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)

Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia-infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia (genomovar III of the B. cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed. B. cepacia (multiplicity of infection > or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection > or =64:1). IL-8 secretion by B. cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia-induced IL-8 secretion was mediated through de novo protein synthesis. Treatment of B. cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia-infected HLE cells was abrogated by > or =80% in the presence of anti-CD14 [specific lipopolysaccharide (LPS) receptor] antibody, thus suggesting that the IL-8-inducing component of B. cepacia was LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia-infected HLE cells (SB203580, > or =80% inhibition; PD98059, > or =30% inhibition). In conclusion, B. cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.
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PMID:Burkholderia cepacia-induced IL-8 gene expression in an alveolar epithelial cell line: signaling through CD14 and mitogen-activated protein kinase. 1276 57

Infection with Helicobacter pylori, a Gram-negative, microaerophilic, flagellated bacteria that adheres to human gastric mucosa, is strongly associated with gastric ulcers and adenocarcinoma. The mechanisms through which gastric epithelial cells recognize this organism are unclear. In this study we evaluated the interactions between the Toll-like receptors (TLRs) and H. pylori-mediated NF-kappa B activation and the induction of chemokine mRNA expression. By reverse transcriptase-PCR we determined that MKN45 gastric epithelial cells express low but detectable amounts of TLR2, -4, and -5 but no MD-2. To determine which, if any, TLRs may play a role in the response of epithelial cells to H. pylori, HEK293 cells were cotransfected with the NF-kappa B-Luc reporter, CD14 and MD2 expression plasmids, and expression plasmids for TLR2, TLR4, or TLR5. Infection of the cultures with H. pylori (strain 26695) induced NF-kappa B activity in cells transfected with TLR2 and TLR5, but not TLR4. Consistent with the HEK293 experiments, H. pylori-induced NF-kappa B activation was decreased in MKN45 gastric epithelial cells by transfection of dominant-negative versions of TLR2 and TLR5 but not TLR4. Highly purified lipopolysaccharide from H. pylori strain 26695 activated NF-kappa B in HEK293 via TLR2 but not TLR4. Partially purified flagellin from H. pylori was also capable of inducing NF-kappa B activation in HEK cells transfected with TLR5. Additionally, chemokine gene expression was induced by H. pylori in HEK293 cells following stable transfection with TLR2 or TLR5 expression plasmids. These studies demonstrate that gastric epithelial cells recognize and respond to H. pylori infection at least in part via TLR2 and TLR5. Furthermore, the unique lipopolysaccharide of H. pylori is a TLR2, not a TLR4 agonist.
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PMID:Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells. 1280 70

In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce adh-DCs was significantly increased when monocytes were incubated with antiCD14 antibody coupled beads, anti-CD14 antibodies, or lipopolysaccharide (LPS), reaching transduction efficiencies up to 86.6%, 53.3%, and 80.9%, respectively. We showed that this enhanced transduction was correlated to an activation of the monocytes, characterized by the up regulation of the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and the de novo synthesis of IL-6 and IL-10. However, the enhanced transduction of immature CD14-DCs was not correlated with a progression in the cell cycle from G(0) to G(1). We further showed that CD14-DCs were phenotypically comparable to adh-DCs. Functional analysis revealed that there were no differences in allostimulatory capacity, production of IL-12 p70 on CD40 ligation or expression of IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha as evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, we showed that lentivirally transduced CD14-DCs were equally capable as adh-DCs in stimulating MAGE-A3 antigen-specific CD4(+) and CD8(+) T cells in vitro.
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PMID:Activation of monocytes via the CD14 receptor leads to the enhanced lentiviral transduction of immature dendritic cells. 1521 15

Peripheral inflammation induces central sensitization characterized by the development of allodynia and hyperalgesia to mechanical and thermal stimuli. Recent evidence suggests that activation of glial cells and a subsequent increase in proinflammatory cytokines contribute to the development of behavioral hypersensitivity after nerve injury or peripheral inflammation. In the present study, we examined mRNA and protein expression of glial markers and proinflammatory cytokines at the lumbar spinal cord, brainstem and forebrain following intraplantar administration of complete Freunds adjuvant (CFA) in rats. Gene expression studied by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for microglial markers (Mac-1, TLR4 and CD14) showed a significant increase in their expression during all phases (acute, subacute and chronic) of inflammation. Conversely, up-regulation of astroglial markers [glial fibrillary acidic protein (GFAP) and S100B] was observed only at the subacute and chronic phases of inflammation. Increased immunoreactivity for OX-42 (CR3/CD11b) and GFAP at various brain regions was also observed after the acute and subacute phases of the inflammation, respectively. Quantification of proinflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) at the mRNA (by real-time RT-PCR) and protein level (by ELISA) revealed enhanced expression during the acute, subacute and chronic phases of CFA-induced peripheral inflammation. This study demonstrates that CFA-induced peripheral inflammation induces robust glial activation and proinflammatory cytokines both spinally and supraspinally. In addition, similar to nerve injury-induced behavioral hypersensitivity microglial activation preceded astrocytic activation following CFA-induced peripheral inflammation, supporting a role of microglia in the initiation phase and astrocytes in maintaining hypersensitivity. These findings further support a unifying theory that glial activation and enhanced cytokine expression at the CNS have a role in eliciting behavioral hypersensitivity.
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PMID:Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the CNS. 1523 55

