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

---

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

As a preliminary to transducing human melanoma cells with lymphokine genes, we sought for constitutive gene expression and production of eight interleukins, tumour necrosis factors and granulocyte-colony stimulating factor in 19 human melanoma cell lines. Conversion of RNA into cDNA by reverse transcriptase and polymerase chain reaction (RT-PCR) were employed to evaluate gene expression while enzyme-linked immunosorbent assays (ELISA) or biological assays were used to assess the presence of proteins. No expression of interleukins (IL) 3, 4, and 5 or interferon-gamma RNA was found, while the other cytokines were variably expressed in melanoma lines, with IL-1 alpha, IL-1 beta, IL-6, IL-8, being detectable in most of the lines. At protein level, 10 melanoma cells were tested with ELISA and all were found to produce IL-8, five produced IL-6, two tumour necrosis factor (TNF)-alpha, one IL-1 alpha and two TNF beta. The levels of TNF beta were at the limit of test sensitivity. The amount of various cytokines released by the different lines varied widely. Biological assay with the D10-G4 clone confirmed the presence of IL-1 alpha in the supernatant of melanoma (ME) 10221 and revealed an IL-1 activity in the supernatant of Me 4024/1. The proliferating activity of melanoma supernatants on D10-G4 was inhibited by treatment with polyclonal antibodies against IL-1 alpha but not with antibodies against IL-1 beta. TNF biological activity was tested against the TNF-susceptible fibrosarcoma WEHI 164 clone 13.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Expression of cytokine genes, including IL-6, in human malignant melanoma cell lines. 145 Jun 72

Interferon gamma (IFN-gamma), a lymphokine that exerts multiple immunoregulatory effects, has been found to be elevated in the plasma, cerebrospinal fluid, and lymph nodes of human immunodeficiency virus (HIV)-infected individuals and has shown variable effects on HIV replication in acutely infected cells. In the present study, we have demonstrated that IFN-gamma is a potent modulator of HIV expression in persistently infected U1 promonocytic cells in which virus production is characterized by a constitutive state of relative latency. Direct stimulation of U1 cells with IFN-gamma (10-1,000 U/ml) activated HIV expression, as measured by reverse transcriptase (RT) activity in the culture supernatant and increased levels of cell-associated viral protein and mRNAs. These effects on virus expression were not accounted for by the induction of endogenous TNF-alpha secretion, as previously described in U1 cells stimulated with phorbol myristate acetate (PMA). At the ultrastructural level, the stimulatory activity of IFN-gamma was correlated with HIV particle production in intracytoplasmic vacuoles along with the differentiation of U1 into macrophage-like cells. Furthermore, costimulation of U1 cells with IFN-gamma and PMA significantly increased the accumulation of vacuole-associated HIV concomitant with decreasing membrane-associated particles and RT activity production, as compared with cells stimulated with PMA alone. No evidence of spontaneous secretion of intracellular vacuole-associated virus was obtained by kinetic analysis of the RT activity released in the supernatants throughout the culture period unless cells were deliberately disrupted. These findings suggest that vacuole-associated virions likely represent a relatively stable intracellular reservoir of HIV, as previously described in primary macrophages infected in vitro or in infected macrophages in the brains of patients with acquired immune deficiency syndrome. The reduced levels of RT activity observed in the culture supernatants of U1 cells stimulated with PMA in the presence of IFN-gamma were not indicative of a suppressive effect of IFN-gamma on PMA-induced expression of HIV proteins and mRNAs, either directly or mediated by the release of IFN-alpha/beta. This study suggests that IFN-gamma may play an important role as an inducer of HIV expression in infected mononuclear phagocytes.
...
PMID:Interferon gamma induces the expression of human immunodeficiency virus in persistently infected promonocytic cells (U1) and redirects the production of virions to intracytoplasmic vacuoles in phorbol myristate acetate-differentiated U1 cells. 151 39

