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)

Regulation of interleukin (IL)-12 production by coexpression of tumor necrosis factor (TNF)-alpha, IL-10, and transforming growth factor (TGF)-beta in human monocytes infected with Mycobacterium tuberculosis H37Ra was analyzed. Also, since IL-12 induces interferon (IFN)-gamma, the effect of IFN-gamma on IL-12 expression was examined. IL-12 mRNA was measured by reverse transcriptase-polymerase chain reaction and IL-12 protein by ELISA. IL-12 p35 mRNA was constitutive and inducible. IL-12 p70 protein paralleled IL-12 p40 protein expression. TNF-alpha protein expression occurred earlier than IL-12 p40 protein but was not required for IL-12 induction. Addition or neutralization of TGF-beta did not significantly alter IL-12 induction. In contrast, recombinant IL-10 reduced IL-12 and neutralization of IL-10 minimally enhanced IL-12. A pronounced increase in IL-12 followed IFN-gamma pretreatment, which selectively up-regulated IL-12 p35 mRNA. Further understanding of operative cytokine networks during M. tuberculosis infection may improve strategies for vaccine development and immunotherapy.
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PMID:Regulation of interleukin-12 by interleukin-10, transforming growth factor-beta, tumor necrosis factor-alpha, and interferon-gamma in human monocytes infected with Mycobacterium tuberculosis H37Ra. 980 41

To understand biological function of IL-6 in the skin in vivo, we constructed a vector that strongly expressed human IL-6 in keratinocytes and introduced it into rat keratinocytes in vivo by the naked DNA method. The overexpression of IL-6 induced macroscopic erythema and histologically evident keratinocyte proliferation and lymphocytic infiltration in the treated area of rat skin. Since previous studies using IL-6 transgenic mice have not shown skin inflammation of these mice, our result provides the first evidence that IL-6 is related to the pathogenesis of inflammatory skin diseases. ELISA suggested that a certain degree of transgenic IL-6 expression in keratinocytes was required for inducing skin inflammation. Cytokine profile in rat keratinocytes after the gene introduction was examined by reverse transcriptase-PCR assay and revealed that gene expression of rat IL-1alpha and TNF-alpha showed no marked change until 24 h, whereas that of rat IL-6 and TGF-alpha increased with time. We then introduced and expressed the IL-6 mutant genes, which were designed to behave as IL-6Ralpha antagonists, and found that their ability to induce erythema was lower than that of the wild-type gene. Furthermore, preintroduction of some mutant genes delayed the erythema induced by postintroduction of the wild-type IL-6 gene, suggesting that the mutant forms of IL-6 prevent wild-type IL-6 from binding to IL-6Ralpha. This result indicates that keratinocyte gene therapy may be possible for inflammatory skin diseases using IL-6 mutant genes.
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PMID:Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes. 982 May 43

We have previously shown that abdominal surgery (explorative laparotomy) reduces the ability of lipopolysaccharide (LPS)-triggered spleen macrophages to secrete TNF-alpha. In this study we characterize possible mechanisms which could be responsible for the reduction in splenic production of TNF-alpha. Post-operative and control (unoperated) rat splenocytes or enriched splenic macrophages were cultured with LPS. Steady-state levels of TNF-alpha mRNA were determined by Northern and slot blot analyses, and validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The amount of TNF-alpha protein was measured by Western blot analysis, and its biological activity was determined by the fibroblast L-929 cytotoxicity assay. Surgery induced a 12-fold inhibition in TNF-alpha activity (P < 0.02), caused up to two-fold reduction in the accumulation of TNF-alpha mRNA (P < 0.01), and suppressed TNF-alpha protein maturation into its 17-kD form in cellular extracts. Post-surgical spleen supernatants revealed mainly a band of a lower molecular weight (14 kD). Our data suggest a multilevel regulation of post-operative inhibition of TNF-alpha response to LPS, at the accumulation of mRNA, translational and secretory levels. We also suggest that the reduced bioactivity could be partially caused by a proteolytic cleavage of TNF-alpha. Since TNF-alpha is an important participant in immune responses, its reduced production and activity may be a central mechanism of post-operative immunosuppression.
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PMID:Abdominal surgery reduces the ability of rat spleen cells to synthesize and secrete active tumour necrosis factor-alpha (TNF-alpha) by a multilevel regulation. 993 16