A novel human tumor-associated antigen, receptor-binding cancer antigen expressed on SiSo cells (RCAS1), induces apoptosis in normal human erythroid progenitor cells, which express putative RCAS1 receptors. In the present study, we investigated a possible role of RCAS1 produced by human peripheral blood monocytes (CD14-positive cells) and monocyte-derived macrophages. RCAS1 was immunohistochemically detected in monocytes as well as macrophages. When macrophages were stimulated with lipopolysaccharide (LPS), the expression of RCAS1 was remarkably enhanced. An increased production of RCAS1 mRNA was observed in LPS-stimulated macrophages by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Soluble RCAS1 molecules were only detected in the culture supernatants obtained from LPS-stimulated macrophages. Moreover, LPS-stimulated macrophages induced cell death of erythroid progenitor cells through RCAS1 production. These results suggest that macrophages may negatively regulate erythropoiesis at least in part through the production of RCAS1 molecules, and this may contribute to the pathogenesis of the anemia seen in patients with inflammatory disorders.
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PMID:A novel mechanism in suppression of erythropoiesis during inflammation: a crucial role of RCAS1. 1581 9

Pattern recognition receptors, which include the toll-like receptors (TLRs), are considered to play an important role in the response against lipopolysaccharide (LPS). In this study, we performed a reverse transcriptase/polymerase chain reaction (RT-PCR) study, Western analysis, immunohistochemical staining, and RT-PCR-amplified in situ hybridization of TLR2 and TLR4 in the case of LPS-induced lung injury. The expression of TLR2 and TLR4 increased in the lung rapidly after LPS inhalation and peaked at 24 h, followed by a gradual decrease. TLR2 and TLR4 expression was observed on the bronchial epithelium and tissue macrophages. In the early hours after inhalation of fluorescein-isothiocyanate (FITC)-labeled LPS, LPS was detected mainly on the bronchial epithelium and on a few of tissue macrophages. One day after inhalation, the LPS signals disappeared in the lungs of the mice, except for a few alveolar macrophages. The expression of TLR2, TLR4, and CD14 was coincident with the signals of FITC-labeled LPS. Instillation of liposome-encapsulated dichloromethylene diphosphonate induced a significant decrease in alveolar macrophages. In the macrophage-depleted mice, however, expression of TLR2 and TLR4 mRNA or protein was slightly suppressed in the lung after LPS inhalation. These data suggest that the bronchial epithelium and macrophages play crucial roles in LPS-induced lung injury through TLR2 and TLR4.
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PMID:Expression of toll-like receptor 2 and 4 in lipopolysaccharide-induced lung injury in mouse. 1590 99

Acute promyelocytic leukemia (APL) is a human cancer generated by a chromosomal translocation t(15;17) involving the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARalpha) genes. The PML/RARalpha oncoprotein expressing blasts show two of the most important biological features of neoplastic progression: block of differentiation, at the promyelocytic state, and increased survival. Although PML/RARalpha interferes with the normal maturation of myeloid precursors to granulocytes, pharmacological doses of retinoic acid are sufficient to restore the differentiation processes. We designed an assay based on the Real-Time reverse transcriptase polymerase chain reaction (RT-PCR) to experimentally follow the differentiation response of leukemic cells even after short-time differentiating treatments. Amplifying CD11b, CD11c, and CD14 mRNAs, as specific markers of differentiation, by the real-time RT-PCR assay we could detect both retinoic acid (RA) and vitamin D3 and human transforming growth factor beta1 (VitD3/TGFbeta1) induced cellular maturation more precociously than the canonical flow-cytofluorimetric assay. Moreover, by amplifying CD14 mRNA it was possible to monitor the ability of PML/RARalpha oncoprotein to block VitD3/TGFbeta1 induced differentiation in U937-PR9 promonocytic inducible model systems.The proposed real-time quantitative RT-PCR approach is a reproducible and highly sensitive assay and can be considered a valid method to study both cellular maturation state and differentiation response.
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PMID:Differentiation response of acute promyelocytic leukemia cells and PML/RARa leukemogenic activity studies by real-time RT-PCR. 1598 48

