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)

In an attempt to characterize genes participating in the allergic late phase reaction, we have isolated a novel intercrine/chemokine (called MARC) from a cDNA library of the stimulated mouse mast cell line, CPII. As measured by Northern blotting, it is strongly upregulated at the mRNA level after the physiological challenge of the cells with immunoglobulin (Ig)E plus antigen. Unstimulated cells completely lack significant, stable expression, as do a number of other, different cell lines (uninduced and induced) and mouse tissues. In contrast to the Northern blot analysis, a polymerase chain reaction (PCR) analysis, performed on CPII cells and on Percoll gradient purified mouse peritoneal mast cells, revealed a basal level of transcription in the uninduced stage. After 2 h of IgE plus antigen challenge, a quantitative reverse transcriptase-PCR, using a spiked in MIMIC, showed a level of transcripts more than 100-fold higher in the CPII cells and 5-20-fold higher in purified mouse peritoneal cavity mast cells. This rapid induction after the Fc epsilon RI challenge, the identification of the gene as a member of the chemokine family, and its upregulated expression in peritoneal mast cells, all suggest an involvement in certain acute and chronic pathological mast cell-driven diseases.
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PMID:Immunoglobulin E plus antigen challenge induces a novel intercrine/chemokine in mouse mast cells. 128 Dec 19

We present evidence for RNA transcripts encoding two forms of human epsilon immunoglobulin (Ig) heavy chain that differ significantly from those of other isotypes. We previously demonstrated three human epsilon mRNA species, instead of the two, corresponding to membrane and secreted proteins, seen with other heavy chain transcripts. In human genomic DNA downstream of the C epsilon gene, we identified sequences homologous to the two putative murine exons M1 (encoding a hydrophobic, presumably transmembrane region) and M2 (encoding hydrophilic residues). To determine the structures of epsilon transcripts containing these sequences, we amplified epsilon-related RNAs with the reverse transcriptase polymerase chain reaction. RNA was examined from fresh human B cells stimulated to IgE production by interleukin 4 plus anti-CD40, as well as from the human IgE-producing line AF10. Instead of the single CH4-M1-M2 splice product predicted for murine membrane IgE, we found two other RNA species. One form has the structure CH4-M1'-M2, in which M1' includes the human sequence homologous to the murine M1 as well as a unique segment of 52 codons further upstream in the genomic sequence; this RNA species apparently encodes the IgE expressed on the membrane of IgE-producing lymphocytes. The other RNA has the structure CH4-M2', in which M2' is spliced in an alternative reading frame that includes an additional 109 codons downstream of the termination codon of the CH4-M1'-M2 form. Because the CH4-M2' mRNA form does not encode a hydrophobic segment, its translated product should be secreted. A secreted epsilon protein of approximately the size predicted for this form was identified by Western blotting. This novel IgE protein could play a significant and distinctive role in allergic disorders.
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PMID:Two unusual forms of human immunoglobulin E encoded by alternative RNA splicing of epsilon heavy chain membrane exons. 161 58

Human immunodeficiency virus type 1 (HIV-1) exhibits extensive genomic and antigenic diversity, which is thought to contribute to the failure of the host's immune response to control infection and prevent clinical progression. Part of this failure may be due to utilization by the virus of antigenic variation as a means to escape protective immune responses. Antibody-escape variants of HIV-1 were studied here using fresh clinical isolates and autologous plasmas. HIV-1 was isolated from the plasma of seven people who were all seropositive for at least 2 years, and symptomatic sometime during that period. Isolated viruses were confirmed as HIV-1 by the presence of reverse transcriptase activity in infected culture supernatants, and by positive immunofluorescence using human monoclonal antibody to HIV-1 core protein. Plasma from these people were positive by Western immunoblot (DuPont) for most major HIV-1 (strain IIIB) antigens. These plasmas neutralized three laboratory strains of HIV-1 (i.e., IIIB, RF, and MN) but did not neutralize the homotypic strain in five cases, and had greatly reduced neutralizing titers against the homotypic strain in two cases. Homotypic neutralizing antibodies were absent in autologous plasma obtained 3 months later. When antibody titers were measured by fixed-cell indirect immunofluorescence assays (IFAs), high titers of IgG (1:6400 to 1:25,600) were detected against HIV-1 IIIB, while low titers of only 1:20 to 1:160 were detected against homotypic viral antigens at the time of virus isolation, and remained low 12 and 16 weeks later. No class IgA, IgD, IgE, or IgM antibodies to homotypic viral antigens, as possible IgG-blocking antibodies, were detected by fixed-cell IFAs. Cross-reactions with heterologous donor's plasmas were observed in some cases, and in these cases the cross-reactions were unidirectional. Live-cell IFAs detected IgG in patient's plasma to HIV-1 IIIB-infected cells but not to cells infected with homotypic isolates. These results suggest that it is common for neutralization-resistant HIV-1 variants to appear during the course of infection, and that all or most antigens of these variants are capable of escaping antibody recognition.
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PMID:Homotypic antibody responses to fresh clinical isolates of human immunodeficiency virus. 170 33

