Home About us Contact
|
Home About us Contact | ||
|
|
||
Real-time Quantitative Reverse Transcription (real-time + quantitative_reverse_transcription)
Selected AbstractsReduced expression of the Rassf1a gene and its aberrant DNA methylation in pancreatic duct adenocarcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamstersMOLECULAR CARCINOGENESIS, Issue 2 2008Kyoko Shimizu Abstract Alterations of the Rassf1a gene were investigated in pancreatic duct adenocarcinomas (PDAs) induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters. Female Syrian golden hamsters received 70 mg/kg BOP, followed by repeated exposures to an augmentation pressure regimen consisting of a choline-deficient diet combined with a sequential course of DL -ethionine, L -methionine, and 20 mg/kg BOP. A total of 15 PDAs were obtained, and total RNAs were assessed by real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR). Expression of the Rassf1a was significantly reduced in PDAs (P,<,0.005) compared with normal pancreatic tissues. For analysis of methylation status, bisulfite sequencing was performed. Normal tissues were all unmethylated in the 5, upstream region of Rassf1a. In contrast, four PDAs were highly methylated, correlating with reduced expression of the Rassf1a gene. Using reverse transcription (RT)-polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, mutations were detected in 3 out of 15 PDAs (20%). These results suggested that alterations of the Rassf1a gene may be involved in development of PDAs induced by BOP in hamsters. © 2007 Wiley-Liss, Inc. [source] Chondrocyte innate immune myeloid differentiation factor 88,dependent signaling drives procatabolic effects of the endogenous toll-like receptor 2/toll-like receptor 4 ligands low molecular weight hyaluronan and high mobility group box chromosomal protein 1 in miceARTHRITIS & RHEUMATISM, Issue 7 2010Ru Liu-Bryan Objective Toll-like receptor 2 (TLR-2)/TLR-4,mediated innate immunity serves as a frontline antimicrobial host defense, but also modulates tissue remodeling and repair responses to endogenous ligands released during low-grade inflammation. We undertook the present study to assess whether the endogenous TLR-2/TLR-4 ligands low molecular weight hyaluronan (LMW-HA) and high mobility group box chromosomal protein 1 (HMGB-1), which are increased in osteoarthritic (OA) joints, drive procatabolic chondrocyte responses dependent on TLR-2 and TLR-4 signaling through the cytosolic adaptor myeloid differentiation factor 88 (MyD88). Methods We studied mature femoral head cap cartilage explants and immature primary knee articular chondrocytes from TLR-2/TLR-4,double-knockout, MyD88-knockout, and congenic wild-type mice. Generation of nitric oxide (NO), degradation of hyaluronan, release of HMGB-1, matrix metalloproteinase 3 (MMP-3), and MMP-13, and protein expression of type X collagen were assessed by Griess reaction and Western blotting analyses. Expression of messenger RNA for type II and type X collagen, MMP-13, and RUNX-2 was examined by real-time quantitative reverse transcription,polymerase chain reaction. Results Interleukin-1, and TLR-2 and TLR-4 ligands induced both HMGB-1 release from chondrocytes and extracellular LMW-HA generation in normal chondrocytes. TLR-2/TLR-4,/, and MyD88,/, mouse cartilage explants and chondrocytes lost the capacity to mount procatabolic responses to both LMW-HA and HMGB-1, demonstrated by >95% suppression of NO production (P < 0.01), and attenuated induction of MMP-3 and MMP-13. Combined deficiency of TLR-2/TLR-4, or of MyD88 alone, also attenuated release of NO and blunted induction of MMP-3 and MMP-13 release. MyD88 was necessary for HMGB-1 and hyaluronidase 2 (which generates LMW-HA) to induce chondrocyte hypertrophy, which is implicated in OA progression. Conclusion MyD88-dependent TLR-2/TLR-4 signaling is essential for procatabolic responses to LMW-HA and HMGB-1, and MyD88 drives chondrocyte hypertrophy. Therefore, LMW-HA and HMGB-1 act as innate immune cytokine-like signals with the potential to modulate chondrocyte differentiation and function in OA progression. [source] Gene expression analysis of macrophages derived from ankylosing spondylitis patients reveals interferon-, dysregulationARTHRITIS & RHEUMATISM, Issue 6 2008Judith A. Smith Objective To determine whether macrophages, a type of cell implicated in the pathogenesis of ankylosing spondylitis (AS), exhibit a characteristic gene expression pattern. Methods Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1,43 years]) and 9 healthy control subjects over 7 days with the use of granulocyte,macrophage colony-stimulating factor. Cells were stimulated for 24 hours with interferon-, (IFN,; 100 units/ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml). RNA was isolated and examined by microarray and real-time quantitative reverse transcription,polymerase chain reaction analysis. Results Microarray analysis revealed 198 probe sets detecting the differential expression of 141 unique genes in untreated macrophages from AS patients compared with healthy controls. Clustering and principal components analysis clearly distinguished AS patients and controls. Of the differentially expressed genes, 78 (55%) were IFN-regulated, and their relative expression indicated a "reverse" IFN signature in AS patient macrophages, where IFN,,up-regulated genes were underexpressed and down-regulated genes were overexpressed. Treatment of macrophages with exogenous IFN, normalized the expression of these genes between patients and controls. In addition, the messenger RNA encoded by the IFN, gene was ,2-fold lower in AS patient macrophages at baseline (P = 0.004) and was poorly responsive to LPS (P = 0.018), as compared with healthy controls. Conclusions Our findings reveal consistent differences in gene expression in macrophages from AS