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)

Recent reports point to a role for interleukin-12 (IL-12) in regulating T- and NK-cell function, macrophage activation and initiation of Th1-type cell responses. We sought to determine whether CD1a+ dendritic cells of the skin, as major antigen-presenting cells, are a source of IL-12 and therefore important in the initiation of Th1-type cell responses. To investigate this hypothesis, we cannulated microsurgically a skin-draining lymph vessel in the lower legs of five healthy volunteers. Altogether, ten different samples, each consisting of 1 x 10(6) lymph cells, were investigated. In four of the ten samples. CD1a+ dendritic lymph cells were isolated and purified by positive selection using mouse anti-CD1a monoclonal antibodies and sheep anti-mouse antibody-coated Dynabeads. Messenger RNA levels were estimated using a nested reverse transcriptase polymerase chain reaction (nRT-PCR) method. Total RNA was extracted from the cells, reverse transcribed to cDNA and amplified using specific primers for the target gene. Amplified products were sized by electrophoresis and visualized by ethidium bromide. Expression of IL-12 p40 and p35 mRNA was detected in all samples, both whole lymph samples and the highly enriched CD1a+ dendritic cell population. Our findings demonstrate that human skin-derived CD1a+ dendritic lymph cells produce IL-12 mRNA and may therefore be an important source of IL-12. Thus one might speculate that these CD1a+ dendritic cells, through their IL-12-producing capacity, might significantly influence the balance of Th1 versus Th2 reactions ultimately occurring.
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PMID:IL-12 gene expression in human skin-derived CD1a+ dendritic lymph cells. 893 85

Interferon-gamma (IFN-gamma) is implicated as a mediator of islet beta-cell destruction in autoimmune, insulin-dependent diabetes mellitus (IDDM). Because interleukin 12 (IL-12) is a potent inducer of IFN-gamma production, we sought evidence implicating IL-12 in IDDM development. In the present study, we used a reverse transcriptase polymerase chain reaction (RT-PCR) assay to measure IL-12 mRNA expression levels in islets from nonobese diabetic (NOD) mice. Expression of mRNA encoding the p40 chain of IL-12 (IL-12 p40) in mono-nuclear leukocytes isolated from islets of female NOD mice increased progressively from age 5 weeks to diabetes onset (> 13 weeks). By contrast, IL-12 p40 mRNA levels were significantly decreased in islet mononuclear leukocytes, but not spleens, from female NOD mice protected from diabetes by administration of complete Freund's adjuvant (CFA) in early life. In addition, mRNA levels of IL-12 p40, IFN-gamma and IL-2 were significantly decreased in syngeneic islet grafts, but not spleens, from female NOD mice protected from diabetes recurrence by CFA administration at the time of islet transplantation. These findings show that IL-12 gene expression in the insulitis lesion correlates with both primary and recurrent diabetes development in NOD mice, possibly via induction of T helper (Th) 1-type cytokines, IL-2 and IFN-gamma.
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PMID:Interleukin 12 mRNA expression in islets correlates with beta-cell destruction in NOD mice. 893 80

Calcitonin gene-related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP-containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme-linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)-10 and IL-1 beta protein in the LC-like cell line XS52 as well as the reverse transcriptase-polymerase chain reaction (RT-PCR) to examine levels of mRNA for IL-10, IL-1 beta, and the 40-kDa subunit (p40) of IL-12. CGRP augmented the lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) -induced release of IL-10 protein and the induced expression of IL-10 mRNA in these cells. However, it suppressed the induction of release of IL-1 beta protein and the induction of mRNA for IL-12 p40 and IL-1 beta by LPS and GM-CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS-induced expression of IL-10 was augmented by CGRP, whereas the induction of IL-1 beta was suppressed. Northern analysis demonstrated augmentation of LPS-induced IL-10 mRNA levels and inhibition of LPS-induced IL-1 beta mRNA by CGRP. CGRP inhibited the LPS-induced induction of IL-12 mRNA as assessed by RT-PCR. Up-regulation of B7-2 expression by LPS and GM-CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co-culture with neutralizing antibodies to IL-10. Furthermore, the presence of neutralizing antibodies to IL-10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP-mediated suppression of EC presentation of tumor-associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed-type hypersensitivity in S1509a-immune mice. These data suggest that suppression of antigen-presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell-mediated immunity.
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PMID:Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide. 902 28

