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)

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

The pleiotropic immunoregulatory cytokine transforming growth factor beta (TGF-beta) potently suppresses production of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome, in the chronically infected promonocytic cell line U1. TGF-beta significantly (50-90%) inhibited HIV reverse transcriptase production and synthesis of viral proteins in U1 cells stimulated with phorbol myristate acetate (PMA) or interleukin 6 (IL-6). Furthermore, TGF-beta suppressed PMA induction of HIV transcription in U1 cells. In contrast, TGF-beta did not significantly affect the expression of HIV induced by tumor necrosis factor alpha (TNF-alpha). These suppressive effects were not mediated via the induction of interferon alpha (IFN-alpha). TGF-beta also suppressed HIV replication in primary monocyte-derived macrophages infected in vitro, both in the absence of exogenous cytokines and in IL-6-stimulated cultures. In contrast, no significant effects of TGF-beta were observed in either a chronically infected T cell line (ACH-2) or in primary T cell blasts infected in vitro. Therefore, TGF-beta may play a potentially important role as a negative regulator of HIV expression in infected monocytes or tissue macrophages in infected individuals.
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PMID:Transforming growth factor beta suppresses human immunodeficiency virus expression and replication in infected cells of the monocyte/macrophage lineage. 170 78

The role of cytokines in vivo has been difficult to assess. This difficulty is due, in part, to the limited number of producer cells and the strict regulation of cytokine production. In order to address this situation, we have developed assays which allow us to quantitate both protein production and steady state mRNA levels from specific in vivo sites. In this report, we present data utilizing these assays on cells obtained from draining LN following specific sensitization with antigen in vivo. In order to determine the relative quantities of cytokine mRNA, we modified the reverse transcriptase-polymerase chain reaction which had been previously described. The modified assay is (1) linear over a large concn range of input template (2) demonstrates a high degree of reproducibility (SE approximately 13%) and (3) is very sensitive. Utilizing this assay, we have measured a constitutive mRNA (DHFR), quantitated both the presence of lymphokine mRNA (IL-2) and the induction of cytokine mRNA (TNF alpha). In this report we have examined the kinetics of TNF alpha mRNA expression and have demonstrated that following epicutaneous sensitization with picryl chloride, there is rapid induction (within 24 hr) of TNF alpha mRNA in the draining LN and that the levels of mRNA remain detectable through d7. In addition, we determined the time course of production of TNF protein by the draining LN cells and found that it was similar to that of the mRNA levels. A potential pathologic role for immune response generated TNF alpha is also discussed. We believe these experiments demonstrate that cytokine production following antigen-specific sensitization in vivo can be analyzed at both the cellular and molecular level. The data suggests that this approach can be used to study cytokine regulation in vivo.
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PMID:Quantitation of cytokine mRNA levels utilizing the reverse transcriptase-polymerase chain reaction following primary antigen-specific sensitization in vivo--I. Verification of linearity, reproducibility and specificity. 171 71

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

We have used the reverse transcriptase-polymerase chain reaction technique to gain insight into the pathogenesis of encephalitis caused by Borna disease virus (BDV). RNA specific for BDV was first detected in the olfactory bulb of intranasally infected rats at 6 days postinfection (p.i.). At 14 days p.i., high levels of BDV RNA were found in all brain regions, and at 26 days p.i., BDV-specific RNA was also present in the eye, nasal mucosa, and facial skin. In the chronic phase of the disease, BDV RNA was identified in many peripheral organs but not in blood. Analysis of brain tissue for the presence of cytokine mRNAs revealed that the mRNA levels of interleukin-6 (IL-6), tumor necrosis factor alpha, and IL-1 alpha had increased sharply at 14 and 26 days p.i. These cytokine mRNAs reached maximum levels at the peak of inflammatory reactions and decreased drastically in the chronic phase of the disease. Although IL-2 mRNA was also found in normal brain, it was markedly increased in BDV-infected brain at 14 days p.i. Expression of gamma interferon (IFN-gamma) mRNA, which was not observed in normal rat brain, was detected at 14 days p.i. and reached a maximum level at 38 days p.i. IL-2 and IFN-gamma mRNA expression correlated with expression of CD4 and CD8 mRNAs, indicating that both CD4+ and CD8+ T lymphocytes are induced in the early stages of BDV infection. Since IFN-gamma and CD8 mRNA levels were still highly elevated in the chronic phase of Borna disease, it is likely that CD8+ T lymphocytes act to reduce inflammation and to ameliorate neurological signs during the chronic phase of infection.
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PMID:Kinetics of virus spread and changes in levels of several cytokine mRNAs in the brain after intranasal infection of rats with Borna disease virus. 173 Nov 17

