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)

Articular chondrocytes from nine arthritic patients, five infants, and Balb/c neonatal mice were analyzed for the presence of various cytokine mRNAs by a reverse transcriptase polymerase chain reaction (RT-PCR). Four cytokine mRNAs, interleukin (IL)-6, IL-8, IL-11, and macrophage colony stimulating factor (M-CSF), were detected in all human chondrocytes, regardless of source. IL-10, IL-12p35, and tumor necrosis factor alpha (TNF-alpha) transcripts were found in at least 12 of the 14 human samples. IL-13, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and TNF-beta mRNAs were found more predominantly in infant samples and in samples from patients with rheumatoid arthritis (RA) compared with samples from patients with osteoarthritis (OA). Another group of cytokine mRNAs, IL-1 (alpha, beta), IL-4, IL-5, and IL-7, were only weakly expressed in some human samples. The cytokine transcripts that were not found were IL-2, IL3, and interferon gamma (IFN-gamma). Because of the large array of cytokine transcripts detected, human chondrocyte preparations were further purified by reacting them with a monoclonal antibody specific to chondrocyte differentiation antigen and subjecting them to fluorescent-activated cell sorting. A similar array of cytokines was found between the sorted and unsorted chondrocytes, although TNF-alpha, G-CSF and GM-CSF transcripts appeared to be upregulated during the sorting process. Human chondrocytes that dedifferentiated into fibroblasts (a 40-day and a 77-day culture) no longer expressed mRNAs for IL-1, G-CSF, GM-CSF, and TNF-alpha, but all other cytokine mRNAs remained detectable. Although certain phenotypic characteristics were lost, including reactivity to chondrocyte-specific monoclonal antibodies and morphological features, chondrocytes in long-term culture still expressed cytokine mRNAs. As expected, more consistent results were obtained when seven preparations of chondrocytes from neonatal Balb/c mice were examined using available cytokine primers. They contained IL-1, IL-5, IL-6, IL-7, IL-12, GM-CSF, M-CSF, transforming growth factor beta (TGF-beta), TNF-alpha, and TNF-beta mRNAs but lacked IL-2, IL-3, IL-4, IL-10, and IFN-gamma mRNAs. Future experiments to define conditions by which these cytokine protein products are expressed are needed to help assess their roles in chondrocyte biology and in disease states.
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PMID:Cytokine mRNA repertoire of articular chondrocytes from arthritic patients, infants, and neonatal mice. 885 28

Acute myeloid leukemia (AML) blast cells frequently produce interleukin-6 (IL-6) and other cytokines such as colony-stimulating factors (CSF: G-CSF, M-CSF, and GM-CSF), tumor necrosis factor (TNF)-alpha, and IL-1. The AML blast cells that produced IL-6 alone could not form autonomous in vitro colonies, whereas the blast cells that coexpressed CSF in addition to IL-6 were able to form such colonies. This suggests that IL-6 acts as a costimulator to enhance CSF-induced clonogenicity of AML blast cells. TNF-alpha and IL-1 that are produced from the blast cells may stimulate the growth of the AML blast cells by inducing production of CSF in bone marrow stromal cells or in the blast cell population itself. Improvement of clinical manifestations by the administration of an anti-IL-6 murine monoclonal antibody in a patient with AML-M5B confirmed an important role of IL-6 in in-vivo growth of the blast cells. The mRNA expression of IL-6 and its related genes in AML and acute lymphoid leukemia (ALL) blast cells was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). IL-6 mRNA expression was common in AML, but rare in ALL, whereas the IL-6 receptor (IL-6R) mRNA was expressed in almost all cases of AML and in more than half of the cases of ALL. In contrast, gp130 was ubiquitously expressed in both AML and ALL. A significant correlation between the levels of IL-6R expression and the responsiveness of the blast cells to exogenous IL-6 was observed. This suggests the possibility of the rapid prediction of the responsiveness of leukemic cells to exogenous IL-6 (IL-6 administration for therapy) by rapid measurement of IL-6R mRNA by RT-PCR.
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PMID:The expression of IL-6 and its related genes in acute leukemia. 890 69

