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Real-time Reverse-transcription Polymerase Chain Reaction (real-time + reverse-transcription_polymerase_chain_reaction)
Selected AbstractsFrequency and prognostic relevance of cyclin D1 dysregulation in multiple myelomaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5-6 2001Thomas Rasmussen Abstract:Objective: Cyclin D1 dysregulation has been found with varying frequencies in multiple myeloma (MM) and has been suggested to be associated with a poor prognosis. The aim of this study was to investigate the frequency of cyclin D1 dysregulation in patients being treated for MM and to test whether cyclin D1 dysregulation is a prognostic factor for MM patients. Methods: To achieve the above aims we designed a highly sensitive and reproducible real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for quantitation of cyclin D1 mRNA. Using this assay, 110 diagnostic bone marrow (BM) samples from patients with MM were screened for cyclin D1 dysfuntion. Results: The real-time assay was able to detect the presence of 0.01% cyclin D1 positive cells allowing a safe detection in MM BM samples. In 42% (46/110) of MM BM samples a ,,3-fold increase in cyclin D1 mRNA was observed compared to the cyclin D1 level in normal BM. In the remaining group of MM patients the cyclin D1 mRNA levels were comparable to normal donors. Follow-up of 76 MM patients showed no significant (P = 0.35) difference in survival between cyclin D1 positive and negative MM patients. In addition, cyclin D1 dysregulation did not correlate with known prognostic factors. Conclusion: The developed real-time RT-PCR assay for detection of cyclin D1 mRNA levels offers a fast and safe screening for cyclin D1 dysfunction. When a large cohort of MM patients was screened, the cyclin D1 gene was found to be frequently dysregulated, but there was no significant correlation to survival or known prognostic parameters. [source] Hepatitis C virus replication is inhibited by 22,-methoxyolean-12-ene-3,, 24(4,)-diol (ME3738) through enhancing interferon-,,HEPATOLOGY, Issue 1 2008Yoichi Hiasa A derivative of soyasapogenol, 22,-methoxyolean-12-ene-3,, 24(4,)-diol (ME3738), ameliorates liver injury induced by Concanavalin A in mice. We examined whether ME3738 has independent antiviral effects against hepatitis C virus (HCV) using an established HCV replication model that expresses the full-length genotype 1a HCV complementary DNA plasmid (pT7-flHCV-Rz) under the control of a replication-defective adenoviral vector expressing T7 polymerase. Hepatocellular carcinoma (HepG2) cells, human hepatoma (Huh7) cells, or monkey kidney (CV-1) cells were transfected with pT7-flHCV-Rz, and infected with adenoviral vector expressing T7 polymerase. ME3738 or interferon-, (IFN-,) was added thereafter and then protein and RNA were harvested from the cells at 9 days after infection. HCV-positive and HCV-negative strands were measured by real-time reverse-transcription polymerase chain reaction and HCV core protein expression was measured using an enzyme-linked immunosorbent assay. The messenger RNA levels of innate antiviral response-related genes were assessed using real-time reverse-transcription polymerase chain reaction. ME3738 dose-dependently reduced HCV-RNA and core protein in hepatocyte-derived cell lines. The antiviral effect was more pronounced in HepG2 than in Huh7 cells. ME3738 increased messenger RNA levels of interferon-, (IFN-,) and of IFN-stimulated genes (2,-5, oligoadenylate synthetase, myxovirus resistance protein A [MxA]). Interferon-, knockdown by small interfering RNA abrogated the anti-HCV effect of ME3738. Moreover, the anti-HCV effects were synergistic when ME3738 was combined with IFN-,. Conclusion: ME3738 has antiviral effects against HCV. The enhancement of autocrine IFN-, suggests that ME3738 exerts antiviral action along the type I IFN pathway. This anti-HCV action by ME3738 was synergistically enhanced when combined with IFN-,. ME3738 might be a useful anti-HCV drug either with or without IFN-,. (HEPATOLOGY 2008.) [source] Platelet-activating factor-induced NF-,B activation and IL-8 production in intestinal epithelial cells are Bcl10-dependentINFLAMMATORY BOWEL DISEASES, Issue 4 2010Alip Borthakur PhD Abstract Background: Platelet-activating factor (PAF), a potent proinflammatory phospholipid mediator, has been implicated in inducing intestinal inflammation in diseases such as inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). However, its mechanisms of inducing inflammatory responses are not fully understood. Therefore, studies were designed to explore the mechanisms of PAF-induced inflammatory cascade in intestinal epithelial cells. Methods: Nuclear factor kappa B (NF-,B) activation was measured by luciferase assay and enzyme-linked immunosorbent assay (ELISA), and interleukin 8 (IL-8) production was determined by ELISA. B-cell lymphoma 10 (Bcl10), caspase recruitment domain-containing membrane-associated guanylate kinase protein 3 (CARMA3), and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) mRNA and protein levels were assessed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. siRNA