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. TDM primes murine macrophages (Mphi) to produce nitric oxide (NO) and to develop antitumoral activity upon activation with low doses of lipopolysaccharide (LPS). In this study, we investigated the ability of TDM to induce interleukin 12 (IL-12) and the role of this cytokine in TDM-induced activation of murine Mphi. RNA isolated from peritoneal exudate cells (PEC) collected at different times after TDM injection was used to determine IL-12 (p35 and p40 subunits) and gamma interferon (IFN-gamma) mRNA levels by semiquantitative reverse transcriptase-PCR. Constitutive expression of IL-12p35 was observed in PEC from untreated as well as from TDM-injected mice. In contrast, expression of the IL-12p40 subunit was almost undetectable in control PEC but was dramatically upregulated in PEC from TDM-injected mice. IL-12p40 expression peaked at 8 h and subsided to baseline levels at 39 h postinjection. TDM was also able to induce IFN-gamma expression; however, kinetics of induction of IFN-gamma was different from that of IL-12p40. Maximal levels of IFN-gamma mRNA were reached by 24 h and did not return to baseline by 4 days. In addition, pretreatment of mice with neutralizing monoclonal antibodies directed against IL-12 (C15.6.7 and C15.1.2) blocked IFN-gamma mRNA induction in PEC from TDM-treated mice. We further determined if the induction of IL-12 and/or IFN-gamma contributes to the in vivo priming effect of TDM on peritoneal Mphi. TDM-injected mice were treated in vivo with anti-IL-12 or anti-IFN-gamma (XMG.1.6) monoclonal antibodies. TDM-primed Mphi were then activated in vitro with LPS and tested for their ability to produce NO and to develop cytostatic activity toward cocultivated L1210 tumor cells. Priming of Mphi by TDM was completely blocked by in vivo neutralization of either IL-12 or IFN-gamma as demonstrated by an absence of tumoricidal activity and NO production by TDM-elicited Mphi in the presence of LPS. Taken together our results show that TDM, a defined molecule from M. tuberculosis, induces in vivo production of IL-12. Moreover, synthesis of IL-12 mediates TDM priming of mouse peritoneal Mphi through IFN-gamma induction.
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PMID:Interleukin-12 synthesis is a required step in trehalose dimycolate-induced activation of mouse peritoneal macrophages. 911 75

It has been reported that the mRNA of the type 1 cytokine, interferon-gamma (IFN-gamma)--but not the type 2 cytokine interleukin-4 (IL-4)--is detected in synovial tissues of rheumatoid arthritis (RA) patients, whereas both IFN-gamma and IL-4 mRNA are detected in reactive arthritis (ReA). To evaluate such data more extensively, we obtained 208 synovial specimens in a prospective study of 52 early synovitis patients (13 RA, 11 ReA, 28 undifferentiated oligoarthropathy) and analyzed type 1 and type 2 cytokine mRNA expression in specimens containing sufficient mRNA. Using a nested reverse transcriptase polymerase chain reaction technique, we measured the relative mRNA levels of 10 cytokines and CD3 delta chain. We detected IL-10, IL-15, and CD3 delta chain mRNA in all RA and ReA patients and frequently detected tumor necrosis factor-alpha, IL-1 beta, and IFN-gamma mRNA. IL-6 and IL-12 p40 mRNA were detected in approximately one-half of the patients. We also detected greater amounts of IL-2 and IFN-gamma mRNA in ReA than were detected in RA. However, we rarely detected IL-4 or IL-13 mRNA. Similar cytokine profiles were observed in undifferentiated oligoarthropathy. The amounts of cytokine mRNAs, except for IL-10, in specimens from the patients taking prednisone or second-line antirheumatic drugs tended to be less than in specimens from the patients taking neither prednisone nor second-line antirheumatic drugs. These results suggest that cytokine mRNA profiles in patients with RA, ReA, and undifferentiated arthritis in their early stages are skewed toward proinflammatory macrophage-derived and type 1 cytokines. IL-10--not IL-4 or IL-13--mRNA appears to be the major antiinflammatory cytokine mRNA. Drug therapy is associated with depressed proinflammatory and type 1 cytokine mRNA production. The differences in the expression of IL-2 and IFN-gamma mRNA between RA and ReA may reflect unique etiological or host factors associated with the early stages of these diseases.
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PMID:In vivo gene expression of type 1 and type 2 cytokines in synovial tissues from patients in early stages of rheumatoid, reactive, and undifferentiated arthritis. 915 45

The cytokine mRNAs expressed in the foot pads and spleens of BALB/cAJcl mice infected with Mycobacterium leprae were studied by the reverse transcriptase-polymerase chain reaction (RT-PCR) method using cytokine-specific primers for interleukin-1 alpha (IL-1 alpha), -2, -4, -6, -10, -12-(p40), gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and TNF-beta, and then for CD4 and CD8 markers. The pattern of cytokine gene expression in the foot pad which supports M. leprae growth was different from the expression in the spleen which does not permit M. leprae multiplication in mice. Before BALB/cAjcl mice were infected with M. leprae, IL-1 alpha and TNF-beta mRNAs were expressed physiologically in the foot pad while all of the cytokine genes examined were expressed in the spleen. In the foot pads of mice inoculated with M. leprae, in addition to the physiological appearance of IL-1 alpha and TNF-beta mRNAs, these signals were intensified. TNF-alpha expression was induced by the infection. On the other hand, in the spleens of mice inoculated with M. leprae, CD4 mRNA expression disappeared on day 1 of the infection, which was accompanied by the reduced expression of IL-2, -4, -6, and -12 mRNAs. The recovery of CD4 mRNA expression at a latter stage was accompanied by a corresponding increase of the cytokine mRNA expression. It was suspected that these results might permit restricted growth of M. leprae in the foot pads of normal mice. Furthermore, our study suggests that tissue-specific, local, immunologic characteristics are important in M. leprae growth.
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PMID:Cytokine gene expression in the foot pad and spleen of BALB/cAJcl mice infected with M. leprae. 920 57

