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)

Direct recognition of viral gene sequences can be used to detect human immunodeficiency virus (HIV-1) in clinical specimens. A modification of the polymerase chain reaction (PCR) for amplification of gene sequences was used for detection of HIV-1-specific RNA prepared from peripheral blood mononuclear cells (PBMC). The RNA served as a template for reverse transcriptase using primers derived from both the 3'ORF and the LTR regions of HIV-1, as well as from the control cellular sequences encoding beta-actin and T cell receptor. The resultant DNA was amplified with DNA polymerase. A transcriptional step using the bacteriophage T7 promoter recognition sequences, incorporated into the primers, was used to enhance the efficiency of the amplification process. This assay detects as few as 100 RNA copies of cloned HIV-1 genome. Starting with 1 microgram RNA isolated from PBMC, we were able to detect HIV-1 sequences in patients with symptomatic and asymptomatic HIV-1 infection. The inclusion of T cell-specific primers permitted simultaneous evaluation of an immunologic parameter. The PCR can be applied to RNA samples for detection of viral and cellular sequences and is a rapid and efficient means for detection of HIV-1 sequences as well as potentially informative cellular sequences.
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PMID:Confirmation of HIV infection using gene amplification. 252 May 45

Human retroviruses have recently been linked with T cell lymphoproliferative disorders and with the acquired immune deficiency syndrome. We investigated the mechanisms for acquired pure red cell aplasia and cutaneous anergy in a patient with the chronic T gamma-lymphoproliferative disease (T gamma-LPD) syndrome. Patient marrow erythroid progenitors (BFU-E) were 17 +/- 9% of control and were selectively increased to 88-102% of control after marrow T cell depletion. Patient Leu 2+ suppressor T cells spontaneously produced high titers of human gamma-interferon and resulted in a concentration-dependent selective inhibition (74-91%) of BFU-E when co-cultured with autologous or allogeneic marrow. Conditioned media (CM) derived from patient Leu 2+ T cells similarly inhibited growth of autologous or allogeneic marrow BFU-E. The inhibitory factor derived from patient CM was acid-labile (pH 2) and sensitive to trypsin; prior treatment of patient T cells with anti-HLA-DR monoclonal antibody plus complement abrogated the suppressive effect of T cell-derived CM. Patient peripheral blood mononuclear cells (PBMC) were unable to support growth of cultured interleukin 2 (IL 2)-dependent T cells, but responded to exogenous IL 2 in vitro with a 16-21-fold augmentation, relative to control, in mitogen-induced proliferation. Antibodies to HTLV-I core proteins p19 and p24 but not to HTLV-III proteins were detected in patient serum by Western blotting; patient cultured PBMC stained (7-11%) with antibodies to p19 and p24. Patient cultured PBMC demonstrated integrated HTLV-I genomic sequences by the Southern technique and expressed both specific HTLV-I genomic sequences by RNA dot blot plus reverse transcriptase activity. Utilizing a cloned DNA probe for the beta chain of the T cell receptor gene, patient PMBC demonstrated gene rearrangements providing presumptive evidence for clonality. The presence in serum of HTLV-I p19 and p24 antibodies, the expression of p19 and p24 core antigens on patient mononuclear cells, the evidence of HTLV-I proviral integration sequences and the expression of HTLV-I genomic sequences in patient cells, indicates infection with HTLV-I and raises the possibility of an etiologic link between human retrovirus infection and some instances of large granular lymphocytic leukemia (T gamma-LPD).
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PMID:Human T cell leukemia virus-I-associated T-suppressor cell inhibition of erythropoiesis in a patient with pure red cell aplasia and chronic T gamma-lymphoproliferative disease. 289 60

Experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), is a paralytic disease of the central nervous system (CNS) mediated by T-lymphocytes reactive to myelin basic protein (MBP). Lewis rats actively immunized with fragment 68 to 82 of guinea pig MBP develop a monophasic disease with spontaneous recovery. Lymphocyte recognition of the primary encephalitogenic sequence of MBP (fragment 68 to 82) is V beta 8.2 T cell receptor (TCR) skewed [1-3]. Lewis rats in clinical remission at 1 month and 3 months after spontaneous resolution of EAE retain V beta 8.2 T-lymphocytes in the CNS when analyzed by reverse transcriptase polymerase chain reaction or in situ hybridization. In contrast, 1 and 3 months after clinical remission from syngeneic bone marrow transplantation, V beta 8.2 T lymphocytes are absent from the CNS. During clinically active EAE and inflammatory breakdown of the blood-brain barrier, immune ablation and reconstitution with syngeneic bone marrow results in clinical tolerance of the new immune system to myelin.
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PMID:Syngeneic bone marrow transplantation eliminates V beta 8.2 T lymphocytes from the spinal cord of Lewis rats with experimental allergic encephalomyelitis. 747 84