The objective of this study was to identify single nucleotide polymorphisms (SNPs) within four functionally related immune response genes in the horse, and to develop genotyping techniques that could be useful for future genomic studies of horse infectious and allergic diseases. The genes analysed were: the lipopolysaccharide (LPS) receptor gene CD14, the toll-like receptor 4 gene TLR4, the gene Cepsilon encoding the IgE heavy chain molecule and the gene FcepsilonR1 alpha coding for the alpha subunit of the IgE receptor molecule. Horse-specific primers amplifying selected gene regions were designed and SNPs were searched by selective resequencing and/or by PCR-SSCP (polymerase chain reaction-sequence specific conformational polymorphism) or PCR-RFLP (PCR-restriction fragment length polymorphism). Gene expression was analysed by RT-PCR (reverse transcriptase-PCR) of all four genes examined. For CD14, the cDNA sequence was determined and a novel sequence of the 5'UTR region was identified. The protein-coding sequence was identical to that previously deposited in GenBank. 5'UTR, intronic and both synonymous and non-synonymous exonic SNPs were identified. Three SNPs were found in the CD14 gene, four in the TLR4 gene; two SNPs were identified in the Cepsilon gene, and one SNP was found in the FcepsilonR1 alpha gene. PCR-RFLP was developed for genotyping eight of the SNPs identified. The RT-PCR assay showed that all the SNPs reported here are parts of expressed genes. The results showed that important immunity-related genes in horses are polymorphic and that even non-synonymous SNPs with potential functional impact may occur. The methods developed for genotyping and haplotyping the SNPs identified represent, along with markers described previously, a potentially useful tool for genomic analysis of the function and role of these genes in immunity and in mechanisms of disease.
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PMID:Single nucleotide polymorphisms in four functionally related immune response genes in the horse: CD14,TLR4, Cepsilon, andFcepsilon R1 alpha. 1616 94

We recently demonstrated that the pattern recognition receptors (PRRs) toll-like receptor 2 (TLR2), TLR4, and CD14 are expressed in mouse colonic epithelium in a compartmentalized manner. Here we report the localization of TLR5, the receptor for bacterial flagellin, and its distinctive down-regulation during experimental colitis. Guts from normal BALB/c mice and those with dextran sodium sulfate (DSS)-induced colitis were compared. Each gut was divided into seven segments (stomach, small intestine [three parts], and colon [three parts]), and epithelial cells and crypt units were collected by scraping and EDTA treatment, respectively. Northern blotting showed that TLR5 mRNA was preferentially expressed in the epithelium of the proximal colon in normal mice. Laser capture microdissection coupled to reverse transcriptase PCR confirmed this localization. TLR5 protein expression reflected mRNA expression, as evidenced by Western blotting. In mice with acute colitis, inflammation occurred mainly in the distal colon. Interestingly, while TLR2, TLR4, and CD14 were up-regulated in the inflamed colon, TLR5 was down-regulated at both the mRNA and protein levels. Decreased TLR5 expression was more evident during chronic colitis. Additional in vitro studies using a mouse cell line, Colon-26, showed that gamma interferon (IFN-gamma) time- and dose-dependently down-regulates TLR5. In conclusion, epithelial cells, mainly in the proximal colon, constitutively express TLR5. TLR5 expression is down-regulated in vivo during acute and chronic DSS-induced colitis, in contrast to the expression of TLR2, TLR4, and CD14. The mechanism governing TLR5 regulation may therefore differ from that controlling other PRRs. Finally, IFN-gamma may be involved in down-regulating TLR5 expression.
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PMID:Epithelial toll-like receptor 5 is constitutively localized in the mouse cecum and exhibits distinctive down-regulation during experimental colitis. 1642 10

Earlier reports on a putative precursor cell population in adipose tissue showed differentiation along several mesodermal lineages, leading some to think that adipose tissue can be a source of cells applicable in regenerative medicine. However, characterizations of these adipose-derived precursor cells (ADPC) in the 3-dimensional (3-D) environment, especially within the area of bone-specific composite scaffolds, have been lacking. In this study, ADPC plated on culture flasks or seeded on medical grade polycaprolactone-tricalcium phosphate scaffolds (mPCL-CaP) were able to differentiate along the osteogenic lineages in both 2-D and 3-D environments as assessed with immunohistochemistry of osteo-related proteins, reverse transcriptase-polymerase chain reactions and alkaline phosphatase assay. The mPCL-CaP scaffolds provided adipose-derived cells (ADC) with a suitable environment as determined by DNA and metabolic assays, light, confocal and scanning electron microscopy. Flow cytometry revealed ADC to be CD29+, CD44+, CD73+, CD90+ and CD14-, CD31-, CD34-, CD45-, CD71-, and therefore showed the absence of hematopoietic stem cells but possibly the presence of pericytes and mescenchymal stem cells with osteogenic potential. The results of this study demonstrated the potential of using ADPC in combination with mPCL-CaP scaffolds for bone regenerative medicine.
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PMID:Characterization of osteogenically induced adipose tissue-derived precursor cells in 2-dimensional and 3-dimensional environments. 1665 24


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