Crohn's disease (CD) is characterized by granulomatous inflammation of the intestinal mucosa, but the etiology and pathogenesis of the inflammatory lesions are unknown. The aim of this study was to determine whether T-cell activation and lymphokine production occurs in the mucosal lesions of this disease. Total cellular RNA was isolated from peripheral blood lymphocytes and from colonoscopic mucosal biopsies of normal individuals and patients with CD of the colon or ulcerative colitis (UC). Levels of interleukin-2 (IL-2) messenger RNA (mRNA) transcripts in samples were determined using a quantitative reverse transcriptase polymerase chain reaction method. IL-2 mRNA transcripts were detected in histologically normal intestinal mucosal biopsies obtained from control subjects. In CD, higher levels of IL-2 mRNA transcripts were detected in the mucosa from areas of active inflammation, but in areas that were histologically normal, levels were similar to control subjects. The levels of IL-2 mRNA transcripts in biopsies from active and inactive UC were similar to control subjects. Levels of IL-2 mRNA in peripheral blood lymphocytes were low and not significantly different in all groups of subjects. In conclusion, the normal intestinal mucosa contains IL-2 mRNA transcripts and may be an important source of IL-2. Furthermore, the inflammatory lesions of CD, but not UC, have higher levels of IL-2 mRNA transcripts, suggesting that T-cell activation and lymphokine secretion in the intestine may be important in the pathogenesis of CD. These data provide further evidence that the pathogenesis of CD and UC are different.
...
PMID:Increased interleukin-2 messenger RNA in the intestinal mucosal lesions of Crohn's disease but not ulcerative colitis. 156 72

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.
...
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

Interleukin 5 (IL-5) is a T-cell lymphokine known to stimulate development, functional activity, and in vitro survival of eosinophils. Tissue and blood eosinophilia occurring during allergic responses of the immune system are potentially mediated by IL-5 secreting T-cells. To test this hypothesis a series of allergen-specific T-cell clones were established from peripheral blood and skin lymphocytes of patients with atopic dermatitis and house dust mite sensitization. In addition, alloreactive T-cell clones were also prepared from peripheral blood lymphocytes of healthy donors. Cloned T-cells were analyzed for IL-5 mRNA expression and IL-5 secretion by means of in vitro gene amplification using the reverse transcriptase polymerase chain reaction and IL-5 specific oligonucleotide hybridization, as well as IL-5-specific ELISA. A majority of allergen-specific long-term cultured T-cell clones (84%) of different donors and of either phenotype (CD8+ or CD4+) disclosed IL-5 transcripts on stimulation with lectins. Almost all clones exhibiting IL-5 transcripts also released immunoreactive IL-5 protein into their culture supernatants. In contrast, only 2% of alloreactive T-cell clones obtained from healthy donors and none of alloreactive T-cell clones of one atopic patient investigated expressed detectable amounts of IL-5 mRNA in response to lectin stimulation, all of whom were CD4+. These results suggest that eosinophilia observed in allergic responses in the peripheral blood and in tissues at the site of induced late-phase cutaneous reaction may be associated with IL-5 release by allergen-specific T-cells.
...
PMID:Interleukin 5 expressing allergen-specific T-lymphocytes in patients with house dust mite sensitization: analysis at a clonal level. 941 Apr 77

Little is known about the activation level of tumor-infiltrating lymphocytes (TIL) in human lung adenocarcinoma. We investigated the activation of fresh TIL at cellular and molecular levels and compared it with autologous and healthy normal peripheral blood lymphocytes (PBL) for baseline level. TIL were extracted from 12 primary lung adenocarcinomas by mechanical disruption without enzyme use and isolated by double-density Ficoll gradients. Flow-cytometry analysis of TIL subset distribution revealed that the majority was composed of T lymphocytes, and double labeling with alpha-CD3 and adhesion/activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, each of which was almost absent in autologous T peripheral blood lymphocytes (T-PBL). Moreover, the proportions of T-TIL expressing CD58, CD65, or CD25 were increased severalfold compared to T-PBL. Lymphokine gene activation in TIL was assessed by mRNA reverse transcriptase/polymerase chain reaction (RT-PCR) and primers for interleukin(IL)-2, IL-4, interferon (IFN) gamma, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF) beta. Semiquantitative comparisons between patients' TIL and PBL and healthy normal and activated PBL were performed by computerized image analysis. RT-PCR gel band products were quantified in relative units as a function of their size and intensity. TIL expressed detectable lymphokine mRNA but seemed poorly activated with respect to the total number of lymphokine genes and the amount of mRNA compared with alpha-CD3-activated healthy PBL. IL-2, IFN gamma, and TNF beta did not appear to be expressed at higher levels in TIL than in autologous or healthy normal PBL. However, two-thirds of the patients had TIL distinguishable from autologous PBL by specific expression of GM-CSF and from healthy normal PBL by expression of IL-4. These results show that lung adenocarcinoma TIL populations had little lymphokine gene activation despite the presence of several T cell subsets expressing different adhesion/activation markers. The lack or deficient combination of lymphokine production may be a factor that prevented efficient activation of TIL in these tumors.
...
PMID:High expression of adhesion molecules/activation markers with little interleukin-2, interferon gamma, and tumor necrosis factor beta gene activation in fresh tumor-infiltrating lymphocytes from lung adenocarcinoma. 764 Dec 14