Human immunodeficiency virus (HIV) infection leads to a progressive loss of CD4+ T helper (Th) type 1 cell-mediated immunity that is associated with defective in vitro CD4+ T cell proliferation and abnormal T cell death by apoptosis in response to T cell receptor (TCR) stimulation. Quantification of interleukin (IL)-2, interferon gamma, IL-4, IL-5, and IL-10 secretion by immunoassays, and of interferon gamma, IL-4 and IL-10 messenger RNA expression by competitive reverse transcriptase polymerase chain reaction after in vitro stimulation of the TCR revealed a similar Th1 cytokine profile in T cells from HIV-infected persons and from controls. These data indicated that the loss of CD4+ Th1 cell function in HIV-infected persons is not related to a Th1 to Th2 cytokine switch as previously proposed, but to a process of activation-induced death of CD4+ Th1 cells. Despite the absence of elevated levels of Th2 cytokines, apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented in vitro by antibodies to IL-10 or IL-4, two Th2 cytokines that downregulate Th1 cell responses, or by the addition of recombinant IL-12, a cytokine that upregulates Th1 functions. TCR-induced apoptosis of T cell hybridomas and preactivated T cells has been shown to involve the CD95 (Fas/Apo-1) molecule. CD4+ and CD8+ T cells from HIV-infected persons expressed high levels of the CD95 molecule, and, in contrast to T cells from controls, were highly sensitive to antibody-mediated CD95 ligation, which induced apoptosis in a percentage of T cells similar to that induced by TCR stimulation. As TCR-induced apoptosis, CD95-mediated apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented by the addition of recombinant IL-12. Together, these findings suggest that apoptosis of CD4+ T cells from HIV-infected persons involves an abnormal sensitivity to CD95 ligation, and to TCR stimulation in the presence of normal levels of Th2 cytokines. The preventive effect of IL-12 on both mechanisms has potential implications for the design of immunotherapy strategies aimed at the upregulation of CD4+ Th1 cell functions in AIDS.
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PMID:T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons. 750 20

The use of reverse transcriptase in conjunction with the polymerase chain reaction (RT-PCR) has proven invaluable in the analysis of the T cell receptor (TCR) repertoire of different populations of T cells. However, the presence of a variable region in the T cell receptor has hindered the design of primers for the 5' end of the TCR cDNA. We describe the design and use of a degenerate consensus primer that allows amplification of both the alpha and beta chains of the human TCR. We have used this primer in the analysis of the TCR distribution of T cell clones, peripheral blood lymphocytes and lymphocytes residing in tissue. In addition, the primer has allowed the identification of an alternative splice site in the beta chain constant region which cannot translate into a functional constant region. We have found the primer to be easy to use, sensitive and specific.
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PMID:A consensus primer to amplify both alpha and beta chains of the human T cell receptor. 751 Jul 55

Cytokine mRNA expression was analyzed by reverse transcriptase (RT)/PCR in extensively purified normal peripheral CD4+CD45R T cell subsets. Both CD45RA+ and CD45 RO+ populations produced mRNAs for interleukin (IL)-2, IL-2 receptor (alpha chain), IL-6 receptor and tumour necrosis factor (TNF)-beta within 3-4 h of activation. Whilst IL-3 and RANTES were also expressed in both subsets, CD45RO+ cells were clearly the major producers of these cytokines. In contrast, mRNA transcripts for IL-1 alpha, IL-4, IL-5, IL-6, IL-10, interferon gamma (IFN-gamma) and the T cell receptor for IL-1 were almost exclusively induced in CD45RO+ T cells. A population of CD4+ T cells co-expressing intermediate levels of both CD45RA and CD45RO, namely CD45RA+/CD45RO+, appeared to be the major producers of IL-6. Addition of cycloheximide (CHx) 4 h after T cell activation resulted in substantial superinduction of IL-2 mRNA in the CD4+CD45RO+ population but had little effect on CD4+CD45RA+ cells. Taken together, these results show that normal CD4+CD45R T cell subsets exhibit distinct cytokine mRNA profiles and that these differ from the patterns displayed by Th1 and Th2 type T helper clones. Furthermore, they suggest for the first time that IL-2 mRNA turnover is differentially regulated in CD45R T cell subsets.
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PMID:Differential expression and regulation of cytokine mRNAs in normal human CD45R T cell subsets. 751 60

