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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The "stromal" or adherent cells of long-term murine Dexter explant bone marrow cultures provide the best in vitro model of the bone marrow microenvironment. Colony-stimulating factor-1 (CSF-1) is produced constitutively by these cells and is easily detected, but most investigators have not found constitutive production of the other hemolymphopoietic cytokines. We have previously reported the detection of granulocyte-macrophage-CSF (GM-CSF) in murine stromal cultures and its induction by the lectin Pokeweed mitogen. The present studies analyzing stromal cytokine messenger RNA (mRNA) production by standard Northern blot analysis show constitutive production of mRNAs for CSF-1, GM-CSF, granulocyte-CSF (G-CSF), c-kit ligand (KL), and interleukin-6 (IL-6), but not IL-3, IL-4, or IL-5 by 3-week irradiated or nonirradiated murine Dexter stromal cells. Exposure of stromal cells to Pokeweed mitogen or IL-1 16 hours before RNA harvest induces the messages for GM-CSF, G-CSF, KL, and IL-6, but not IL-3, IL-4, IL-5, or CSF-1. Polymerase chain reaction amplification of cDNA made with reverse transcriptase from stromal RNA using two separate sets of IL-3-specific primers shows the presence of IL-3 message in irradiated stromal cells, which is only detectable with this more sensitive technique. The factor-dependent cell lines FDC-P1 and 32D are supported by the stromal cells without the addition of exogenous growth factors, demonstrating a cytokine activity in these cultures that is inhibited by the addition of anti-IL-3 or anti-GM-CSF antibodies. These data indicate that murine Dexter stromal cells constitutively produce CSF-1, GM-CSF, G-CSF, IL-6, KL, and IL-3. This growth factor production could explain the support of granulocyte, macrophage, and megakaryocyte production and stem cell maintenance in Dexter-type long-term murine bone marrow cultures.
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PMID:Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma. 137 43

Experimental autoimmune encephalomyelitis (EAE) serves as an important animal model for understanding the events that lead to immune-mediated inflammation and tissue destruction within the central nervous system. We have utilized a murine adoptive transfer model of EAE and semiquantitative reverse transcriptase-polymerase chain reaction analysis to examine cytokine mRNA expression within the central nervous system in relation to the onset and resolution of paralysis associated with EAE. Spinal cord samples, obtained from mice as they progressed through discrete clinical stages of EAE, were examined for the expression of six cytokine genes (IL-1 alpha, IL-2, IL-4, IL-6, IL-10, and IFN-gamma). Distinct patterns of cytokine gene expression were observed during the acute, recovery, and chronic phases of EAE. The acute phase of disease was characterized by rapid increases in the levels of mRNA for IL-2, IL-4, IL-6, IFN-gamma, and IL-1 alpha. In fact, peak expression of several cytokine mRNA (e.g., IL-2, IL-4, IL-6, and IFN-gamma) occurred before the peak in clinical severity. In contrast, IL-1 alpha mRNA levels were elevated throughout the initial disease course. IL-10 mRNA demonstrated only modest increases during the acute phase of EAE. Stabilization of the clinical symptoms was characterized by rapid declines in the mRNA levels of IL-2, IL-4, IL-6, and IFN-gamma. The decreases in these four cytokine mRNA levels occurred concomitant with a dramatic rise in IL-10 mRNA. Finally, of the six cytokine mRNA examined, only IL-1 alpha, IFN-gamma, and IL-10 mRNA remained elevated during the early chronic stage. These results suggest that local cytokine production varies significantly during the course of EAE and that increases in discrete sets of cytokines are associated with the acute response and the recovery/chronic phase of disease.
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PMID:Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. 152 89

Thuja polysaccharide g fraction (TPSg) was shown to be an inducer of the CD4+ fraction of the human peripheral blood T-cell subset (1,2). Furthermore, it could be demonstrated that TPSg is a potent inhibitor of the expression of HIV-1-specific antigens and of the HIV-1-specific reverse transcriptase (3). This report deals with the cytokine pattern induced by TPSg in human peripheral blood lymphocyte (PBL) and purified monocyte/macrophage cultures. In addition, a further characterization of the CD4+ T-cell fraction stimulated by TPSg was performed by FACS analysis. TPSg is induces IL-1 beta, IL-2, IL-3, IL-6, gamma-IFN, G-CSF, GM-CSF, and TNF-beta production in PBL cultures; and IL-1 beta and IL-6 in monocyte/macrophage cultures. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that no IL-4 was produced by PBL cultures under TPSg influence.
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PMID:Mitogenic activity of high molecular polysaccharide fractions isolated from the cuppressaceae Thuja occidentalis L. enhanced cytokine-production by thyapolysaccharide, g-fraction (TPSg). 160 22