When mast cells are activated through their immunoglobulin (Ig)E receptors, release of low molecular weight mediators like histamine is followed by secretion of multiple cytokines, including interleukin (IL)-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor. Here we report that stimulated mast cells also synthesize IL-13 mRNA and protein; secretion of this cytokine may be of particular importance because of its ability to stimulate IgE expression. IL-13 transcripts detected by a semiquantitative reverse transcriptase-mediated polymerase chain reaction assay were induced within 30 min after stimulation of mast cells by dinitrophenyl plus monoclonal IgE anti-dinitrophenyl, and peaked at about 1 h. Within 3 h of IgE stimulation, secreted IL-13 bioactivity, estimated by proliferation of an IL-13-dependent cell line, reached levels equivalent to 1-2 ng/ml of IL-13. When added to human B lymphocytes, the mast cell-derived IL-13 activity (like bone fide IL-13) induced Ig C epsilon transcripts, DNA recombination characteristic of the isotype switch to C epsilon, and the secretion of IgE protein. These results suggest a model of local positive feedback interactions between mast cells and B cells, which could play a role in the pathogenesis of atopy.
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PMID:Activated mast cells produce interleukin 13. 753 36

By using the reverse transcriptase (RT)-PCR and in situ hybridization we have studied the expression of mRNA for IL-5 and IL-4 in human lung mast cells induced by cross-linkage of high affinity Fc epsilon Rs. Lung mast cells were purified using affinity magnetic selection with mAb YB5.B8 against c-kit to achieve a final mast cell purity > 93%. Purified mast cells were precultured with stem cell factor (SCF) (10 ng/ml) and myeloma IgE (3 micrograms/ml) for 16 h before challenge with anti-IgE (1 or 10 micrograms/ml). IgE-dependent activation of lung mast cells caused expression of IL-5 mRNA, which was evident by 2 h and persisted for up to 48-72 h in all of 12 experiments, whereas IL-4 mRNA expression was of a shorter duration and was demonstrable in 6 of 13 experiments. We confirmed that mast cells, and not T cells, were the source of these cytokine messages by using reverse transcriptase-PCR in cell preparations containing known numbers of mast cells and T cells, in situ hybridization in enriched mast cell preparations, and double in situ hybridization-immunocytochemical staining. IL-5 mRNA expression did not require the pretreatment of cells with SCF, whereas expression of IL-4 mRNA seemed to require both anti-IgE and SCF. The strength of IL-5 mRNA signal was related to anti-IgE concentration. Immunoreactive IL-5 was detectable 8 h after anti-IgE challenge, and 10(6) mast cells generated a mean of 731 +/- 400 pg of IL-5 into the supernatant during 48-h culture, but no IL-4 product was detectable. These findings demonstrate the capacity of human lung mast cells to transcribe IL-4 and IL-5 after IgE-dependent activation and to synthesize and release immunoreactive IL-5.
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PMID:IgE-dependent expression of mRNA for IL-4 and IL-5 in human lung mast cells. 754 33

Despite pathophysiologic effects including diarrhea, cholera toxin (CT) is a potent mucosal immunogen and adjuvant. We investigated the influence of CT on T helper (Th)-type 1 (Th1) and Th2 cell-regulated Ag-specific B cell isotype and IgG subclass Ab responses elicited when the toxin was co-administered orally with different protein Ags. When mice were orally immunized with tetanus toxoid (TT) and CT as adjuvant, this regimen induced TT-specific secretory IgA responses in the gastrointestinal tract as well as serum IgG, including IgG1 and IgG2b subclasses, and IgA responses. This oral regimen also induced TT- and CT-B-specific IgE responses. In addition, CT also elicited adjuvant effects for Ag-specific IgG1, IgE, and IgA responses when two other protein Ags, OVA and hen egg white lysozyme, were given by the oral route. Quantitative reverse transcriptase-PCR was performed to assess levels of mRNA for Th1 (IFN-gamma) and Th2 (IL-4) cytokine expression in TT-stimulated CD4+ T cell cultures. Both Peyer's patches and splenic CD4+ T cells expressed markedly increased levels of IL-4-specific message, but did not result in changes in IFN-gamma mRNA expression. To determine whether the route of immunization influenced IgE responses, mice were immunized s.c. with TT and CT as adjuvant. Significant increases in total and TT-specific IgE Abs were induced when CT was co-administered. Taken together, these results show that CT acts as a mucosal adjuvant to enhance Th2-type responses and in particular, the IL-4 produced results in a characteristic Ab isotype pattern associated with this cytokine.
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PMID:Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4. 759 61

