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)

Neutrophils and Langerhans cells participate in inflammatory reactions within the human cornea. Because granulocyte-macrophage (GM)-CSF is a chemotactic and activating factor for these two cell types, we investigated whether this cytokine is produced by human corneal epithelial cells and corneal fibroblasts. Cultures of each cell type were exposed to increasing concentrations of IL-1 alpha or TNF-alpha. Culture supernatants were assayed for GM-CSF by using ELISA and cytokine mRNA levels were monitored by using reverse transcriptase-PCR. IL-1 alpha treatment of both cell types resulted in the appearance of GM-CSF mRNA and the production of > 480 pg protein/10(6) cells. However, TNF-alpha treatment yielded divergent results. Stimulation of epithelial cells with TNF-alpha resulted in the appearance of > 560 GM-CSF mRNA molecules per cell and production of > 1300 pg GM-CSF/10(6) cells. In contrast, stimulation of corneal fibroblasts resulted in < 16 GM-CSF mRNA molecules/cell and < 60 pg GM-CSF/10(6) cells. Binding studies with 125I-labeled TNF-alpha revealed that corneal fibroblasts had as many receptor sites as did corneal epithelial cells. Furthermore, corneal fibroblasts could respond to TNF-alpha-receptor-mediated signal transduction because they produced nanogram amounts of IL-6 after being treated with this cytokine. The results suggest that both cell types synthesize GM-CSF in response to IL-1 alpha, but that only corneal epithelial cells produce significant amounts of GM-CSF after TNF-alpha exposure. Differences in the responses of the two cell types to TNF-alpha may reflect a means of limiting accumulation of neutrophils and Langerhans cells and, thus, minimize corneal damage.
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PMID:Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by pro-inflammatory cytokines. 820 39

Peritoneal injection of thioglycollate medium (TM) to mice results in a dramatic increase in total number of peritoneal macrophages within 48 to 72 hours. Unlike resident macrophages, a fraction (10 to 20%) of these newly arrived young macrophages, designated as macrophage colony-forming cells (M-CFC), are highly proliferative and formed macrophage colonies in vitro in the presence of either macrophage or granulocyte-macrophage colony-stimulating factor (M-CSF or GM-CSF). Using a reverse transcriptase polymerase chain reaction (RT-PCR) technique, peritoneal exudate macrophages (PEM) obtained 2 to 5 days after a single TM injection actively expressed mRNA for recombinant murine macrophage inflammatory protein-1 alpha (rmMIP-1 alpha). Yet none or only a trace amount of mRNA for MIP-1 alpha was detected in normal resident macrophages or PEM obtained 7 days after TM treatment. The effect of rmMIP-1 alpha on the induction of exudate M-CFC was investigated. Multiple intraperitoneal (IP) administration of rmMIP-1 alpha caused a marked increase in the total number of peritoneal M-CFC and macrophages similar to but weaker than the increase in TM-injected mice. The total number of neutrophils, mast cells, and eosinophils also increased, but with different kinetics, following multiple injections of rmMIP-1 alpha. rmMIP-1 alpha alone did not stimulate the proliferation of M-CFC, nor did it potentiate their responsiveness to either rmGM-CSF or recombinant human (rh) M-CSF in vitro. Taken together, our results suggest that MIP-1 alpha released by exudate macrophages is a major chemoattractant responsible for the migration of M-CFC from the circulation to the peritoneal cavity during a TM-induced inflammatory response.
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PMID:Induction of murine peritoneal macrophage colony-forming cells by peritoneal administration of macrophage inflammatory protein-1 alpha. 840 40

Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross-reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down-modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the "distal" GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR ("pure" progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase-polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the "proximal" HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL-6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain-potentiation mechanism.
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PMID:Cascade transactivation of growth factor receptors in early human hematopoiesis. 845 93