silencing of Bcl10 was used to examine its role in PAF-induced NF-,B activation and IL-8 production. The promoter region of the Bcl10 gene was cloned with the PCR method and promoter activity measured by luciferase assay. Results: The adaptor protein Bcl10 appeared to play an important role in the PAF-induced inflammatory pathway in human intestinal epithelial cells. Bcl10 was required for PAF-induced I,B, phosphorylation, NF-,B activation, and IL-8 production in NCM460, a cell line derived from normal human colon, and Caco-2, a transformed human intestinal cell line. PAF also stimulated Bcl10 interactions with CARMA3 and MALT1, and upregulated Bcl10 expression in these cells via transcriptional regulation. Conclusions: These findings highlight a novel PAF-induced inflammatory pathway in intestinal epithelial cells, requiring Bcl10 as a critical mediator and involving CARMA3/Bcl10/MALT1 interactions. The proinflammatory effects of PAF play prominent roles in the pathogenesis of IBD and this pathway may present important targets for intervention in chronic inflammatory diseases of the intestine. (Inflamm Bowel Dis 2009;) [source] Effect of natural commensal-origin DNA on toll-like receptor 9 (TLR9) signaling cascade, chemokine IL-8 expression, and barrier integritiy of polarized intestinal epithelial cellsINFLAMMATORY BOWEL DISEASES, Issue 3 2010Darab Ghadimi Abstract Background and Aim: The intestinal epithelium is constantly exposed to high levels of genetic material like bacterial DNA. Under normal physiological conditions, the intestinal epithelial monolayer as a formidable dynamic barrier with a high-polarity structure facilitates only a controlled and selective flux on components between the lumen and the underlining mucosa and even is able to facilitate structure-based macromolecules movement. The aim of this study was to test the effect of natural commensal-origin DNA on the TLR9 signaling cascade and the barrier integrity of polarized intestinal epithelial cells (IECs). Methods: Polarized HT-29 and T84 cells were treated with TNF-, in the presence or absence of DNA from Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum. TLR9 and interleukin-8 (IL-8) mRNA expression was assessed by semiquantitative and TaqMan real-time reverse-transcription polymerase chain reaction. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (I,B,), and p38 mitogen-activated protein kinase (p38 MAP) phosphorylation were assessed by Western blotting. To further reveal the role of TLR9 signaling, the TLR9 gene was silenced by siRNA. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Nuclear factor-kappa B (NF-,B) activity was assessed by the electrophoretic mobility shift assay (EMSA) and NF-,B-dependent luciferase reporter gene assays. As an indicator of tight junction formation and monolayer integrity of epithelial cell monolayers, transepithelial electrical resistance (TER) was repetitively monitored. Transmonolayer movement of natural commensal-origin DNA across monolayers was monitored using qRT-PCR and nested PCR based on bacterial 16S rRNA genes. Results: In response to apically applied natural commensal-origin DNA, polarized HT-29 and T84 cells enhanced expression of TLR9 in a specific manner, which was subsequently associated with attenuation of TNF-,-induced NF-,B activation and NF-,B-mediated IL-8 expression. TLR9 silencing abolished this inhibitory effect. Apically applied LGG DNA attenuated TNF-,-enhanced NF-,B activity by reducing I,B, degradation and p38 phosphorylation. LGG DNA did not decrease the TER but rather diminished the TNF-,-induced TER reduction. Translocation of natural commensal-origin DNA into basolateral compartments did not occur under tested conditions. Conclusions: Our study indicates that TLR9 signaling mediates, at least in part, the anti-inflammatory effects of natural commensal-origin DNA on the gut because TLR9 silencing abolished the inhibitory effect of natural commensal-origin DNA on TNF-,-induced IL-8 secretion in polarized IECs. The nature of the TLR9 agonist, the polarity of cells, and the tight junction integrity of IECs has to be taken into account in order to predict the outcome of TLR9 signaling. (Inflamm Bowel Dis 2010) [source] Plant sterol guggulsterone inhibits nuclear factor-,B signaling in intestinal epithelial cells by blocking I,B kinase and ameliorates acute murine colitisINFLAMMATORY BOWEL DISEASES, Issue 12 2006Jae Hee Cheon MD Abstract Background/Aims: The plant sterol guggulsterone has been shown to have anti-inflammatory properties. It remains unknown, however, whether guggulsterone is effective for the treatment of inflammatory bowel disease (IBD). Therefore, we investigated anti-inflammatory effects of guggulsterone on intestinal epithelial cells (IEC) and on experimental murine colitis models and elucidated its molecular mechanisms. Methods: Human Caco-2 cells and rat non-transformed IEC-18 cells were stimulated with interleukin (IL)-1, or lipopolysaccharide (LPS) with or without guggulsterone. The effects of guggulsterone on nuclear factor (NF)-,B signaling in IEC were examined by intercellular