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To identify activated T cell subset in the asthmatic bronchia, we developed a triple-colour immunohistofluorescence labelling technique on cryo-section to discriminate activated CD4+ CD25+ T cells, (effector T cells) from Foxp3+ regulatory T cells ( Treg). Additional coexpression of activation and proliferation markers was also examined in situ. Bronchial biopsies were taken from 20 aluminium potroom workers (12 smokers) with asthma (>12% reversibility), 15 non-asthmatic potroom workers (7 smokers) and 10 non-smoking, non-exposed controls. Non-smoking asthmatics had significantly higher subepithelial density of both Tregs, effector T cells, activated ( HLA- DR+) CD8+ and activated CD4+ T cells. Moreover, both Tregs, effector T cells and CD8+ T cells proliferated in the non-smoking asthmatics, only. Although smoking asthmatics had no asthma-associated increase in bronchial T cell, both had a significantly increase in effector T cell to Treg ratios. The significantly increased bronchial density of Tregs, effector T cells, proliferative T cells and activated CD8+ T cells in non-smoking asthmatics clearly showed that both the effector T cells and the inhibitory Treg system were activated in asthma. [ABSTRACT FROM AUTHOR]

It has been suggested that epithelial cyclooxygenase- 2 (COX-2) promotes oral carcinogenesis and carcinoma malignancy through increased prostaglandin E2 (PGE2) production. Although oral squamous cell carcinomas (OSCC) often express COX-2, they may also produce PGE2 in a COX-1-dependent manner. We used 6 isolated cell lines to investigate which COX isoforms OSCC may use for PGE2 production. COX-1 and -2 expression patterns divided the 6 OSCC cell lines into 3 distinct groups: both COX isoforms low, only COX-1 high, or both COX isoforms high. Multicolor immunohistofluorescence staining confirmed the COX-expression profiles in organotypic 3D cultures and the COX-2 dominance in OSCC tumors. Epidermal growth factor (EGF) stimulation induced COX-2 (but not COX-1) expression and increased PGE2 production, which was attenuated by COX-2 (but not COX-1) specific inhibition or siRNA-mediated COX-2 gene knockdown. Thus, PGE2 production in OSCC cell lines was COX-2- dependent. Abbreviations: COX, cyclooxygenase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; NSAID, non-steroidal antiinflammatory drug; OSCC, oral squamous cell carcinoma; PG, prostaglandin; PGE2, prostaglandin E2; and PGES, prostaglandin E2 synthase. [ABSTRACT FROM AUTHOR]

Salivary gland specimens from 10 patients with primary Sjögren's syndrome (pSS) were examined by two-colour immunofluorescence with various combinations of monoclonal and polyclonal antibody reagents of the following specificities; human leucocyte antigen (HLA) class I and II (DR, DP and DQ), CD3. CD45 (leucocyte common antigen), various cytokeratins, and factor VIII-related antigen. Tissue specimens from 10 normal glands and 10 glands with obstructive sialadenitis (no known autoimmunity) served as controls. Only some intercalated ducts and scattered acini of the normal major glands expressed HLA class II determinants( < 5% of total epithelial area); the relative proportion of positive elements indicated differential expression (DR > DP > DQ). SS glands contained substantial T cell infiltrates and increased numbers of activated (DR+ ) T cells; adjacent epithelium showed extensive differential expression of HLA class II determinants (DR >> DP > DQ). Glands with obstructive sialadenitis showed similarly increased epithelial expression of H LA-DR but with surprisingly small amounts of concomitant HLA-DP and -DQ expression. Epithelial HLA class 11 expression probably depends on cytokines as an inductive event, which is not unique for SS but particularly prominent in this disorder. Our results suggest that epithelial expression of HLA-DP or -DQ. rather than -DR. might be a prerequisite for the autoimmune process of SS to develop in genetically susceptible individuals. [ABSTRACT FROM AUTHOR]

