Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Distinct cytokine-producing T cell subsets are well known to play a major role in IgE production and to be differentially regulated in allergic patients, although the characterization of the type 1/type 2 cytokine pattern in PBMC during allergic responses remains to be clearly defined. The aim of this study was to determine whether different cytokine profiles are observed directly in PBMC of atopic donors. We attempted to study several cytokines (IL-2, IFN-gamma, IL-4, IL-10 and IL-13) using not only ELISA but also polymerase chain reaction (PCR) techniques, because the frequency of cytokine-producing cells in peripheral blood is very low. All the patients were selected during their acute symptomatologic phase. Data showed a significantly higher production of IL-4 (P = 0.05) and IL-10 (P < 0.005) as determined by ELISA in phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA)-stimulated mononuclear cells of atopic donors compared with controls, although spontaneous IL-4 production without stimulation was never detected within either atopic or control groups. The reverse-transcriptase (RT)-PCR technique appeared to be advantageous in that it allowed the detection of the spontaneous expression of cytokine mRNA in cells without stimulation. We found a clear expression of IL-4 mRNA spontaneously in all atopic patients, whereas normal donors in most cases did not show specific signals (P < 0.0001). Less differences between atopic subjects and controls were found in IL-10 mRNA expression. Although the technique of RT-PCR amplification used in this study is semiquantitative, a reproducible and significant (P < 0.001) enhancement of IL-10 mRNA expression was observed in atopic donors. A heterogeneous expression of IL-13 mRNA was observed in individuals from the two groups studied, although mean levels in atopic donors were slightly enhanced compared with controls (P = 0.02). Furthermore, we did not observe any alteration in the expression of the type 1-derived cytokines such as IFN-gamma and IL-2. In addition, we showed a lack of correlation between the expression of serum IgE (total or specific) and spontaneous IL-4 mRNA expression. This study showed a tendency of PBMC from atopic donors to express a type 2-like cytokine pattern, with IL-4 as the most discriminatory cytokine. Additionally, as the level of serum IgE has a low predictive value in allergic disease, and as the elevated expression of IL-4 that we found was not correlated with serum IgE, we could strongly suggest that the measurement of IL-4 in blood mononuclear cells may be of great value in the analysis of allergic responses in atopic donors.
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PMID:Differential spontaneous expression of mRNA for IL-4, IL-10, IL-13, IL-2 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cells (PBMC) from atopic patients. 856 69

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM-1), CD49a (VLA-1), CD49b (VLA-2), CD49c (VLA-3), CD49e (VLA-5), CD49f (VLA-6), CD29, CD51, CD44 and produce vinculin, beta-tubulin, alpha-actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up-regulated CD54 (ICAM-1) and induced HLA-DR and CD106 (VCAM-1). HTSC constitutively produce interleukin (IL)-6 which is enhanced upon activation with TNF-alpha. IL-8 and granulocyte/macrophage colony-stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL-1 alpha, leukemia inhibitory factor and IL-7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL-2-activated B lymphocytes cocultured in direct cell-cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non-lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.
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PMID:In vitro cultured stromal cells from human tonsils display a distinct phenotype and induce B cell adhesion and proliferation. 856 62