/We have studied the effect of interferon alpha (IFN-alpha) on MHC class II expression by human buccal epithelial cells (BEC), and mRNA expression by BEC and mucosal-associated mononuclear cells (MAMC). In 6 experiments, freshly collected BEC were suspended at a concentration of 1.0 x 10(5)/ml in RPMI 1640 and incubated in the presence of 0-10,000 IU/ml of human lymphoblastoid IFN-alpha (HuIFN-alpha). Zero and six hour samples were analyzed by single color flow cytometry using FITC-labeled murine IgG1 monoclonal antibody to HLA-DR. Preparations were also analyzed for expression of cytokine transcripts (IL-2, IL-4, IL-5, IL-6, IL-8, IFN-gamma, GM-CSF) by reverse transcriptase polymerase chain reaction (RT-PCR). Increasing concentrations of IFN-alpha resulted in proportionate increases in the percentage of HLA-DR + BEC (r = 0.7897, p = 0.0627) and in the percentage of HLA-DR+ staining at higher intensities (10(1) to 10(2) log fluorescence intensity) (LFI) (r = 0.4010, p = 0.0424). The percentage of HLA-DR + BEC rose from a mean of 1.5% with no IFN-alpha to 7% with 10,000 IU/ml IFN-alpha (p < 0.05). The percentage of HLA-DR + BEC staining at 10(1) to 10(2) LFI rose from a mean of 8.3% with no added IFN-alpha to 19.2% with 10,000 IU/ml IFN-alpha (p < 0.05). Unstimulated BEC constitutively expressed IL-8 and GM-CSF. IFN-alpha stimulated preparations also expressed IFN-gamma, possibly due to the presence of MAMC, which comprised 2-9% of the total cell population. These data indicated that HuIFN-alpha upregulates MHC class II expression by human BEC, possibly by enhancing IFN-gamma production by MAMC present in the culture preparations.
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PMID:Effect of interferon alpha on HLA-DR expression by human buccal epithelial cells. 891 10

GM-CSF together with IL-1 beta and TNF-alpha has been shown to play a key role in the maturation of LC in vitro. To investigate the presence of GM-CSF, IL-1 beta and TNF-alpha in human skin-derived lymph, we cannulated microsurgically a superficial lymph vessel on the lower leg of six healthy volunteers. Messenger RNA levels were estimated by a reverse transcriptase polymerase chain reaction (RT-PCR) method. From a total of 20 different samples, each consisting of 10(6) lymph cells, total RNA was extracted, reverse transcribed to cDNA and amplified using specific primers for the target gene. Amplified products were sized by electrophoresis and visualized by ethidium bromide. Specific transcripts for GM-CSF were detected in all lymph samples, indicating that circulating human skin-derived lymph cells express GM-CSF mRNA. A mean level of 11.5 +/- 2.1 pg/ml GM-CSF was detected in the lymph samples examined, as determined by a sensitive ELISA. In contrast to GM-CSF, occasional weak mRNA signals together with a mean level of 2.7 +/- 2.2 pg/ml were found for IL-1 beta, and neither specific transcripts nor protein were detected for TNF-alpha. Thus, our results demonstrate that afferent skin lymph cells constitutively express GM-CSF.
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PMID:GM-CSF mRNA and protein in human skin-derived lymph. 893 64

These studies tested the hypothesis that the cerebral vasospasm that follows subarachnoid hemorrhage (SAH) is due to alterations in endothelin (ET) and ET receptor expression. Eight monkeys underwent cerebral angiography and induction of SAH. Angiography was repeated 7 days later to confirm the presence of cerebral vasospasm, and animals were killed. RNA was isolated from right (vasospastic) and left (control) side middle cerebral arteries and surrounding cerebral cortex. The levels of prepro (PP) ET-1 (ppET-1) and ppET-3 and ETA and ETB receptor MRNAs were determined using a quantitative reverse transcriptase polymerase chain reaction-based assay. ET-1 peptide was also measured in CSF at baseline and after 7 days. Specific agonist binding to ETA and ETB receptors in both middle cerebral arteries and in surrounding brain cortex was measured in three animals by autoradiographic binding assays. Levels of ETB receptor mRNA were 3.4 +/- 2.2-fold higher in the right than in the left cerebral arteries (p < 0.01). There were no significant differences in the levels of ppET-1, ppET-3, or ETA receptor mRNA in cerebral arteries. ET-1 peptide was not elevated in CSF. Levels of ETA and ETB receptor mRNAs were 2.6 +/ 1.1- and 2.1 +/ 1.3-fold higher, respectively, in the right than in the left cerebral cortex, while the level of ppET-3 mRNA was 2.1 +/- 1.0-fold lower. There were no differences in ppET-1 mRNA levels between right and left cerebral cortex. Binding to ETA and ETB receptors in cerebral arteries and cortex did not differ significantly between right and left sides. These results do not support the hypothesis that overexpression of ET-1 is principal cause of vasospasm, but rather they suggest that SAH causes complex changes in the ET system that together are responsible for the cellular response to SAH.
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PMID:Increased expression of endothelin B receptor mRNA following subarachnoid hemorrhage in monkeys. 896 9