The specialized M cells in the follicle-associated epithelium (FAE) of Peyer's patches (PP) represent an intimate interphase between luminal antigens and gut-associated lymphoid tissue (GALT). M cells form pockets that contain clusters of leucocytes probably involved in the first encounter with antigens from the gut lumen. Three-colour immunofluorescence in situ phenotyping of these leucocytes in humans revealed about equal numbers of B (CD19/20+) and T (CD3+) lymphocytes, the latter mainly CD4 + (median 73%, range 40-90%), but relatively few macrophages (CD68+). Most B cells (90%) were positive for surface IgM (sIgM) and often co-expressed sIgD (median 34%, range 6-60%). Occasional B cells (median 2%) did not express CD45RA (range 0-15%) and 13% virtually lacked HLA-DR (range 0-40%). Some B and T lymphocytes expressed the nuclear proliferation marker Ki-67 (range 1-10%). The M-cell pockets also contained occasional cells with cytoplasmic lgA or IgM. These sites thus contained a heterogeneous B-cell population with features of both follicular mantle (sIgD+ sIgM+) and marginal zone (sIgD- sIgM+) B lymphocytes. Adjacent T lymphocytes were generally of the memory phenotype (CD45RO+). Our findings suggest that the M-cell-associated B lymphocytes represent local extensions of B-cell follicles towards the gut lumen, developed topically to facilitate antigen presentation and diversification of mucosal immune responses. [ABSTRACT FROM AUTHOR]

Jejunal biopsy specimens from 10 patients with treated coeliac disease and seven non-coeliac controls were challenged in vitro with peptic-tryptic gluten digest. Mucosal T cells were examined in situ by three-colour immunofluorescence staining for expression of the activation marker CD25 (the p55 α-chain of interleukin-2 receptor) and the nuclear proliferation marker revealed by monoclonal antibody Ki-67. Intraepithelial T cells expressed CD25 rarely whereas the proportion of activated lamina propria T cells increased (P < 0.002) from median 2.8% (cultured with 20% fetal calf serum alone for 24-48 h) to 10.0% after 24 h with gluten (n = 10; range 1.1-17.4%) and to 10.4% after 48 h (n = 7; range 1.4-17.5%). Such gluten-induced increase of CD25+ T cells was not observed in specimens from non-coeliac control subjects. Crypt-cell hyperplasia and T-cell proliferation (Ki-67+) were observed neither in the coeliac nor in the control mucosae after gluten stimulation. Three-colour staining combining a polyclonal antibody reagent to CD3 and a monoclonal antibody to CD25 with a monoclonal antibody to CD45R0, CD4, CD8, the p75 β-chain of interleukin-2 receptor, integrin αEβ7, or HLA-DR showed that most of the CD25+ T cells (> 90%) were CD4+CD8-, co-expressed CD45R0 and the p75 β-chain, and often also the integrin αEβ7 but not HLA-DR. In addition to these activated T cells, a dominating population of CD25+ CD3- CD4+ subepithelial pan-HLA-class II+ macrophages (CD68+) with variable expression of the p75 β-chain was often induced by gluten challenge. [ABSTRACT FROM AUTHOR]

Expression of the γ/δ T-cell receptor (TcR) for antigen on CD3+ intraepithelial lymphocytes (IEL) was studied in situ by two-colour immunofluorescence on jejunal tissue sections from 24 patients with celiac disease and 17 controls. The proportion of intraepithelial TcRγ/delta; + cells was significantly increased (P<0.002) in untreated (median 20%, range 11-53%) as well as in treated (gluten-free diet) celiac disease (median 23%, range 16-55%) compared with controls (median 2%, range 0-39%). Although TcRα/β + IEL dominated both in controls and celiac disease, T cells expressing the TcRγ/δ were preferentially located within the epithelium rather than in the lamina propria. Paired staining for TcRγ/δ + and CD8 revealed that most (sim;90%) intraepithelial TcRγ/δ + lymphocytes in celiac disease were CD8-. A remarkably large fraction (median 67%, range 58-94%) of intraepithelial TcRγ/δ + cells expressed the Vδ1/Jδ1-encoded epitope revealed by monoclonal antibody &deta;TCSI. Our results suggested that increase of the intraepithelial TcRγ/δ + CD8- subset of T cells is particularly related to celiac disease. [ABSTRACT FROM AUTHOR]