Neuroblastoma (NB) is a major-histocompatibility-complex(MHC)-negative neuroectodermal tumour that is often infiltrated with lymphocytes. A detailed characterization of NB-associated tumour-infiltrating lymphocytes (TIL) has never been carried out. Here we have investigated the immunophenotype and the cytotoxic activities of TIL from nine and seven NB patients respectively. Furthermore, the T cell receptor (TcR) variability and the patterns of cytokine gene expression of fresh versus recombinant (r) interleukin (IL)-2-cultured TIL were studied in four NB cases. The results obtained showed the following: (1) freshly isolated TIL were comprised of a mixture of CD4+ and CD8+ T cells partially expressing HLA-DR and/or CD25. The CD4/CD8 ratio ranged from 0.5 to 5 in the different cases. Upon culture of TIL with rIL-2, an increased proportion of CD56+ and CD8+ lymphocytes was consistently observed; (2) IL-2-expanded TIL lysed natural-killer(NK)sensitive and lymphokine-activated-killer(LAK)-sensitive target cell lines; (3) reverse-transcriptase/polymerase-chain-reaction (RT-PCR) experiments showed that most TcR V beta genes were expressed both in fresh and in cultured TIL, suggesting that such cell populations were polyclonal; (4) interferon gamma, IL-4, IL-5, tumour necrosis factor (TNF) alpha, IL-8, IL-10 mRNA and, to a lesser extent, IL-2 mRNA were expressed by cultured TIL, as assessed by RT-PCR; the corresponding tumour samples consistently contained TNF alpha, IL-8 and IL-10 mRNA, whereas IL-2 and IFN gamma mRNA were faintly expressed in some NB tumours and IL-4 and IL-5 mRNA were never detected. A total of 90 clones were subsequently raised from IL-2-expanded TIL from six NB patients; 87/90 clones were of T cell lineage with a CD4+ or CD8+ immunophenotype, whereas the 3 remaining clones were of NK cell origin. Upon triggering of the CD3-TcR complex, 64% CD4+ and 77% CD8+ T cell clones killed the murine P815 mastocytoma cell line. Virtually no T cell clone lysed a LAK-sensitive NB cell line whereas 15% CD4+ and 17% CD8+ clones mediated NK-like activity against the K562 cell line. Finally, the patterns of cytokine production by CD4+ clones were roughly consistent with those of a T helper (TH) 1 profile and similar to those observed in CD8+ clones.
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PMID:Functional and molecular characterization of tumour-infiltrating lymphocytes and clones thereof from a major-histocompatibility-complex-negative human tumour: neuroblastoma. 864 Aug 45

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
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PMID:Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. 878 Nov 19

We have analysed the expression of interleukin-2 receptor (IL-2R) on a panel of small-cell lung cancer (SCLC) cell lines. None of the 11 SCLC cell lines studied expressed detectable surface IL-2R alpha or beta chains by indirect immunofluorescence. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses indicated that only one out of 11 cell lines expressed detectable IL-2R beta mRNA while two expressed a weak positivity for IL-2R gamma. Five SCLC cell lines were transfected with the plasmid vector RSV.5 neo containing IL-2 cDNA coding sequence. Stable transfectants secreted biologically active IL-2 (ranging from 25 to 100 U ml-1 in the culture supernatant). IL-2 transfection did not produce significant modifications in the expression of surface molecules such as IL-2R alpha and beta chains, intercellular adhesion molecule-1 (ICAM-1), CD44, HLA class I and II or in IL-2R beta or gamma mRNA. More importantly, IL-2-transfected N592 and NCI H69 cell lines completely lost their tumorigenic potential in nude mice after subcutaneous injection, whereas experimental controls transfected with RSV.5 neo vector only, displayed an in vivo growth pattern identical to that of untransfected cells. In addition, in the N592 model, IL-2-producing N592 inhibited the growth of wild-type N592 injected at the same site, while injection of parental cells on the opposite side did not significantly affect the growth of wild-type tumour cells. Histopathological analysis of the rejection process of IL-2-transfected cells demonstrated the presence of MAC-1+, MAC-3+ macrophages and of RB68C5+ granulocytes, whereas T cells were undetectable and NK cells were scarcely represented. In addition, a reduction of the tumour blood vessels was observed. The possible relevance of these data for the development of vaccination strategies using cytokine-engineered tumour cells in SCLC is discussed.
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PMID:Analysis of IL-2 receptor expression and of the biological effects of IL-2 gene transfection in small-cell lung cancer. 879 83

