, no such increases were seen in the mice with a disrupted IL-2 gene. Instead, the volume densities of enteroglucagon, serotonin cells and substance P (SP), vasoactive intestinal polypeptide (VIP) and total myenteric nerves were lower in the older IL-2+/- and IL-2-/- mice compared with the wild type. The other was disease-related. Polypeptide YY (PYY) cells and tissue levels of PYY, SP and VIP were significantly decreased in the IL-2-/- mice during the course of bowel inflammation compared with the healthy IL-2+/- and IL-2+/+ controls. These findings indicate that colonic neuroendocrine alterations did occur in the mice with a disrupted IL-2 gene and diminished local IL-2 level, suggesting a role of IL-2 in the regulation of the neuroendocrine system and a prevalent interaction between the immune and neuroendocrine systems in normal colon. On the other hand, there were some changes that seemed to correlate with the bowel inflammatory process. They might be associated with the impaired function in inflamed gut and contribute to the development and/or prolongation of disease.
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