An in vitro culture system was developed that facilitates detailed studies of the interaction of Human Immunodeficiency Virus (HIV) with dendritic cells (DC). Cultured immature DC were generated from adherent peripheral blood mononuclear cells in the presence of GM-CSF and IL-4. These cells were non-adherent, non-phagocytic and had a veiled surface appearance. They expressed high levels of MHC class I and II proteins, CD1a, B7/BB1 and low levels of CD4, and were known to possess a potent soluble antigen presenting capacity. Upon infection with the HIV-1 strains Lai (lymphocytotropic) and BaL (monocytotropic), the viral RNA was reverse transcribed to complete DNA provirus. However the infection was non-productive as judged from measuring the activity of the virus encoded reverse transcriptase in the culture supernatant. Thus HIV infection was restricted at a step post entry.
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PMID:Infection of cultured immature dendritic cells with human immunodeficiency virus type 1. 852 22

We investigated the profiles of cytokine mRNA expression in muscle in 15 cases of inflammatory myopathy (IM) (5 each of polymyositis, inclusion body myositis, and dermatomyositis) and in 10 controls (5 of Duchenne dystrophy and 5 non-weak subjects). Expressions of the predominantly T cell-derived cytokines (interleukin (IL)-2, IL-4, IL-5, and interferon-gamma (IFN-gamma), of the predominantly macrophage-derived cytokines (IL-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha)), as well as cytokines that can be of either T cell or macrophage origin (granulocyte-macrophage colony stimulating factor (GM-CSF) and transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2), were monitored by the reverse transcriptase-PCR method. The expression of T cell cytokine mRNAs for IL-2, IL-5, and IFN-gamma was generally weak or inconsistent. IL-4 mRNA expression was consistently moderate to strong in polymyositis but generally weak or absent in the other IMs. The expression of macrophage cytokine mRNAs for IL-1 alpha and IL-1 beta was weak or absent in all cases. Variable TNF-alpha mRNA expression was observed in 12 of 15 IM cases and faint or weak expression in 5 of 10 controls. Very strong GM-CSF expression was detected, but only on boosted PCR, in 12 of 15 cases of IM but in none of the controls. IL-6 was expressed only weakly or inconsistently. In contrast to the variable expression of several of the above mentioned cytokine mRNAs, all IM specimens strongly expressed TGF-beta 1 mRNA and 12 of 15 strongly expressed TGF-beta 2 mRNA. Thus, with the exception of IL-4 expression in polymyositis, a similar pattern of cytokine mRNA expression exists in the different types of IMs. Moreover, this pattern resembles that detected in non-weak and DD controls, although expression is generally weaker in the non-weak controls. The findings suggest that in IM muscle a sustained secretion of cytokines by T cells or of IL-1 by macrophages is not a prerequisite for operation of the immune effector response and that muscle may not be the site of ongoing sensitization.
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PMID:Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls. 855 29

During the process of placental implantation, sessile villous trophoblast cells migrate from the villi into the decidua as isolated motile extravillous trophoblast cells. There is differential expression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villous and extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblast by fluorescence activated cell sorting. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the following cytokines: CSF-1, GM-CSF, TNF-alpha, TGF-beta1, IFN-gamma, IL-2, LIF and also for LIF-receptor. TNF-alpha and TGF-beta 1 were present in all trophoblast populations. GM-CSF and CSF-1 were only found in some samples, with preferential expression of CSF-1 in villous populations. IFN-gamma, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptor mRNA. These cytokines (CSF-1, TGF-beta, TNF-alpha and GM-CSF) are likely to influence trophoblast growth and differentiation in an autocrine manner, since their receptors are also present on trophoblast. These results illustrate a quick and simple method to analyse for the presence of cytokine and other transcripts in trophoblast subpopulations during early pregnancy.
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PMID:Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT-PCR). 858 67

In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.
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PMID:CD40 expression by human peripheral blood eosinophils. 860 42