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important modulator of hematopoietic cells. However, the role of GM-CSF in nonhematopoietic cells remains unclear. We have determined whether GM-CSF is an autocrine mitogenic factor for human osteoblastic (hOB) cells. We found by reverse transcriptase-polymerase chain reaction (RT-PCR) that hOB cells express constitutively both GM-CSF and the alpha and beta chains of the GM-CSF receptor (GMR). Immunocytochemistry showed that serum-starved hOB cells express both GM-CSF and GMR alpha chain. Recombinant human (rh) GM-CSF induces a dose-dependent stimulation of hOB cell proliferation, showing that hOB cells have functional GMRs. A specific neutralizing GM-CSF antibody decreased the basal growth rate and suppressed cell proliferation induced by media conditioned by hOB cells, indicating that GM-CSF released by hOB cells is biologically active. Treatment of hOB cells with GM-CSF antisense (AS) oligonucleotide inhibited the endogenous GM-CSF production as shown by ELISA and immunocytochemistry, whereas a random (R) sequence had no effect. AS oligonucleotides markedly inhibited hOB cell growth reversibly, whereas R oligonucleotides had no effect. AS was more effective than the anti-GM-CSF antibody, and the addition of rhGM-CSF did not rescue the inhibitory effect of AS on cell growth. The findings that human osteoblastic cells produce GM-CSF and express functional GMR constitutively and that suppression of endogenous GM-CSF results in inhibition of cell growth demonstrate that GM-CSF is involved as an autocrine growth factor for human osteoblastic cells.
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PMID:Endogenous GM-CSF is involved as an autocrine growth factor for human osteoblastic cells. 901 83

The macrophage scavenger receptor (SR) plays a leading role in atherogenesis, but little is known about the relevance of SR to atherosclerosis in uremia. In this study, the impact of uremic serum on SR expression and activity was examined in the human monocytic cell line U937. The cells were cultured with serum from ten healthy subjects, ten hemodialysis (HD) and ten continuous ambulatory peritoneal dialysis (CAPD) patients. SR mRNA expression was examined using reverse transcriptase-polymerase chain reaction followed by Southern blot. SR protein amount was evaluated by ligand blot. SR activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using flow cytometry. Uremic serum dose-dependently enhanced SR activity primarily by increasing the amount of receptor protein. Heat-inactivated uremic serum had a stimulatory effect, but ultrafiltrate of uremic serum, which included molecules with a molecular weight less than ten kDa, had no effect. The serum levels of macrophage-colony stimulating factor (M-CSF), an activator of SR, were fourfold higher in uremia and significantly correlated with SR activity in cells treated with uremic serum. Pre-treatment of uremic serum with a neutralizing antibody to M-CSF abolished the effect of uremic serum on SR activity. In conclusion, uremic serum contains a factor(s) that enhances SR expression and activity in U937 cells. Elevated M-CSF in uremic serum could be responsible for this enhancement.
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PMID:Uremic serum enhances scavenger receptor expression and activity in the human monocytic cell line U937. 906 11

Recently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.
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PMID:Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia. 932 53

Three clonally related lymphoma lines (Mac-1, Mac-2A and Mac-2B) derived from progressive stages of CD30+ cutaneous T-cell lymphoma were found to constitutively secrete GM-CSF. The secretion of GM-CSF was identified by the ability of cell line supernatants to stimulate growth of megakaryoblastic cell line M-07e. This supernatant-mediated stimulation was inhibited by anti-GM-CSF MoAb (>98% inhibition for Mac-1 and Mac-2B lines, and >95% for Mac-2A line). Synthesis of GM-CSF was confirmed, at the mRNA level, by reverse transcriptase PCR and, at the protein level, by ELISA. Quantification of GM-CSF in supernatants by ELISA showed that the Mac-1 line, derived from an early, clinically indolent stage of the lymphoma, produced much more GM-CSF (>1600 pg/ml) than Mac-2A and Mac-2B lines which were derived from a late, aggressive stage (30-50 and 50-120 pg/ml, respectively). Lack of inhibition of cell growth by anti-GM-CSF MoAb as well as lack of response to exogenous GM-CSF of cells cultured at low concentration have demonstrated that GM-CSF does not act directly as a growth factor for these lines. ELISA studies showed that GM-CSF concentration in serum and urine of the patient were not elevated (<5 pg/ml). From several other cell lines tested (two primary CD30+ ALCL, 2 CD30- non-lymphoblastic T-cell lymphomas and 4HD), only two HD lines with a T-lymphocyte phenotype secreted detectable amounts of GM-CSF. Our data show that cells lines from a patient with cutaneous T-cell lymphoma constitutively secrete GM-CSF, although this capacity is relatively diminished in lines developed from more advanced disease.
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PMID:Constitutive secretion of GM-CSF by three different cell lines derived from a single patient with a progressive cutaneous lymphoproliferative disorder. 916 23

Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.
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PMID:Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation. 919 Sep 16


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