Activated human and rat T cells as well as mouse T-cell clones have been reported to synthesize and express major histocompatibility complex (MHC) class II molecules. However, the capacity of class II+ antigen (Ag) presenting T cells to induce proliferation of Ag-specific cloned T cells has been controversial. We analysed whether the failure of some T-cell clones to proliferate in response to Ag presented by class II+ T cells is because of a lack of costimulatory cytokine production by the antigen-presenting cells (APC). As a model system the mouse class II+ cloned BI/O4.1 T cells were used as APC in order to activate the T cell clone KIII5. This T-helper 1 (Th1) type, GAT (synthetic copolymer of L-glutamic acid, L-alanine and L-tyrosine)-specific clone is characterized by an efficient downregulation of interleukin-2 receptor (IL-2R) with time following antigenic stimulation. KIII5 cells respond to GAT-presenting splenic antigen-presenting cells (APC) by IL-2 production, IL-2R upregulation and proliferation. When BI/O4.1 T cells were used as APC, KIII5 cells produced IL-2, but did not proliferate. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a lack of IL-12 production by BI/O4.1 cells. Addition of IL-12 to a coculture of Ag-presenting BI/O4.1 cells and KIII5 cells fully reconstituted a proliferative response. IL-12 in synergy with IL-2 upregulated IL-2R alpha chain expression and enhanced proliferation of KIII5 cells. Our data suggest, that class II+ T cells are not functional in inducing Ag-mediated expansion of resting Th1 cells owing to their failure to produce IL-12, but rather that they play a role in amplification loops during an ongoing immune response.
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PMID:Costimulatory signalling potential of murine MHC class II-positive T-clone cells. 895 51

The recently cloned cytokine interleukin-15 (IL-15) shares several functional activities with IL-2 in different cell systems. Although IL-15 does not show sequence homology with IL-2, it uses components of the IL-2 receptor (IL-2R) for binding and signal transduction, namely, p75 (beta) and the p64 (gamma) chains of IL-2R. To evaluate whether IL-15 is involved in the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), we evaluated the ability of IL-15 to stimulate GL proliferation, cytotoxic function, and the role of IL-2R beta and gamma molecules on relevant cells. Our results show that IL-15 stimulates cell proliferation and cytotoxic activity of GL in LDGL patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) and phenotypic analyses using the anti-IL-2R gamma-chain-specific TUGh4 monoclonal antibody (MoAb) indicate that both CD3+ and CD3- GL express the p64 IL-2R, a result previously unknown. IL-15 activity was inhibited by antibodies against p75 and p64 IL-2R chains, while no inhibitory effects are detectable with anti-p55 IL-2R antibody. The association of anti-p75 and anti-p64 IL-2R MoAbs resulted in a nearly complete (95%) inhibition of IL-15-induced GL proliferation. Using RT-PCR analysis, we demonstrated that highly purified CD3+ and CD3- GL did not express mRNA for IL-15 or IL-2. By contrast, a clear-cut IL-15 mRNA signal was detected by RT-PCR in patients' peripheral blood mononuclear cells, with monocytes likely accounting for the source of IL-15 in LDGL patients. However, even in concentrated supernatants from enriched monocyte populations, we could not demonstrate the presence of IL-15 protein. Using anti-IL-15 specific MoAbs, a membrane-bound form of this cytokine was demonstrated both on CD3+ and CD3- LDGL cells. By RT-PCR analysis, purified GL from these patients were found to express the message for IL-15 receptor alpha chain. Taken together, these results indicate that both CD3+ and CD3- GL are stimulated by IL-15 and that this cytokine mediates its activity through the beta and gamma chains of the IL-2R, providing further suggestions for the interpretation of the mechanisms that lead to cell expansion in patients with LDGL.
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PMID:Interleukin-15 triggers the proliferation and cytotoxicity of granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes. 897 93