Interleukin-12 (IL-12) is a heterodimeric cytokine implicated in the early differentiation of naive T-lymphocytes into the Th1 subset. IL-12 is important for induction of the cellular immune response against viruses, intracellular parasites and neoplasms. Its role in alloresponsiveness has not been fully elucidated. Preliminary data in the literature point toward the prevalence of Th1 lymphocytes in processes of allograft rejection. In attempt to further investigate the expression of this cytokine during episodes of cellular rejection of renal allografts, we searched for IL-12 message in human kidney allograft biopsies using the reverse transcriptase-polymerase chain reaction technique. Twenty-three allograft core biopsies from 19 patients were obtained percutaneously for clinical indications in 18 cases, and as part of an investigational protocol in five cases. A portion of the tissue was used for RNA extraction using the guanidium-thiocyanide phenol-chloroform method. Histology was performed on the remaining core material. Ten mg of total RNA were used for reverse transcription. PCR of the c-DNAs was done for 40 cycles using primers for the p40 subunit of IL-12 and GAPDH which was used as a control. PCR products were photographed after electrophoresis, transferred to a nylon membrane and hybridized with a radiolabelled cloned human IL-12 p40 1 kb c-DNA fragment. Autoradiographies were developed after 20-min exposure. All samples were run in triplicate. IL-12 p40 m-RNA was expressed in all 17 biopsies showing acute cellular rejection as well as in all three biopsies showing focal interstitial fibrosis. No message was found in the presence of normal allograft histology. This is the first in vivo report of IL-12 p40 subunit m-RNA expression during renal allograft rejection in humans. The role of this Th1 cytokine in the alloresponse deserves further investigation.
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PMID:Interleukin-12 p40 m-RNA expression in human kidney allograft biopsies. 940 86

Cytokines are signalling glycoproteins mediating acute inflammation, chronic inflammation, and connective tissue destruction. The present study was designed to characterize the profile of cytokine message in normal human articular cartilage and from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), by means of the reverse transcriptase-polymerase chain reaction (RT-PCR). Message RNA (mRNA) was extracted from fresh or frozen cartilage. The results showed expression of mRNA for IL-6, IL-6R, IL-7, IL-8, IL-10, and IL-12 (p35 and p40) exclusively in the RA cartilage. Except for mRNA for IL-8 and IL-10, no other cytokine or cytokine receptor was expressed in OA and control cartilage. mRNA for IL-1beta, IL-4, TNF-alpha, and TNFR-p75, was not detected in any cartilage sample except for one RA specimen expressing IL-1beta mRNA. However, the expression of message for pro-inflammatory cytokines was far more prominent than anti-inflammatory cytokines. This may suggest a disturbed balance of pro- and anti-inflammatory activity in RA cartilage.
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PMID:Detection of cytokine mRNA in human, articular cartilage from patients with rheumatoid arthritis and osteoarthritis by reverse transcriptase-polymerase chain reaction. 950 80

Two adeno-associated virus (AAV)-derived plasmids were constructed with portions of the N-methyl-d-aspartic acid-R1 (NMDA-R1) receptor subunit downstream from the AAV p40-(pJDT95dlk-aR1) or cytomegalovirus (CMV) promoter (pTRUF3-aR1) in an antisense orientation. Each plasmid drove expression of antisense NMDA-R1 in primary rat neocortical neuronal cultures 4 days after transfection as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Transfection with pTRUF3-aR1 (2x4 microgram) but not with pJDT95dlk-aR1 decreased neuronal [3H]MK-801 binding in a dose-dependent manner.
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PMID:Reduced MK801 binding in neocortical neurons after AAV-mediated transfections with NMDA-R1 antisense cDNA. 951 73

Recent reports suggest that production of interleukin-12 (IL-12) by dendritic cells and keratinocytes may play an important part in contact hypersensitivity reactions. In the present study we investigated mRNA and protein expression of IL-12 in human skin lymph derived from normal untreated skin (n = 5) and from the induction phase of allergic contact dermatitis (CD) (n = 5). mRNA levels were determined at various time points in the lymph cells by a nested reverse transcriptase-polymerase chain reaction method. Time course analysis reproducibly revealed a constitutive expression of both IL-12 p40 and p35 mRNA in the migrating lymph cells in all volunteers. However, no enhancement of the IL-12 mRNA signal was found during the induction phase of allergic CD. Furthermore, as determined by a sensitive ELISA technique, IL-12 protein was not detectable in 60 lymph samples derived from normal untreated skin or in 68 lymph samples obtained during the induction phase of allergic CD at any time point of the lymph cannulation. In conclusion, our findings indicate that no significant protein levels of IL-12 are washed out from the skin into the afferent lymph or are produced and released by migrating lymph cells during the induction phase of allergic CD in vivo.
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PMID:Interleukin-12 expression in human afferent lymph derived from the induction phase of allergic contact dermatitis. 960 78


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