To investigate the pathogenicity of T cells infiltrating in the rheumatoid joints, mononuclear cells (MNC), predominantly T cells, isolated from either synovial fluid or synovial tissues of the patients with RA were transferred into severe combined immunodeficient (SCID) mice by intraarticular injections. According to our observations in this experimental system, patients with RA could be classified into at least two groups. In one group of patients, the infiltrating MNC induced synovial hyperplasia in the recipient SCID mice (the positive group). Whereas, in the other group no synovial hyperplasia was observed (the negative group). The induction of synovial hyperplasia observed in the positive group was prevented by an anti-human CD3 antibody (OKT3), indicating T cell mediation. Analysis of T cell receptor (TCR) V beta usage by reverse transcriptase polymerase chain reaction in the infiltrating MNC transferred into SCID mice revealed a marked skew towards the preferential use of certain V beta genes, which was not seen in the peripheral blood MNC, in only the positive group. The patterns of TCR/V beta skew were not uniform among the patients. The analysis of the PCR-amplified genes of such skewed TCR/ V beta by single strand conformational polymorphism showed distinct bands, indicating that the T cell populations expanding in rheumatoid joints of the positive group were oligoclonal. Furthermore, the enrichment of the T cell populations expressing such skewed TCR/V beta by in vitro stimulation of peripheral blood MNC of the patients with the relevant superantigen enabled the induction of synovial hyperplasia in the SCID mice. These results suggest that the pathogenic T cells could be activated locally in rheumatoid joints by certain antigens in some, but not in all patients with RA.
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PMID:Transfer of rheumatoid arthritis into severe combined immunodeficient mice. The pathogenetic implications of T cell populations oligoclonally expanding in the rheumatoid joints. 756 66

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

Cytotoxic T lymphocytes (CTL) specific for human immunodeficiency virus type 1 (HIV-1) are thought to play an important role in controlling HIV-1 infection. HIV-1-specific CTL are readily demonstrated in unstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected adults but less frequently in PBMC from vertically infected children. HIV-1-specific CTL lines were derived from a long-term survivor of vertical HIV-1 infection using PBMC stimulated with a CD3-specific monoclonal antibody and interleukin-2; these lines had Gag- or reverse transcriptase (RT)-specific cytotoxicity. Cytotoxicity was restricted by major histocompatibility complex class I antigen and blocked by antibody to the T cell receptor complex. Fluorescence-activated cell sorting analysis demonstrated their phenotype to be CD3+CD4-CD8+. Unstimulated PBMC from this patient had no detectable HIV-1-specific cytotoxicity when tested against autologous HIV-1 envelope-, Gag-, or RT-expressing target cells. Thus, this child with vertically acquired HIV-1 infection likely has HIV-1-specific CTL precursors despite the absence of circulating, activated HIV-1-specific CTL.
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PMID:Cytotoxic T lymphocyte lines specific for human immunodeficiency virus type 1 Gag and reverse transcriptase derived from a vertically infected child. 768 62

T lymphocytes in bronchoalveolar lavage (BAL) from idiopathic interstitial pneumonia (IIP) patients are considered to recognize unknown antigens, such as dust, fume, virus or degenerated autoantigens. To analyse the nature of these T lymphocytes, we investigated T cell receptor (TCR) V beta gene usage (22 kinds) in BAL Lymphocytes and Peripheral blood lymphocytes from 10 11P patients and 9 normal controls, using reverse transcriptase-polymerase chain reaction method. In BAL lymphocytes, predominant usage of V beta genes (> 15% of the sum of all V beta transcripts) was recognized in 7 of 10 IIP patients, which appeared to vary in individuals (Case 1: V beta 14, case 2: V beta 3, V beta 5.1, case 5: V beta 2, case 6: V beta 6, case 7: V beta 8, case 8: V beta 7, V beta 20, case 9: V beta 6), whereas no predominant V beta usage was demonstrated in normal controls. It remains to be elucidated whether the BAL lymphocytes expressing predominant V beta genes are involved in the activation of alveolar macrophages, fibroblasts, thereby inducing the production of autoantibodies.
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PMID:[Analysis of T cell receptor V beta gene expression in bronchoalveolar lavage fluid lymphocytes from patients with idiopathic interstitial pneumonia]. 770 52


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