We present evidence for RNA transcripts encoding two forms of human epsilon immunoglobulin (Ig) heavy chain that differ significantly from those of other isotypes. We previously demonstrated three human epsilon mRNA species, instead of the two, corresponding to membrane and secreted proteins, seen with other heavy chain transcripts. In human genomic DNA downstream of the C epsilon gene, we identified sequences homologous to the two putative murine exons M1 (encoding a hydrophobic, presumably transmembrane region) and M2 (encoding hydrophilic residues). To determine the structures of epsilon transcripts containing these sequences, we amplified epsilon-related RNAs with the reverse transcriptase polymerase chain reaction. RNA was examined from fresh human B cells stimulated to IgE production by interleukin 4 plus anti-CD40, as well as from the human IgE-producing line AF10. Instead of the single CH4-M1-M2 splice product predicted for murine membrane IgE, we found two other RNA species. One form has the structure CH4-M1'-M2, in which M1' includes the human sequence homologous to the murine M1 as well as a unique segment of 52 codons further upstream in the genomic sequence; this RNA species apparently encodes the IgE expressed on the membrane of IgE-producing lymphocytes. The other RNA has the structure CH4-M2', in which M2' is spliced in an alternative reading frame that includes an additional 109 codons downstream of the termination codon of the CH4-M1'-M2 form. Because the CH4-M2' mRNA form does not encode a hydrophobic segment, its translated product should be secreted. A secreted epsilon protein of approximately the size predicted for this form was identified by Western blotting. This novel IgE protein could play a significant and distinctive role in allergic disorders.
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PMID:Two unusual forms of human immunoglobulin E encoded by alternative RNA splicing of epsilon heavy chain membrane exons. 161 58

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay is described that allows the rapid detection and quantitation of mRNA encoding the cytokines interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma). Analysis of cytokine production by defined CD4+ T cell clones and the thymoma cell line EL4, demonstrates that the oligonucleotide primers used in this assay are specific for the genes encoding the individual cytokines, generating PCR products of different sizes. This allows the simultaneous and unambiguous detection of all three cytokine mRNAs in the same cDNA sample. The assay is sensitive enough to reproducibly detect cytokine mRNA expressed in as few as ten cells and requires 10,000-fold less cells for the detection of IL-2 production than that required for its detection using a conventional bioassay. Reverse transcribed mRNA is quantitated in the PCR assay by amplifying in the presence of a known amount of competitive genomic DNA (gDNA) template containing a small intron using the same primers. The PCR products obtained form the target cDNA and gDNA templates, which are distinguished by size, are processed by Southern analysis and quantitated by scanning densitometry of autoradiographs. As little as two-fold differences in cytokine mRNA can be reliably detected using this assay. We have demonstrated the successful application of this assay to the quantitation of pg amounts of IL-2 mRNA that is constitutively produced at low levels by fetal thymocytes in vivo during T cell ontogeny. The sensitivity, specificity, reliability and speed of this assay will facilitate the analysis of cytokine production in in vivo-derived or, in vitro propagated cells which are not available in sufficient numbers for analysis using more conventional molecular and biochemical assays.
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PMID:A polymerase chain reaction assay for the detection and quantitation of cytokine gene expression in small numbers of cells. 162 16

Recombinant interleukin 4 (IL-4) stimulated extracellular (EC) and intracellular (IC) production of human immunodeficiency virus (HIV) from infected human blood-derived monocytes and macrophages when incubated with the cells after but not before virus inoculation. Significant stimulation was observed in 20 of 27 experiments with monocytes (inoculated with HIV immediately after adherence) and 10 of 13 experiments with macrophages (inoculated after 5 days adherence) using a total of 30 normal donors of monocytes and macrophages, and 11 recent isolates of monocytotropic HIV strains (after one passage in mononuclear cells). Marked increases in EC and IC HIV antigen were observed in some experiments, which were comparable with the maximal stimulatory effects of other cytokines such as IL-2. IL-4 also had similar effects on infectious HIV concentration as measured by reverse transcriptase and TCID50 assays. Antibody to IL-4 prevented the stimulatory effect of the cytokine. The proportion of monocytes and macrophages infected by HIV, as determined by in situ hybridization, also increased after incubation with IL-4 for 7 days. The most marked effects were observed with HIV-infected macrophages, for which the proportion of unstimulated infected cells was lower (35 to 45% increasing to 66 to 70% with IL-4 treatment). There was also an increased proportion of cells with high granule concentrations, suggesting that IL-4 increases the intracellular concentration of viral nucleic acids. This was supported by semi-quantitative hybridization experiments showing that total HIV RNA increased in IL-4-stimulated monocytes 48 to 96 h after HIV inoculation. A marked increase in aggregates was observed on day 7 in HIV-infected monocytes treated with IL-4, compared to that in HIV-infected cells alone or IL-4-treated uninfected monocytes. These findings suggest that IL-4 stimulates HIV replication in the early phases of infection and may also facilitate virus transmission by aggregate formation.
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PMID:Recombinant interleukin 4 stimulates human immunodeficiency virus production by infected monocytes and macrophages. 163 80

Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy.
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PMID:Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody. 171 59