patients, with evidence of a striking "reverse" IFN signature. Together with poor expression and responsiveness of the IFN, gene, these results suggest that there may be a relative defect in IFN, gene regulation, with autocrine consequences and implications for disease pathogenesis. [source] SPARC, an upstream regulator of connective tissue growth factor in response to transforming growth factor , stimulationARTHRITIS & RHEUMATISM, Issue 12 2006X. D. Zhou Objective To differentiate the effects of inhibition of specific small interfering RNA (siRNA) of SPARC (secreted protein, acidic and rich in cysteine) and siRNA of connective tissue growth factor (CTGF) in cultured human fibroblasts, and to identify potential interrelationships between SPARC and CTGF. Methods Fibroblasts from skin biopsy specimens of 2 normal individuals were transfected with siRNA of SPARC and siRNA of CTGF. The fibroblasts were stimulated with or without transforming growth factor ,1 (TGF,1) and examined by real-time quantitative reverse transcription,polymerase chain reaction to determine the transcription levels of several extracellular matrix genes. Results After exogenous TGF,1 stimulation, both SPARC siRNA and CTGF siRNA showed a protective role against overexpression of collagen genes. Following TGF,1 stimulation, SPARC siRNA,transfected fibroblasts showed a greater reduction in expression of the collagen genes compared with CTGF siRNA,transfected fibroblasts, as well as a significantly decreased expression of CTGF (P < 0.05). Using linear structure equations to quantitatively model a genetic network based on expression levels of each gene, a positive regulatory role of SPARC on CTGF, COL1A2, COL3A1, COL11A1, and TIMP3 was observed. However, the regulatory role of CTGF on SPARC appeared to be negative and very small, while the positive regulatory effects of CTGF on COL1A2, COL3A1, COL11A1, and TIMP3 were less than those of SPARC. Conclusion The results of this quantitative comparison support the hypothesis that in these cultured fibroblasts, the regulatory effects of SPARC on some major extracellular matrix structural components are greater than those of CTGF. In addition, SPARC appears to regulate CTGF in a predominantly positive manner, while CTGF may act as a negative feedback control on SPARC following TGF, stimulation. [source] Inflammatory cytokine regulation of transgene expression in human fibroblast-like synoviocytes infected with adeno-associated virusARTHRITIS & RHEUMATISM, Issue 7 2006Russell S. Traister Objective An ideal gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis (RA) would provide local transgene expression only when the disease is active. To determine whether adeno-associated virus (AAV) possesses this ability, the effects of inflammatory cytokines on transgene expression were evaluated in human RA fibroblast-like synoviocytes (FLS). Methods Human FLS were infected with AAV in the presence or absence of inflammatory cytokines or synovial fluid obtained from patients with RA. Transgene expression was monitored by either enzyme-linked immunosorbent assay or flow cytometry. Transgene messenger RNA (mRNA) was measured by real-time quantitative reverse transcription,polymerase chain reaction. Results Inflammatory cytokines increased transgene expression in FLS by up to 60-fold. Synovial fluid from patients with RA, but not from patients without arthritis, was also able to increase expression in synoviocytes. Protein expression correlated with transgene mRNA levels. The enhanced expression required the continued presence of cytokines because, upon removal, transgene expression returned to baseline levels. Expression could be repeatedly reinduced by reexposure to cytokines. The effect was not promoter specific and was demonstrated to be phosphatidylinositol 3-kinase,dependent. Conclusion These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV gene transfer, making it an attractive vector for chronic inflammatory diseases such as RA. [source] Prognostic significance of peritoneal minimal residual disease in gastric cancer detected by reverse transcription,polymerase chain reactionBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 4 2004K. Oyama Background: A sensitive method for detecting minimal residual disease in the peritoneal cavity by quantifying carcinoembryonic antigen (CEA) mRNA using real-time quantitative reverse transcription,polymerase chain reaction (RQ-RT,PCR) was developed. The clinical value of the method for predicting peritoneal recurrence in patients with gastric cancer was evaluated. Method: A total of 195 patients with gastric cancer and 20 with asymptomatic cholecystolithiasis were included in the study. CEA mRNA expression in peritoneal washings (p- CEA mRNA) was measured by RQ-RT,PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA expression. The cut-off level of p- CEA mRNA for gastric cancer was determined by examining p- CEA mRNA levels in patients with asymptomatic cholecystolithiasis. Results: Fifty-five (28·2 per cent) of the 195 patients were p- CEA mRNA positive. The rate of p- CEA mRNA positivity correlated significantly with clinicopathological factors. In 163 patients who underwent curative surgery, overall survival and disease-free survival were significantly poorer in p- CEA mRNA-positive patients than in p- CEA mRNA-negative patients (P < 0·001). Cox regression analysis revealed that only p- CEA mRNA was a significant independent prognostic factor (P = 0·034). Multivariate logistic regression analysis showed that p- CEA mRNA was a significant independent risk factor for peritoneal recurrence (P = 0·027). Conclusion: These results suggest that p- CEA mRNA is a reliable prognostic factor and predictor of peritoneal recurrence in gastric cancer. Copyright © 2004 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] | ||||||||||