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)

Immunosuppressive drugs used in clinical transplantation block cytokine mRNA transcription in vitro, but clinical rejection episodes are common. An understanding of what cytokine message is transcribed would be helpful in determining what contributes to the success of immunosuppression and provide directions for further research aimed at targeting specific cytokines. Previous studies have examined cytokine mRNA in rejecting solid organ biopsies by the reverse transcriptase polymerase chain reaction (RT-PCR) with variable results. We used nonradioactive in situ hybridization with cytokine-specific riboprobes to determine the frequency of cells expressing cytokine mRNA in the allograft infiltrate. Kidney biopsies were obtained from patients receiving protocol biopsies and with clinical evidence of rejection. Fourteen biopsies with a pathologic diagnosis of rejection were studied. Eight showed no cytokine staining, 2 expressed IL-2, and 3 expressed IL-4 and IFN-gamma. The positive cells were present at a low frequency (mean 2, range 1-5 per 10 high-power fields). The proportion of kidney biopsies expressing detectable message for interleukin-2 (IL-2), interleukin-4 (IL-4), and interferon-gamma (IFN-gamma) by in situ hybridization were similar to those reported using RT-PCR. The novel finding is that these cytokines are expressed in a few strongly positive cells in the allograft infiltrate. The vast majority of infiltrating cells are negative. This suggests that either the biopsies were performed when cytokine message was not expressed at a high level or that in human allograft recipients the sustained expression of the cytokines IL-2, IL-4, and IFN-gamma may not be necessary for graft rejection.
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PMID:Low frequency of infiltrating cells intensely expressing T cell cytokine mRNA in human renal allograft rejection. 753 48

Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.
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PMID:Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages. 754 98

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type 1 (Th1) (interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV+ patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-gamma mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n = 30) as compared to patients with > 400 CD4+ T cells/mm3 (n = 6) and normal controls (n = 16). In addition, IL-10 mRNA levels were inversely associated with IFN-gamma expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-gamma produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV+ individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable to that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with < 400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-gamma by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulated and mitogen-stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.
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PMID:Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients. 755 96

The effects of staphylococcal enterotoxin B (SEB), a Staphylococcus aureus-derived bacterial superantigen, on expression of intercellular adhesion molecule-1 (ICAM-1) were examined in cultured normal and transformed (DJM-1 cells) human keratinocytes by flow cytometry, confocal microscopy, digital image processing, and reverse transcriptase-polymerase chain reaction. SEB significantly upregulated ICAM-1 expression in the interferon-gamma (IFN-gamma)-pretreated, HLA-DR-positive normal keratinocytes and DJM-1 cells in a dose-dependent manner, but not in the untreated, HLA-DR-negative cells. Other toxins such as diphtheria and pertussis toxins did not have the effect. The distribution of SEB and HLA-DR molecules was identical on the IFN-gamma-treated, HLA-DR-positive DJM-1 cells by confocal microscopy. Digital image processing analysis demonstrated that SEB induced a transient increase of intracellular calcium concentration only in the IFN-gamma-treated DJM-1 cells. Pretreatment of the IFN-gamma-treated DJM-1 cells with anti-major histocompatibility complex class II monoclonal antibody completely blocked the effect of SEB. Furthermore, ICAM-1 mRNA was detected in the IFN-gamma-pretreated, SEB-exposed normal keratinocytes by reverse transcriptase-polymerase chain reaction. Our results demonstrate that SEB binds to keratinocytes, presumably via major histocompatibility complex class II molecules such as HLA-DR, triggers calcium mobilization, and induces the synthesis of ICAM-1 molecules. We speculate that, in various cutaneous disorders, SEB penetrates the epidermis and interacts with HLA-DR-positive keratinocytes to upregulate ICAM-1 expression, thus modulating the course of the inflammatory process.
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PMID:Staphylococcal enterotoxin B upregulates expression of ICAM-1 molecules on IFN-gamma-treated keratinocytes and keratinocyte cell lines. 756 Nov 55