CD43/leukosialin is a major sialoglycoprotein of the dendritic cell (DC) surface, which can regulate cell adhesion and has the potential to mediate cell activation signals. Monocyte-derived DC transiently incubated with the anti-CD43 mAb, MEM-59, or with F(ab')2 fragments, but not with monovalent Fab fragments or control IgG, 24 h later showed increased levels of membrane HLA-DR, CD54, CD40, CD80, CD86, and CD83. In parallel, CD43 cross-linking induced synthesis and release of IL-1beta, IL-6, TNF-alpha, IL-12, and IL-10. CD43 ligation inhibited the endocytic activity of DC, and enhanced the capacity of DC to stimulate T cell proliferation in the primary allogeneic and autologous MLR assay. In addition, anti-CD43-treated DC were less efficient at presenting native HIV-1 reverse transcriptase to a specific CD4+ T cell clone, whereas presentation of the reverse transcriptase 55-72 peptide to the same clone was increased. Finally, MEM-59 or its F(ab')2 fragments elicited a rise in intracellular free calcium and tyrosine phosphorylation of a 25-kDa protein in DC. The results thus indicate that CD43 cross-linking with specific ligands induces activation and functional maturation of DC.
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PMID:Cross-linking of membrane CD43 mediates dendritic cell maturation. 1035 44

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.
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PMID:Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages. 1041 Sep 80

Interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha, and the secreted form of the IL-1 receptor antagonist (sIL-1RA) are involved in the inflammatory response to inhaled grain dust. Previously, we found considerable production of these cytokines in the lower respiratory tract of workers exposed by inhalation to aqueous extracts of corn dust extract. Alveolar macrophages (AM) have long been considered the cell type responsible for producing these cytokines, and only recently has it been realized that airway epithelial cells may also be involved in cytokine production. In order to determine whether airway epithelia are involved in the inflammatory response to inhaled corn dust extract and to compare the magnitude of response of bronchial epithelial cells (BE) and bronchoalveolar lavage (BAL) cells, we used the reverse transcriptase/polymerase chain reaction (RT/PCR) technique in a semiquantitative manner to evaluate the concentration of IL-1beta, IL-6, IL-8, TNF-alpha, and sIL-1RA. Alveolar cells were obtained by BAL, and BE were obtained by endobronchial brush biopsy from 15 grain handlers 6 h after experimental inhalation of saline or an aqueous corn dust extract. After inhalation of saline, BE expressed low but detectable levels of IL-6, IL-8, and IL-1beta (> 1 complementary DNA [cDNA] molecule/cell). After inhalation of corn dust extract, the expression of messenger RNA (mRNA) for IL-1beta and IL-8 in the BE were significantly increased, whereas no change was seen in IL-6, sIL-1RA, and TNF-alpha mRNA expression. Comparing cytokine mRNA levels in BE and BAL cells from the same subjects after inhalation of corn dust extract, BE and BAL cells expressed equivalent amounts of IL-8 mRNA; IL-1beta was 11-fold higher in BAL cells; and TNF-alpha and sIL-1RA were expressed exclusively by BAL cells. Immunostaining for the cytokines in BAL cells showed cytokine protein expression in AMs but not in polymorphonuclear cells (PMNs). On the other hand, sIL-1RA was strongly expressed in both AMs and PMNs. Analysis of cytokine protein levels in endobronchial lavage (EBL) fluid demonstrated that only IL-8 was released in detectable amounts into the airway lumen, whereas all the other cytokines of interest were exclusively found in the BAL fluid. Thus, within 6 h after inhalation exposure to corn dust extract, BE appear to contribute to airway inflammation by producing IL-8. AMs are responsible for most of the IL-1beta and IL-6 production in the alveolar region, whereas AMs and PMNs both produce sIL-1RA. Our findings suggest that the inflammatory response to inhaled grain dust is compartmentalized, involving specific mediators of inflammation released by macrophages, neutrophils, and airway epithelial cells.
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PMID:Compartmentalization of the inflammatory response to inhaled grain dust. 1050 23