Three clonally related T-cell lymphoma lines (PB-1, 2A, and 2B) were examined for expression of IL-2, IL-4, and their receptors. All three lines were derived from a single patient who had an atypical, progressive T-cell lymphoproliferative disorder involving primarily skin (Davis, T.H. et al. 1992, N. Engl. J. Med. 326:1115). The PB-1 cell line was obtained from a relatively early, clinically indolent stage of the cutaneous lymphoma, whereas the 2A and 2B lines were established from a late, aggressive stage of the lymphoma. Reverse-transcriptase PCR performed with primer pairs specific for IL-2 and IL-4 showed that no mRNA coding for these cytokines was present in any of the lines with the exception of IL-4 mRNA in the 2A line. No IL-4 protein, however, was found in any of the cell lines including 2A by immunocytochemical staining with anti-IL-4 mAb. Accordingly, no bioactive IL-4 was present in the supernatants of these lines. In contrast, all three T-cell lymphoma lines contained mRNA for IL-2R alpha, IL-2R beta, IL-4R and common gamma chain. Immunocytochemical analysis revealed that only the PB-1 line stained strongly with mAbs specific for IL-2R alpha, IL-2R beta, and IL-4R whereas the 2A and 2B lines showed only limited staining with these mAbs. In contrast to expression of IL-2R alpha and IL-4R primarily on the cell surface, IL-2R beta was localized mainly in the cell cytoplasm. Testing supernatants of the cell lines by ELISA for the presence of soluble alpha chain of the IL-2R (sIL-2R) has shown that only PB-1 secreted a large amount of sIL-2R, whereas the 2A and 2B lines secreted lesser amounts. Furthermore, the PB-1 cells expressed a relatively large number of IL-4R as determined by IL-4 binding studies using an IL-4-alkaline phosphatase fusion protein. The remaining two lines displayed only limited binding of IL-4. Addition of IL-2 and/or IL-4 to the culture medium did not modulate growth of PB-1 and the other two lines. These findings may indicate that at least some types of T-cell lymphoma evolve from cells which lose the capacity to synthesize T-cell autocrine growth factors such as IL-2 and IL-4, and show progressive loss of receptors for these cytokines.
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PMID:Analysis of IL-2, IL-4 and their receptors in clonally-related cell lines derived from a patient with a progressive cutaneous T-cell lymphoproliferative disorder. 902 95

To compare the cytokine profile with the degree and composition of cellular infiltration in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium, synovial membranes from patients with RA (n = 14) and OA (n = 5) were examined, employing immunohistochemistry and competitive reverse-transcriptase polymerase chain reaction (RT-PCR), for interleukin (IL)-I beta, IL-2, IL-4, IL-5, IL-6, and IL-10, and tumour necrosis factor-alpha (TNF-alpha) gene expression. It was found that the strength of cytokine gene expression within the synovial membranes of patients with RA was not significantly correlated with the degree of synovial infiltration of T-cells, B-cells, or macrophages. No IL-2, IL-4, or IL-5 RNA was detected in the synovium of either RA or OA. Quantitative cytokine determination showed a similar pattern in RA and OA, although the two diseases differed in total synovial infiltration and the composition of infiltrating cellular elements. Thus the number of cell types known to produce certain cytokines does not appear to determine the strength of synovial cytokine expression measured by quantitative RT-PCR. Furthermore, the pattern of T-cell specific cytokines found in RA synovium does not accord with the concept of the TH0, TH1, and TH2.
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PMID:Evaluation of synovial cytokine patterns in rheumatoid arthritis and osteoarthritis by quantitative reverse transcription polymerase chain reaction. 903 18

NK cells migrate in response to C-C chemokines, including monocyte chemotactic protein-1 (MCP-1) and MCP-3. Increased migration was observed in IL-2-activated NK cells. It was therefore of interest to define the expression in resting and activated NK cells of the MCP-1 receptor (CCR2) for which two cDNAs (A and B) have been described. Specific oligonucleotides and reverse-transcriptase PCR revealed the presence in activated NK cells and mononuclear phagocytes of the fragments expected on the basis of the reported cDNAs. In addition, amplification with a common A/B- and an A-specific oligonucleotide yielded an unexpected, abundant, 1649-bp fragment. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated NK cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B, followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A-specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. The augmented migratory capacity of IL-2 activated vs resting NK cells was associated with increased CCR2 transcript levels.
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PMID:IL-2-regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells: characterization of a predominant 3.4-kilobase transcript containing CCR2B and CCR2A sequences. 905 2


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