The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is composed of at least two chains (alpha and beta). The alpha chain binds GM-CSF specifically with low affinity, and the binding is converted to high affinity when the alpha chain is associated with the beta chain. To date, there are at least six isoforms described for the GM-CSFR alpha, all involving alternative splicing at the 3' end, which alters the coding region and hence the protein produced. To detect variants at the 5' end of the GM-CSFR alpha mRNA, RNAse protection and reverse transcriptase polymerase chain reaction (RT-PCR) assays were performed using a probe spanning nucleotides 102-392 and pairs of primers covering exons 1-4. in addition to the expected full-length transcript, two mRNAs were detected, one containing a deletion of 24 nucleotides by alternative splicing at the 3' end of exon 2 (exon 2b-deleted isoform) and another in which exon 2 was completely deleted (exon 2-deleted isoform). Together, the isoforms were more highly expressed form). Together, the isoforms were more highly expressed than the full-length sequence (TF-1 cells: full-length 36 +/- 2.8% vs. exon 2-deleted isoforms 64 +/- 5.5%). These isoforms were detected in primary hematopoietic cells, blasts from patients with acute myeloid leukemia (AML), and malignant cell lines and the relative mRNA expression for the isoforms, was always similar to that of TF-1 cells. As sequences in the 5'untranslated region can be involved in the modulation of translational efficiency, translation of constructs constructs corresponding to these exon 2 deleted isoforms was assessed using an in vitro reticulocyte lysate system. Deletion of exon 2 resulted in significantly lower in vitro translation of the receptor protein relative to the full-length sequence (53, 56, and 76% in three separate batches of reticulocytes), while deletion of exon 2b resulted in higher translation of the sequence (164, 128, and 305%; p = 0.01). These data suggest a mechanism by which expression of the GM-CSFR alpha protein may be regulated by alternatively spliced transcripts with different translational efficiencies.
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PMID:Expression of two alternatively spliced forms of the 5' untranslated region of the GM-CSF receptor alpha chain mRNA. 863 32

Mycobacterium avium subspecies paratuberculosis is an intracellular parasite of intestinal macrophages and causes a chronic granulomatous enteritis in sheep and other ruminants (paratuberculosis or Johne's disease). Macrophages can be produced a variety of immunoregulatory cytokines that may influence mycobacterial killing and produce disordered inflammation within the gut. In this study, messenger RNA (mRNA) was extracted from intestinal tissue from control and multibacillary diseased sheep and profiles for the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, transforming growth factor-beta1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF) were semi-quantified using reverse transcriptase polymerase chain reactions (RT-PCR). Infected intestinal tissues had significantly increased mRNA for TNF-alpha, IL-1beta and IL-6 but TGF-beta1 and GM-CSF mRNA levels were significantly different from controls. Supernatants from in vitro intestinal cultures were assayed for TNF-alpha activity using the PK(15)-1512 cytotoxicity bioassay and levels were significantly raised in diseased samples. TNF-alpha was not detected in any serum samples. Further analysis on intestinal tissues from sheep with the different, paucibacillary, form of the disease showed significant elevation of TNF-alpha mRNA but not other cytokines tested. Increased pro-inflammatory cytokine expression in the intestine coincident with a failed or misdirected immune response may contribute to the pathogenesis of paratuberculosis and the persistence of a chronic inflammatory state.
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PMID:Increased intestinal TNF-alpha, IL-1 beta and IL-6 expression in ovine paratuberculosis. 867 35

Restoration of bone marrow and immune function by means of allogeneic bone marrow transplantation has been attempted in AIDS patients but has not been successful as the donor-derived cells, or their progeny, inevitably became infected. A hairpin ribozyme that specifically cleaves HIV-1 RNA has been developed by F. Wong-Staal et al. and has been demonstrated to confer resistance against HIV-1 infection. Allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced by vectors bearing this ribozyme gene, can protect the stem cells and their progeny from HIV-1 infection and eventually restores immune function. We have provided evidence that long-term repopulating stem cells can be mobilized into peripheral blood by growth factors. The combination of G-CSF and GM-CSF seems to yield a high frequency of pluripotent stem cells with a CD34+ subset profile that is similar to placental and umbilical cord blood (PUCB). We have then demonstrated a highly efficient transduction of CD34+ cells from PUCB and mobilized leukapheresis products by retroviral vectors bearing the ribozyme gene. Expression of the ribozyme gene, as shown by reverse transcriptase-polymerase chain reaction, was of similar magnitude (70%-90% of cells that grow into colonies). Challenge of the progeny macrophages from such transduced CD34+ cells with monocyte-trophic strains of HIV-1 showed that they were resistant to infection. Thus allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced with ribozyme gene, can be a promising strategy for the treatment of HIV infection.
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PMID:Stem cells as vehicles for gene therapy: novel strategy for HIV infection. 874 96


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