1 The effects of nepalolide A on the expression of inducible nitric oxide synthase (iNOS) caused by incubation with lipopolysaccharide/interferon-gamma (LPS/IFN-gamma) or tumour necrosis factor-alpha/interleukin-1beta/IFN-gamma (TNF-alpha/IL-1beta/IFN-gamma, mixed cytokines) in C6 glioma cells and primary astrocytes of rat were investigated. The mechanisms by which nepalolide A confers its effect on iNOS expression were also elucidated. 2 Treatment with LPS/IFN-gamma and mixed cytokines for 24 h elicited the induction of iNOS activity as determined by nitrite accumulation in the culture medium and assay of enzyme activity. Nepalolide A at 10 microM abrogated the LPS/IFN-gamma- and mixed cytokines-mediated induction of iNOS by more than 90% in C6 glioma cells, and by 80% for mixed cytokines-induced induction of iNOS in primary astrocytes. The effect of nepalolide A (2-10 microM) was concentration-dependent. 3 The inhibition of iNOS induction by nepalolide A was attributed to decreases in the content of iNOS protein and the level of iNOS mRNA, as measured by immunoblotting and reverse transcriptase-polymerase chain reaction. 4 Electrophoretic mobility shift assay was used to evaluate the effect of nepalolide A on the activation of nuclear factor-kappaB (NF-kappaB). Results showed that nepalolide A diminished the LPS/IFN-gamma-mediated association of NF-kappaB with consensus oligonucleotide in a concentration-dependent manner. The activation of NF-kappaB by mixed cytokines was modulated both in the extent of activation and in its time-course by nepalolide A. 5 The ability of nepalolide A to inhibit NF-kappaB activation was further confirmed by studies on the degradation of the inhibitor of NF-kappaB, IkappaB, as measured by immunoblotting. 6 The present study demonstrates that the attenuation of NF-kappaB activation by nepalolide A was mediated by blockade of the degradation of IkappaB, leading to suppression of the expression of iNOS.
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PMID:Nepalolide A inhibits the expression of inducible nitric oxide synthase by modulating the degradation of IkappaB-alpha and IkappaB-beta in C6 glioma cells and rat primary astrocytes. 1051 Apr 44

Evidence from clinical studies and murine models supports a role for cytokines in the pathogenesis of human cerebral malaria (CM). In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate expression of mRNA for transforming growth factor (TGF)-beta, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha in human postmortem tissue. Immunohistochemistry was used to examine the distribution of cytokine protein. TGF-beta was expressed in normal brain, in CM, and in meningitis and encephalitis. IL-1beta was absent from normal brain but was detected in CM and other cerebral infections. TNF-alpha mRNA was expressed only in CM, although TNF-alpha protein was also seen in meningitis. Cytokine mRNA expression in the brain did not correlate with the density of parasitized erythrocytes detected using RT-PCR for major surface protein-2. This report of RT-PCR on postmortem human tissues infected with CM demonstrates induction of the proinflammatory cytokines TNF-alpha and IL-1beta in the brain.
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PMID:Cytokine expression in the brain in human cerebral malaria. 1051 46

Dendritic cells (DC) have been implicated in the initial selection for macrophage-tropic HIV-1 during transmission and in the generation of high-level virus replication during interactions with CD4 T cells. The role of DC as viral reservoirs and the extent of productive infection is unclear, but the ability to generate large numbers of DC from blood monocytes has produced a tractable model for study of DC-HIV-1 interactions. When cultured in granulocyte-macrophage colony stimulating factor and IL-4, sorted CD14+ monocytes rapidly lost phagocytic function for both 93 nm and 977 nm latex particles and developed the surface markers and function of DC. After 7 days, when returned to medium containing human serum without cytokines, some monocyte-derived dendritic cells (MDDC) became adherent, but retained the costimulatory markers CD80 and CD86 and continued to express CD83 and CD40. The MDDC stimulated allogeneic CD4 T cells, did not express new macrophage markers and remained non-phagocytic. With or without TNF-alpha, MDDC generated in cytokines were infected by macrophage and T cell-tropic virus and produced higher reverse transcriptase levels than did the autologous monocyte-derived macrophages (MDM). When added to T cells, the infected MDDC were able to infect T cells with a wider range of viral isolates than were MDM.
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PMID:Monocyte-derived dendritic cells as a model for the study of HIV-1 infection: productive infection and phenotypic changes during culture in human serum. 1054 Feb 11

Immunologic injury to heart allografts is an initial and essential event in the pathogenesis of graft coronary artery disease (GAD). A variety of cytokines expressed in heart allografts modify both acute rejection and chronic inflammation, and could contribute to the development of GAD. The present study investigated the gene expression of interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and Fas ligand in chronically rejecting DBA/2-to-B 10.D2 mouse heart allografts at defined intervals of 7, 14, 28, or 70 days after transplantation by semiquantitative reverse transcriptase-polymerase chain reaction. GAD developed gradually, showing the highest value for mean intima/media ratio at day 70. Fas ligand, and the Th1 cytokines IL-2 and IFN-gamma, were vigorously induced in allografts at day 7, when histology showed pronounced parenchymal rejection, and rapidly decreased by day 28. However, the level of mRNA expression of Th2 cytokines, IL-6 and IL-10, and other inflammatory cytokines, TNF-alpha and IL-1beta, were still elevated on day 28. The persistent expression of specific cytokines suggests an important role in chronic inflammation. Thus, a persistently high level expression of inflammatory cytokines could be associated with chronic inflammation in the allografts, which promotes the development of GAD.
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PMID:Cytokine gene expression during the development of graft coronary artery disease in mice. 1055 20


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