We have studied the effect of poly(ADP-ribose) synthetase on the interferon-gamma (IFN-gamma)-inducible expression of major histocompatibility complex (MHC) class II molecules. We constructed an expression plasmid capable of expressing either a sense RNA (MT-ARS) or an antisense RNA (pAS-FL or pAS-5') for poly(ADP-ribose) synthetase. We transfected the plasmid into mouse or human macrophage tumor cells and examined the effect on the expression of MHC class II molecules. The IFN-gamma-inducible expression of MHC class II gene was considerably reduced in transformant clones (A-2, B-2), in which the synthetase was highly expressed, whereas the depletion of the synthetase due to the expression of antisense RNA for the synthetase amplified the expression of MHC class II molecules. The results indicate that the level of the synthetase critically regulates the IFN-gamma-inducible MHC class II molecules. Next, we analyzed DNase I hypersensitive sites (DHS) of mouse MHC class II, I-A beta gene and found two sites, one in the promoter region and the other one in the first intron. The DHS in first intron was less sensitive towards DNase I attack in transformant clones (A-2, B-2) in which the synthetase was synthesized in a large quantity. Thus we constructed two beta-galactosidase reporter genes, one (A beta 2.0kb-lac z) containing the promoter region to a part of the second exon of the class II gene, and the other (A beta pro-lac z) containing the promoter region of the class II gene alone. The expression of the reporter gene was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and found that the expression of A beta 2.0kb-lac z was suppressed in the transformant clones (A-2, B-2) relevant to control cells but the expression of A beta pro-lac z was the same level among those cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of poly(ADP-ribose) synthetase on the expression of major histocompatibility complex (MHC) class II genes. 757 32

Currently only limited information is available as to why dominant IgA isotype responses are supported by mucosal T cells in effector tissues. To address this issue directly, gamma delta and alpha beta T cells were isolated from the submandibular gland (SMG) of mice as an example of mucosal effector tissues. Freshly isolated CD3+ T cells from this tissue contained relatively high numbers of activated cells [approximately 10% interleukin-2 receptor (IL-2R)+ cells and 15% of cells in cycle stages S and G2 + M], of which 25% and 75% were gamma delta and alpha beta T cells, respectively. The cytokine-specific quantitative reverse transcriptase-polymerase chain reaction and enzyme-linked immunospot analyses revealed that, although both gamma delta and alpha beta T cells were capable of producing an array of Th1 or Th2 cytokines following stimulation via the T cell receptor-CD3 complex, these mucosal T cells were mainly committed to IL-5 and IL-6 expression in vivo (Th2 type). Both freshly isolated gamma delta and alpha beta T cells expressed mRNA and contained IL-5 and IL-6 spot-forming cells (SFC); however, only the latter exhibited high mRNA levels and SFC for a Th1 cytokine (interferon-gamma). Taken together, the results show that freshly isolated CD3+ T cells from SMG contain activated gamma delta and alpha beta T cells which are programmed to produce IL-5 and IL-6. Thus, SMG, an example of an IgA effector tissue, can be characterized as a Th2-dominant site. However, although both gamma delta and alpha beta T cells express cytokine profiles consistent with a Th2 phenotype, only the latter subset with a CD4+ CD8- phenotype provided effective help for mucosal B cell responses in vitro.
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PMID:Polarized Th2 cytokine expression by both mucosal gamma delta and alpha beta T cells. 758 66

Cytokine profiles of circulating mononuclear cells were studied with the aim of delineating T-cell subsets in leprosy patients with active disease. Using reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokine mRNA and enzyme-linked immunoassay (ELISA) for the secreted products, interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied. Three antigens, native Mycobacterium leprae, a recombinant antigen LSR/A15 of M. leprae and peptide 624 spanning 58-77 amino acids of the latter, were used to induce cytokine expression and release. Half of the subjects, irrespective of the clinical type or antigen used, showed a mixed T-helper type 0 (Th0)-like cytokine pattern, with evidence of the concomitant presence of IFN-gamma and IL-4. The remainder showed a polarized pattern based on the type of leprosy. Lepromatous patients with disseminated disease had Th2-type cytokines, with IL-4 but not IFN-gamma. In contrast, tuberculoid leprosy patients with localized disease showed a Th1-like profile, with the presence of IFN-gamma but not IL-4. Of interest was the stability of the Th phenotype for M. leprae-related antigens. Both the recombinant and the peptide antigens induced the same phenotype as the natural M. leprae bacillus in all except four of 45 leprosy patients.
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PMID:Cytokine profile of circulating T cells of leprosy patients reflects both indiscriminate and polarized T-helper subsets: T-helper phenotype is stable and uninfluenced by related antigens of Mycobacterium leprae. 759 Aug 88