Seven days after activation with concanavalin A and irradiated spleen cells, murine CD4+ T cells were re-stimulated with ionomycin and phorbol 12-myristate 13-acetate (PMA). IL-2 and IL-4 were determined in the supernatant. When cholera toxin, forskolin together with phosphodiesterase inhibitors or dibutyryl-cAMP were added at the time of re-stimulation, a dose-dependent increase of IL-4 and IL-5 release was noted. IL-2 was down-regulated as reported before. The up-regulation of IL-4 and the down-regulation of IL-2 correlated with an increase of IL-4 mRNA and a decrease of IL-2 mRNA as determined by semi-quantitative reverse transcriptase polymerase chain reaction. Similar results were found with prostaglandin E2 using PMA and ionomycin or plate-bound anti-CD3 antibody as re-stimulants. These results suggest that, in activated CD4+ T cells, cAMP-elevating agents induce a switch of lymphokine production towards a Th2-like phenotype through regulation at the transcriptional level. This is supported by the fact that complex formation between a synthetic nuclear factor of activated T cells (NF-AT) binding site from the IL-2 promoter and nuclear extracts was decreased when cholera toxin was added to re-activated CD4+ T cells, suggesting that cholera toxin and cAMP down-regulate IL-2 expression via decreased NF-AT binding. Finally, since IL-4 has been reported to amplify IL-4 release from activated CD4+ T cells, the autoinduction of IL-4 may very well function via cAMP.
...
PMID:cAMP up-regulates IL-4 and IL-5 production from activated CD4+ T cells while decreasing IL-2 release and NF-AT induction. 781 41

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln-->Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.
...
PMID:Expression of interleukin 1 alpha, interleukin 6, and tumor necrosis factor alpha genes in human melanoma clones is associated with that of mutated N-RAS oncogene. 806 79

The mechanisms by which cellular immunity maintains the asymptomatic state after human immunodeficiency virus type 1 (HIV-1) infection are poorly understood. CD4+ T lymphocytes play a complex role in regulating anti-HIV effector pathways, including activation of macrophages, which are themselves implicated in clinical latency and pathogenesis of symptomatic acquired immune deficiency syndrome. We have found that a newly identified T helper type 2 lymphokine, interleukin 13 (IL-13), inhibits HIV-1ADA and Ba-L replication in primary tissue culture-derived macrophages but not in peripheral blood lymphocytes. Viral production in cells was measured by viral protein (p24) and reverse transcriptase levels, while entry was assessed by proviral DNA analysis at timed intervals after infection. Inhibition by IL-13 was dose and time dependent and not mediated through altered viral entry, reverse transcription, or viral release. IL-13 is therefore a candidate cytokine for the suppression of HIV infection within monocytes and macrophages in vivo.
...
PMID:Interleukin 13 inhibits human immunodeficiency virus type 1 production in primary blood-derived human macrophages in vitro. 810 65

In order to better understand the immunoregulation following Mycobacterium tuberculosis infection, cytokine mRNA induction in response to in vitro infection of human monocytes with live virulent M. tuberculosis H37Rv cocultured with autologous lymphocytes was quantitated by reverse transcriptase-PCR. Induced levels of interleukin 1 beta (IL-1 beta), IL-2, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were compared among groups of individuals representing three phases of immunity to infection with M. tuberculosis: naive normal control subjects, purified protein derivative (PPD)-reactive normal donors, and individuals with active tuberculosis (TB [diseased]). Levels of IL-1 beta and tumor necrosis factor alpha mRNA in cocultured cells from TB patients were 51 and 45%, respectively, of those obtained in cells from sensitized healthy volunteers and were comparable to those from naive normal donors. Lymphoproliferative responses to M. tuberculosis and induction of the T-cell cytokine IL-2 were predictably high in the cells of PPD-sensitized donors, low in normal naive individuals, and variable among TB patients. In contrast, the induced level of another lymphokine, IFN-gamma, did not follow the pattern seen in IL-2 induction. Infection with live M. tuberculosis induced high levels of IFN-gamma mRNA in lymphocytes of both PPD-sensitized and normal naive donors compared with those of TB patients. Interestingly, polyclonal stimulation with the mitogen concanavalin A induced similar IFN-gamma levels in cells from all three donor groups. The high level of IFN-gamma induced by the infection of monocytes from naive normal donors suggests a role for natural killer (NK) cells in the production of IFN-gamma in this coculture system. This response appears independent of the role performed by T cells.
...
PMID:Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes. 813 51


1 2 3 Next >>

---