A cytokine-mediated excessive increase in nitric oxide (NO) by macrophages or glial cells via an inducible isoform of NO synthase (iNOS) has been proposed to play an important role in demyelinating diseases. To further investigate the role of iNOS in demyelination, experimental allergic encephalomyelitis (EAE), a known animal model of multiple sclerosis (MS) in mice, was chosen in this study. A semiquantitative reverse transcriptase-polymerase chain reaction (RT/PCR) analysis revealed an increase in the mRNA levels of iNOS and cytokines known to induce iNOS or inflammatory cytokines (interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-6, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and TNF-beta) in the spinal cord corresponding to the severity of the disease without significant change in the mRNA levels of immunoregulatory cytokines (IL-4, IL-10 and transforming growth factor (TGF)-beta) during the course of EAE. An immunohistochemical examination of the spinal cord using an iNOS-specific antibody showed iNOS-positive cells to be mainly inflammatory cells with a higher frequency of iNOS-positive cells at the peak of EAE than in the early phase. These iNOS-positive cells at the peak appeared to be composed of infiltrating macrophages and most of them were located in the necrotic area. These results suggested that cytokine-induced excessive NO via iNOS by macrophages caused tissue damage in the central nervous system in EAE.
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PMID:Expression of the inducible isoform of nitric oxide synthase in the central nervous system of mice correlates with the severity of actively induced experimental allergic encephalomyelitis. 749 86

Human immunodeficiency virus (HIV) infection leads to a progressive loss of CD4+ T helper (Th) type 1 cell-mediated immunity that is associated with defective in vitro CD4+ T cell proliferation and abnormal T cell death by apoptosis in response to T cell receptor (TCR) stimulation. Quantification of interleukin (IL)-2, interferon gamma, IL-4, IL-5, and IL-10 secretion by immunoassays, and of interferon gamma, IL-4 and IL-10 messenger RNA expression by competitive reverse transcriptase polymerase chain reaction after in vitro stimulation of the TCR revealed a similar Th1 cytokine profile in T cells from HIV-infected persons and from controls. These data indicated that the loss of CD4+ Th1 cell function in HIV-infected persons is not related to a Th1 to Th2 cytokine switch as previously proposed, but to a process of activation-induced death of CD4+ Th1 cells. Despite the absence of elevated levels of Th2 cytokines, apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented in vitro by antibodies to IL-10 or IL-4, two Th2 cytokines that downregulate Th1 cell responses, or by the addition of recombinant IL-12, a cytokine that upregulates Th1 functions. TCR-induced apoptosis of T cell hybridomas and preactivated T cells has been shown to involve the CD95 (Fas/Apo-1) molecule. CD4+ and CD8+ T cells from HIV-infected persons expressed high levels of the CD95 molecule, and, in contrast to T cells from controls, were highly sensitive to antibody-mediated CD95 ligation, which induced apoptosis in a percentage of T cells similar to that induced by TCR stimulation. As TCR-induced apoptosis, CD95-mediated apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented by the addition of recombinant IL-12. Together, these findings suggest that apoptosis of CD4+ T cells from HIV-infected persons involves an abnormal sensitivity to CD95 ligation, and to TCR stimulation in the presence of normal levels of Th2 cytokines. The preventive effect of IL-12 on both mechanisms has potential implications for the design of immunotherapy strategies aimed at the upregulation of CD4+ Th1 cell functions in AIDS.
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PMID:T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons. 750 20

Human tumors can constitutively express cytokines and growth factors, but the extent of this expression has not been investigated. Using 44 different probes to cytokines, growth factors, and their receptors, we tested 21 melanoma and 5 melanocyte cultures for RNA transcript expression by reverse transcriptase-polymerase chain reaction. With 30 amplification cycles, expression of the cytokines interleukin (IL)-1 beta, IL-6, leukemia inhibitory factor (LIF), IL-7, gro alpha, IL-8 and the p35 chain of IL-12 was detected in more than 60% of melanomas. Concomitant receptors for IL-6 and IL-7 were also detected. IL-1 alpha, IL-5, Rantes, IL-10, interferon (IFN)-beta, tumor-necrosis factor (TNF)-alpha, G-colony-stimulating factor (CSF) and GM-CSF were expressed at lower levels. Melanocytes showed greatly reduced cytokine RNA transcripts, and only gro alpha was consistently detected. No expression of IL-2, IL-3, IL-4, IL-9, the p40 chain of IL-12, IFN-alpha or IFN-gamma RNA transcripts was detected in melanomas or melanocytes. The growth factors expressed by melanomas and, after further signal amplification, by melanocytes were transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), TGF-beta, endothelial-cell growth factor (ECGF), basic-fibroblast growth factor (bFGF), nerve growth factor (NGF) and steel. The receptors EGFR, FGFR, NGFRp70 and c-kit were also expressed by melanomas and melanocytes. These results point to new possible autocrine and paracrine pathways in melanoma biology.
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PMID:Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes. 750 78


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