Using a cell sorter, CD16-CD56bright natural killer (NK) cells were sorted from decidual mononuclear cells at an early stage of pregnancy. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) method for their expression of mRNA coding for the following 12 cytokines: IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and leukemia inhibitory factor (LIF). Although mRNA coding for every cytokine was detected in decidual mononuclear cells, mRNAs coding for only G-CSF, GM-CSF, M-CSF, TNF-alpha, IFN-gamma, and LIF were detected in CD16-CD56bright NK cells. Also, the supernatant of CD16-CD56bright NK cell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-alpha, IFN-gamma, and LIF. These findings indicate that CD16-CD56bright NK cells produce many different cytokines and that these cytokines may play an important role in a successful pregnancy.
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PMID:Cytokine production by CD16-CD56bright natural killer cells in the human early pregnancy decidua. 768 93

The interaction between lymphoma cells and vascular endothelial cells (EC) is the first critical step in the invasion of lymphoma cells. We found that invasive human CCRF-CEM T lymphoma cells (CEM) released a factor that upregulates the expression of adhesion molecules on vascular EC. The supernatant of CEM (CEM-SUP) increased the expression of both ICAM-1 and ELAM-1 in time- and dose-dependent manners as shown by cell enzyme-linked immunoabsorbent assay (ELISA). In contrast, the induction of VCAM-1 on EC with CEM-SUP was relatively weak. No activity for interleukin-1 alpha (IL-1 alpha), IL-1 beta, interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha), which are known to augment ICAM-1 expression, was detected in CEM-SUP by ELISA. In reverse transcriptase polymerase chain reaction (RT-PCR) assay, CEM expressed a minimum amount of TNF-alpha mRNA, but absolutely no IL-1 beta or IFN-gamma mRNA. In addition, antibodies for cytokines did not inhibit the upregulatory effect of CEM-SUP. Semipurified CEM-SUP further increased the cellular binding between CEM cells and EC in vitro. This factor was stable to heat (65 degrees C, 30 minutes) and labile to acid (pH 2.0). Gel filtration and chromatofocusing estimated its molecular weight at 50 kd, with an isoelectric point of pH 7.2. Production of this factor might contribute to the invasive character of CEM through upregulation of adhesion molecules on EC.
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PMID:Invasive human T lymphoma cells produce a novel factor that upregulates expression of adhesion molecules on endothelial cells. 769 38

It has been reported that the level of Tap-1 (transporter associated with antigen processing) mRNA and the expression of class I on splenocytes are low in NOD mice. Class I expression at 37 degrees C depends on an adequate supply of peptides, so a decrease in Tap could lead to lower class I levels. Since hypoexpression of class I correlated uniformly with the development of diabetes, it has also been suggested that Tap-1nod is diabetogenic. However, others report normal Tap-1 and class I levels in NOD mice. We examined Tap-1 and Tap-2 mRNA levels in NOD/Smrf mice using a reverse transcriptase-polymerase chain reaction method that detects > or = 25% changes in mRNA. We also assessed class I expression with three monoclonal antibodies. No difference in Tap-1 or Tap-2 mRNA levels for females of different ages or between diabetic and nondiabetic animals was observed. Tap-1 mRNA levels were identical between NOD/Smrf and BALB/cJ mice. Kd expression was significantly lower on NOD lymphocytes than in BALB/cJ cells, but the difference was due to the smaller size of the NOD splenic lymphocyte. When cells of the same size were analyzed, no difference in class I levels was observed. Class I levels were also identical in diabetic and age-matched nondiabetic NOD and BALB/c females. Both NOD Tap-1 mRNA and class I were increased by interferon-gamma. We find no evidence for impaired NOD Tap gene activity or class I expression, as previously reported for this strain.
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PMID:Levels of Tap-1 and Tap-2 mRNA and expression of Kd and Db on splenic lymphocytes are normal in NOD mice. 772 18


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