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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

FK-506 is a potent immunosuppressant which is known to decrease the generation of allospecific cytotoxic T cells, possibly through its effect on cytokine production. FK-506 has been reported to have a suppressive effect on cytokine synthesis and secretion by pooled lymphocytes; its action on more selective subsets of lymphocytes is unknown. We currently report the effect of FK-506 on proliferation and IL-4 messenger RNA production by the differentiated mouse T-helper 2 cell line Ly1+2-/9. Ly1+2-/9 cells were grown in culture and treated with (1) media alone, (2) Con A, (3) FK-506, and (4) Con A + FK-506. Proliferation to Con A was determined by treating cells with 10 micrograms/ml Con A +/- FK-506 and measuring [3H]TdR uptake. Reverse transcriptase-polymerase chain reaction amplification was used to quantitate the level of IL-4 mRNA expression. FK-506 markedly inhibits Con A-induced proliferation and IL-4 mRNA production by the T-helper 2 cell line Ly1+2-/9. The ability of FK-506 to block the proliferation and IL-4 production by this helper cell subset suggests that this effect may contribute to its observed marked immunosuppressive properties in vitro and in vivo.
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PMID:FK-506 inhibits proliferation and IL-4 messenger RNA production by a T-helper 2 cell line. 138 12

Peptides of melanosomal proteins have recently been shown to be recognized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are considered to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. Flow cytometric analysis of the cells during a 2-week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a,b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20-fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically active proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reverse-transcriptase-polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.
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PMID:Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. 748 49

Because UV-induced epidermal macrophages (UV-Mph) preferentially activate CD4+ T suppressor-inducer cells and induce tolerance, we hypothesized that they differentially up-regulate T cell early activation genes compared with constitutive epidermal APC, Langerhans cells. We used epidermal cells from UV-exposed (UV-EC) and control (C-EC) human skin to stimulate allogeneic CD4+ T lymphocytes. Reverse transcriptase-PCR revealed that both C-EC (Langerhans cells) and UV-EC (UV-Mph) induced 10(3)- to 10(6)-fold increases in IL-2 mRNA. However, while T cells stimulated by C-EC for 48 h showed a greater than 10(3)-fold increase in IL-2R alpha mRNA, those stimulated by UV-EC did not (n = 5, p = 0.004). Flow cytometry demonstrated that 4.1 +/- 2.3% of unstimulated CD4+ lymphocytes expressed cell surface IL-2R alpha, which increased to 15.7 +/- 1.8% upon stimulation by C-EC for 48 h, but stimulation by UV-EC failed to increase the IL-2R alpha+ population (n = 3, p = 0.038). The addition of neutralizing anti-TGF-beta Abs to UV-EC-stimulated cultures restored CD4+ cell surface IL-2R alpha expression to 12.9 +/- 0.2%. CD4+ T cell activation by UV-Mph is distinct from previously described models of tolerance such as Th2 activation (IFN-gamma mRNA was induced and IL-4 mRNA was not) and Th1 anergy (IL-2 mRNA levels induced by UV-EC and C-EC were similar). Furthermore, costimulatory signals were provided by UV-Mph; CTLA4-Ig and LFA-3-Ig fusion proteins and Abs to CD2, LFA-3, LFA-1, and ICAM-1 inhibited UV-Mph-induced T cell proliferation. Thus, the altered immune outcome induced by UV-Mph (tolerization) compared with Langerhans cells (sensitization) is reflected as a novel mechanism of initial CD4+ T cell early activation gene expression characterized by TGF-beta-dependent deficient IL-2R alpha expression.
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PMID:Suppressor T cell-activating macrophages in ultraviolet-irradiated human skin induce a novel, TGF-beta-dependent form of T cell activation characterized by deficient IL-2r alpha expression. 749 43

Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1-5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals.
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PMID:Mathematical considerations of competitive polymerase chain reaction. 749 79

The anti-CD4 mAb W3/25 inhibits experimental autoimmune encephalomyelitis (EAE) in Lewis rats by blocking Th cell responses to encephalitogenic determinants of myelin basic protein (MBP). However, it has yet to be resolved how W3/25 modulates CD4 to inhibit EAE-associated T cell responses. This study revealed that W3/25 profoundly inhibited MBP-stimulated proliferation by sensitized lymph node cells but only partially inhibited the respective response of uncloned and cloned lines of MBP-specific T cells. That is, low concentrations of W3/25 blocked 30 to 60% of MBP-stimulated proliferation, but 100-fold higher concentrations did not result in additional inhibition. W3/25 also inhibited MBP-induced acquisition of EAE transfer activity, but only in cultures of freshly isolated lymph node cells and not in cultures of continuously propagated T cells. Studies focusing on the GP2.E5 T cell line revealed that the lack of sensitivity to W3/25 in encephalitogenic and proliferative assays was nevertheless associated with an effective blockage of MBP-stimulated IL-2 production. Importantly, W3/25 specifically inhibited antigenic but not mitogenic stimulation of IL-2 production. Reverse transcriptase/polymerase chain reaction analyses revealed that MBP-activated GP2.E5 T cells produced mRNA for both IL-2 and IL-4, and that W3/25 selectively inhibited accumulation of IL-2 as compared to IL-4 mRNA. Thus, GP2.E5 T cells apparently express a IL-4-dependent pathway that confers resistance to the inhibitory activity of W3/25. Studies focusing on two CD4+ T cell hybridomas revealed that W3/25 profoundly inhibited MBP-stimulated IL-2 production but did not affect the alternative response of MBP-induced growth inhibition. Several other hybrids also mediated MBP-stimulated IL-2 production but did not express CD4 and were not affected by W3/25. These results indicate that: 1) interactions of W3/25 with CD4 do not necessarily block class II MHC-restricted recognition of MBP; and 2) expression of CD4 is not necessary for Ag recognition by several clonotypes of MBP-reactive T cells. Rather, the results of this study are consistent with the concept that W3/25 inhibits transduction of costimulatory signals that are required specifically for initiation of IL-2 production. These findings may have important implications for understanding the therapeutic potential of anti-CD4 mAb in autoimmune disease.
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PMID:Differentiation of encephalitogenic T cells confers resistance to an inhibitory anti-CD4 monoclonal antibody. 750 25