To assess changes in epidermis-derived cytokine mRNA levels early in the afferent phase of allergic contact sensitivity, total epidermal mRNA was analyzed at various times after painting skin with haptens. We used a sensitive reverse transcriptase-polymerase chain reaction technique to quantitatively compare the regulation patterns of the following mRNAs: class II major histocompatibility complex I-A alpha, tumor necrosis factor alpha (TNF-alpha), interleukin (IL) 1 alpha, IL-1 beta, interferon (IFN) gamma, granulocyte/macrophage colony-stimulating factor, IFN-induced protein 10, and macrophage inflammatory protein 2. Enhanced Langerhans cell-derived IL-1 beta mRNA signals were detected as early as 15 min after skin painting with allergens. TNF-alpha, IFN-gamma, and granulocyte/macrophage colony-stimulating factor mRNAs were found to be upregulated after application of allergens, irritant, and tolerogens, but class II major histocompatibility complex I-A alpha, IL-1 alpha, IL-1 beta, IFN-induced protein 10, and macrophage inflammatory protein 2 mRNAs were upregulated only after allergen painting. Depletion of specific cell populations demonstrated that Langerhans cells were the primary source of the IL-1 beta and class II major histocompatibility complex I-A alpha mRNAs, keratinocytes were the primary source of TNF-alpha, IL-1 alpha, IFN-induced protein 10, and macrophage inflammatory protein 2, and infiltrating T lymphocytes were the source of IFN-gamma. Relevance of the molecular findings was demonstrated by the identification of biologically active IL-1 alpha and immunoreactive TNF-alpha in culture supernatants. These studies demonstrate that Langerhans cell-derived and certain keratinocyte-derived cytokine mRNAs are selectively upregulated by allergens in the very early afferent phase of contact sensitivity.
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PMID:Early molecular events in the induction phase of contact sensitivity. 174 95

Lentiviruses belong to the retroviruses family (ie RNA viruses with reverse transcriptase activity); they induce inflammatory and/or degenerative slowly progressive diseases, affecting various organs. Some lentiviruses preferentially infect lymphocytes (HIV-1 and HIV-2, SIV and FIV) and are associated with infectious and tumoral disorders. Most lentiviruses induce a pulmonary disease, typically diffuse interstitial pneumonia. The visna/maedi-virus of sheep infects monocyte macrophage cells and the pulmonary lesions are macrophagic and neutrophilic alveolitis, lymphoid infiltration, myomatosis and interstitial fibrosis. Such pulmonary lesions are also induced by the goat and equine lentiviruses. In humans infected by HIV-1 or HIV-2, a diffuse interstitial lung disease also occurs; the histological findings are of alveolitis associated with lymphoid peribronchovascular infiltrates. The mechanism of formation of the lesions involves complex cellular interactions (especially between macrophage and lymphocyte, via cytokine production). These interactions are well modelled by small ruminant lentivirus induction of interstitial pneumonia.
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PMID:[Diffuse interstitial pneumopathies caused by lentivirus (HIV-1) in humans and animals]. 198 Jan 53

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.
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PMID:Direct and cytokine-mediated activation of protein kinase C induces human immunodeficiency virus expression in chronically infected promonocytic cells. 220 Aug 85

We have previously described model systems for cytokine-induced regulation of chronically HIV-infected promonocyte and T cell clones. Using these systems, we have shown that monokines contained in supernatants from LPS-stimulated human monocyte/macrophages (MO) up-regulate HIV expression, reflected by an increase in reverse transcriptase activity, viral RNA levels, and expressed viral proteins. Current studies were designed to determine whether viral Ag can interact with MO and secondarily affect HIV1 expression by stimulating monokine production. We found that certain herpes-group viruses, including CMV and EBV, augment HIV1 expression by inducing monokine production, whereas others, such as HSV1, HSV2, varicella-zoster virus, and human herpes virus 6 were unable to function in this capacity. The HSV1 and HSV2 Ag which failed to stimulate monokine production did not interfere with MO stimulation by CMV Ag, suggesting that failure to induce HIV expression was not attributable to MO suppression. When nonherpes group viruses were tested, we found that human adenovirus, hepatitis B virus, and vaccinia virus all failed to stimulate the production of monokines capable of activating HIV in the chronically infected cell lines. In contrast, HIV1 can augment its own expression by inducing the secretion of monokines which up-regulate HIV expression in the infected cells. The viral Ag-induced MO supernatants capable of up-regulating HIV expression did so in a dose-dependent manner, whereas viral Ag alone produced no significant change. Monokine production mediated by viral Ag was not attributable to contaminating endotoxin. These studies provide a model to determine whether other opportunistic infections may induce the expression of HIV by indirect mechanisms, such as the stimulation of cytokine production.
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PMID:Viral antigen stimulation of the production of human monokines capable of regulating HIV1 expression. 254 45


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