An early step in immunoglobulin isotype switching involves the initiation of active transcription of downstream heavy chain loci while they are still in germ-line configuration. This results in the production of 'sterile' germ-line RNA transcripts that do not contain VDJ sequences, the role of which in isotype switching is under intense scrutiny. We investigated whether such human epsilon germ-line transcripts employ the full complement of complex alternative 3' RNA splicing, splicing we have recently reported occurring with productive epsilon mRNA transcripts. Using a reverse transcriptase polymerase chain reaction (RT-PCR) strategy, in which the 5' primer was located in the I region of the epsilon gene, a region expressed in germ-line but not productive (VDJ containing) epsilon transcripts, we showed that the full range of alternative 3' epsilon splices occur with germ-line transcripts. These results demonstrate that epsilon 3' splicing events are independent of 5' isotype DNA switching. Additionally, we showed that, just as with mature epsilon mRNA, the relative production of the various epsilon germ-line mRNA isoforms was responsive to modulation by stimuli such as interleukin-10 (IL-10). Thus B cells, when stimulated to produce epsilon germ-line transcripts, generate a family of germ-line mRNA that differ not only in their initiation sites but, more importantly, also differ in their 3' sequences, sequences that could be important in regulation of the parent gene itself. Furthermore, by discontinuous or trans-splicing, cells could utilize these various epsilon germ-line transcripts to produce the full range of mature IgE proteins prior to undergoing deletional recombination of isotype switching.
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PMID:Germ-line human epsilon heavy chain gene RNA transcripts utilize the full range of alternative 3' splicing seen in productive epsilon mRNA. 764 11

Purified human basophils have been examined for secretion of IL-4 protein and expression of IL-4 mRNA after stimulation with several secretagogues. In general, these studies used a 15-min preincubation with IL-3, before challenge with secretagogues. Under these conditions, IL-4 release averaged 30 pg/10(6) basophils (range 4-70) after challenge with anti-IgE Ab. FMLP and C5a led to somewhat lower levels of secretion. A direct comparison of basophils at 88 to 99% purity with basophils from the same preparations, but at lower purities, showed that the amount of IL-4 secretion was proportional to the purity of the basophils. The presence of mRNA for IL-4 (as determined by reverse transcriptase-PCR, competitive reverse transcriptase-PCR, or Northern blots) was also strictly related to the purity of the basophils. IL-4 mRNA was also found to be constitutively present and was increased after stimulation. The concentration of polyclonal anti-IgE Ab required for optimal IL-4 release was somewhat less than that required for optimal histamine release. IL-4 secretion was slower (t1/2 of 1.5 h) than histamine release and was inhibited by cycloheximide. In a final series of studies, we found that IL-3 was not required for IL-4 secretion; a short 15-min preincubation with IL-3 resulted in the same or slightly less IL-4 release than no treatment with IL-3. In contrast, an 18-h pretreatment with IL-3 resulted in a nearly tenfold increase in IL-4 secretion. We conclude that human basophils secrete IL-4 in response to several secretagogues and that IL-3 priming is not necessary to observe IL-4 secretion.
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PMID:Secretion of IL-4 from human basophils. The relationship between IL-4 mRNA and protein in resting and stimulated basophils. 814 99

In RNA from human IgE-producing lymphocytes, we previously discovered two alternatively spliced epsilon-immunoglobulin mRNA isoforms that encode a novel secreted form of IgE and a membrane-bound species. Further analysis using epsilon-specific reverse transcriptase polymerase chain reactions has elucidated several additional alternatively spliced species of epsilon mRNA. One RNA isoform is generated by splicing the CH4 exon to a novel distal splice acceptor site, forming an epsilon RNA species (designated CH4-M2") that encodes a secreted epsilon protein 6 amino acids larger than the classic secreted epsilon protein. The other three novel epsilon RNAs are generated by splicing from within CH4 to a new exon structure (designated CH5) that is located between CH4 and the membrane exons. Since the three new mRNAs using CH5 share the same stop codon in CH5, they all encode the same novel protein, which is 10 amino acids shorter than the classic secreted epsilon heavy chain. The new alternatively spliced epsilon mRNAs reported here, in addition to the previously reported forms encoding membrane and larger secreted IgE, appear to reflect the normal splicing pattern in humans, as we have detected all these epsilon RNAs in all the human IgE-secreting cells and cell lines tested.
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PMID:Complex alternative RNA splicing of epsilon-immunoglobulin transcripts produces mRNAs encoding four potential secreted protein isoforms. 798 77

The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.
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PMID:Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset. 840 19


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