In nasal biopsies from 17 adult patients with seasonal allergic rhinitis and from 10 healthy controls, cytokines were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). The time-course study during winter included repeated local allergen provocation with subsequent nasal biopsies as well as biopsies taken during pollen season. The RT-PCR for CD44 yielded positive bands in 65 of 71 cases, in which cases mRNA for interleukins 2, 4, and 5 (IL-2, IL-4, and IL-5) were thus investigated by means of seminested PCR. IL-4 mRNA was found almost exclusively in the allergic patients. During provocation a significant increase in IL-4 was noticed compared with controls (p = 0.043). Equally, during the natural pollen season, IL-4 mRNA expression was significantly higher in patients not using nasal corticosteroids compared with those who did (p = 0.011). No differences in IL-2 or IL-5 were observed between the groups. These findings also indicate, together with earlier observations of T-cell activation, a phenotype switch toward T-helper 2 (Th2) cells, and the accumulation (homing) of these T cells in the nasal mucosa, that T cells constitute the main source for IL-4 in the nasal mucosa. Therefore, allergic patients have an increased synthesis of IL-4 when provoked with the allergen, and during natural pollen season this synthesis can be downregulated by corticosteroids. Furthermore, this study exemplifies the versatility of molecular biology in surgical pathology and that even low-copy-number cytokine mRNA can be examined in routinely snap-frozen surgical specimens.
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PMID:Nasal messenger RNA expression of interleukins 2, 4, and 5 in patients with allergic rhinitis. 755 Dec 98

The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand.
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PMID:CD40 expression by human fibroblasts. 755 83

Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet beta-cells following islet infiltration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in beta-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1 beta, IL-2, IL-4, IL-10, and IFN-gamma in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (> 13 wk). However, only IFN-gamma mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-gamma and IL-4 were not different in the four groups of mice. These results suggest that islet beta-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-gamma production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-gamma production in the islets.
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PMID:IFN-gamma gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice. 772 37

The cytokine effector status of CD4+ T cells from lymph nodes (LN) and the central nervous system (CNS) of SJL/J mice immunized with autoantigen in adjuvant for the induction of experimental allergic encephalomyelitis (EAE) was compared. CD4+ T cells were FACS sorted based on the levels of expression of the activation marker CD45RB. Low levels of expression of this surface marker are induced by antigen recognition and are associated with 'effector' T cell function. Reverse transcriptase polymerase chain reaction (PCR) was used to analyze the expression of different T cell cytokine genes in the sorted populations. CD45RBlow cells constituted a minority of CD4+ cells in the LN and expressed elevated levels of IL-2, IFN-gamma, and IL-4 mRNA, whereas the CD45RBlow CD4+ population did not express detectable message for these cytokines under linear PCR conditions. By contrast to the LN, CD4+ cells from the CNS were predominantly CD45RBlow and expressed readily detectable levels of IL-2 and IFN-gamma mRNA, but almost no IL-4 transcription could be detected. IL-4 mRNA levels in CNS were 100- to 250-fold lower than in LN. Also, IL-4 message could not be detected in the CNS 1 week after remission. A cytokine-specific immunocytochemical single cell staining technique was used to enumerate cytokine-producing cells in LN cell populations and in CNS infiltrates. Between 1 and 5% cells in isolated LN cells produced detectable IL-2 and IFN-gamma. By contrast, the frequency of cytokine-producing cells stained in perivascular infiltrates in frozen sections from the brains of animals with active EAE was 10-fold higher.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Selective enrichment of Th1 CD45RBlow CD4+ T cells in autoimmune infiltrates in experimental allergic encephalomyelitis. 791 Apr 82

A biologically relevant mechanism of generating peripheral tolerance in T cells that have escaped thymic deletion is by the oral administration of soluble antigens. Oral tolerance occurs by two distinct mechanisms. Feeding a single high dose of antigen induces anergy of antigen-specific TH1 cells, while multiple low doses of antigen induce regulatory T cells that mediate suppression by producing immunosuppressive cytokines. Although cytokine production orally tolerant animals has been well studied utilizing in vitro assay systems, semi-quantitative characterization of cytokine production in oral tolerance in vivo has not been carried out. In this paper we have developed a system using semi-quantitative RNA polymerase chain reaction to characterize cytokine gene expression in vivo in mice orally tolerized by feeding either a single high dose or multiple low dosages of antigen. We find that measurable differences in interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) gene expression occurred between the tolerized and non-tolerized groups. Qualitatively, IL-4 mRNA was increased in both orally tolerized groups. However, significant differences in IL-4 gene expression between the two groups in both magnitude and kinetics were found. A large but short-lived increase in IL-4 was produced in mice fed a single high dose, while a relatively more moderate, longer-lived increase was produced in mice fed multiple low doses. The increase in IL-4 gene expression was specific only to the draining lymph node following antigen administration. Expression of IFN-gamma was decreased in both orally tolerant groups. These results indicate that tolerance in TH1 cells is induced by both of these feeding regimens while TH2 responses are intact and amplified upon reexposure to antigen.
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PMID:Different kinetic patterns of cytokine gene expression in vivo in orally tolerant mice. 795 64


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