adhesion molecule (ICAM)-1 real-time reverse-transcription polymerase chain reaction, NF-,B transcriptional activity assay, Western blotting for I,B phosphorylation/degradation, electrophoretic mobility shift assay, and in vitro I,B kinase (IKK) assay. For in vivo study, dextran sulfate sodium (DSS)-treated mice were fed with or without guggulsterone. Colitis was quantified by disease activity index and evaluation of macroscopic and microscopic findings. Phosphorylation of I,B and IKK in colon mucosa was assessed by Western blotting and immunohistochemistry. Results: Guggulsterone significantly inhibited LPS- or IL-1,-induced ICAM-1 gene expression, NF-,B transcriptional activity, I,B phosphorylation/degradation, and NF-,B DNA binding activity in IEC. Moreover, guggulsterone strongly blocked IKK activity. Administration of guggulsterone significantly reduced the severity of DSS-induced murine colitis as assessed by clinical disease activity score, colon length, and histology. Furthermore, tissue upregulation of I,B and IKK phosphorylation induced by DSS was attenuated in guggulsterone-treated mice. Conclusion: Guggulsterone blocks NF-,B signaling pathway by targeting IKK complex in IEC and attenuates DSS-induced acute murine colitis, which suggests that guggulsterone could be an attractive therapeutic option in the treatment of IBD. [source] Inverse Association between T-Cell Immunoglobulin and Mucin Domain-1 and T-bet in a Mouse Model of Allergic Rhinitis,THE LARYNGOSCOPE, Issue 6 2007Geng Xu MD Abstract Background: It has been suggested that human hepatitis A virus cellular receptor, also known as T-cell immunoglobulin and mucin domain-1 (TIM-1), plays an important role in the development of allergic diseases on the basis of epidemiologic data, but the molecular mechanism has been unclear. In a murine model of ovalbumin (OVA)-sensitized allergic rhinitis (AR), we examined the expression of TIM-1 and its correlation with T helper1-associated transcription factor, T-bet, as a potential mediator of T-cell immunoglobulin expression. Methods: Mice were challenged intranasally with OVA to elicit AR. The expression of TIM-1 in nasal tissues was examined by real-time reverse-transcription polymerase chain reaction (RT-PCR), and the surface expression of TIM-1 in peripheral blood mononuclear cells was evaluated by means of flow cytometry. In addition, the expression of TIM-1 as well as T-bet in splenic lymphocytes was examined by Western blotting. Results: TIM-1 mRNA was increased significantly in nasal tissues (P < .05) as seen by real-time RT-PCR. Flow cytometry indicated a differential TIM-1 expression of 135.5 ± 34.2 in the AR group versus 51.1 ± 10.9 in the control group (P < .05). The mean values of normalized TIM-1 were 0.43 ± 0.18 and 0.21 ± 0.10 in AR and control groups, respectively, whereas the mean values of normalized T-bet were 0.22 ± 0.13 and 0.67 ± 0.17 in the AR and control groups, respectively. There was a significant difference in the production of TIM-1 as well as T-bet in AR mice versus control mice (P < .05). The increased production of TIM-1 correlated significantly with the decreased T-bet in spleen tissue of AR mice (r = ,0.52, P < .05). Conclusion: Our experimental model recapitulates an increase in lymphocyte TIM-1 expression seen in AR both locally and systemically. Our results also demonstrate an inverse relationship between lymphocyte TIM-1 and T-bet expression, suggesting a possible mechanism that TIM-1 influences the development of AR. [source] Cross-talk between gibberellin and auxin in development of Populus wood: gibberellin stimulates polar auxin transport and has a common transcriptome with auxinTHE PLANT JOURNAL, Issue 3 2007Simon Björklund Summary Both indole acetic acid (IAA) and gibberellins (GAs) stimulate cell and organ growth. We have examined GA/IAA cross-talk in cambial growth of hybrid aspen (Populus tremula×tremuloides). Decapitated trees were fed with IAA and GA, alone and in combination. Endogenous hormone levels after feeding were measured, by mass spectrometry, in the stem tissues below the point of application. These stem tissues with defined hormone balances were also used for global transcriptome analysis, and the abundance of selected transcripts was measured by real-time reverse-transcription polymerase chain reaction. By feeding isotope-labeled IAA, we demonstrated that GA increases auxin levels in the stem by stimulating polar auxin transport. This finding adds a new dimension to the concept that the endogenous GA/IAA balance in plants is determined by cross-talk between the two hormones. We also show that GA has a common transcriptome with auxin, including many transcripts related to cell growth. This finding provides molecular support to physiological experiments demonstrating that either hormone can induce growth if the other hormone is absent/deficient because of mutations or experimental treatments. It also highlights the potential for extensive cross-talk between GA- and auxin-induced responses in vegetative growth of the intact plant. The role of endogenous IAA and GA in wood development is discussed. [source] | ||||||||||