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Quantitative Reverse Transcription (quantitative + reverse_transcription)
Kinds of Quantitative Reverse Transcription Terms modified by Quantitative Reverse Transcription Selected AbstractsHypoxia-inducible factor regulation of ANK expression in nucleus pulposus cells: Possible implications in controlling dystrophic mineralization in the intervertebral discARTHRITIS & RHEUMATISM, Issue 9 2010Renata Skubutyte Objective Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins. Methods Quantitative reverse transcription,polymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity. Results ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1, and HIF-2, resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1,,null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1, and HIF-2, in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxia-responsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state. Conclusion Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions. [source] BAFF synthesis by rheumatoid synoviocytes is positively controlled by ,5,1 integrin stimulation and is negatively regulated by tumor necrosis factor , and toll-like receptor ligandsARTHRITIS & RHEUMATISM, Issue 10 2007Ghada Alsaleh Objective It was recently demonstrated that synoviocytes (FLS) from rheumatoid arthritis (RA) patients express BAFF transcripts that are up-regulated by tumor necrosis factor , (TNF,) and interferon-, (IFN,). Thus, BAFF increases in RA target cells might be related to activation of the receptors of innate immunity. The purpose of this study was to determine whether ligands of Toll-like receptor 2 (TLR-2), TLR-4, TLR-9, and ,5,1 integrin are able to induce BAFF synthesis by RA FLS. Methods Quantitative reverse transcription,polymerase chain reaction analyses and enzyme-linked immunosorbent assays were performed to evaluate BAFF messenger RNA induction and BAFF release from FLS after stimulation by ligands for TLR-2, TLR-4, TLR-9, ,5,1 integrin (bacterial lipopeptide [BLP] palmitoyl-3-cysteine-serine-lysine-4, lipopolysaccharide [LPS], CpG, and protein I/II, respectively), TNF,, and IFN,. Results In contrast to IFN,, neither TNF,, LPS, BLP, nor CpG induced the de novo synthesis and release of BAFF by FLS. Priming of cells with IFN, did not have a synergistic effect on BAFF synthesis by FLS stimulated with bacterial products known as pathogen-associated molecular patterns. Moreover, we found that IFN,-induced BAFF synthesis is inhibited by simultaneous stimulation with either TLR ligands or TNF,. We also showed that interplay between TLRs, TNF receptors, and IFN, signaling induces the expression of suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 and reduces IFN,-dependent STAT-1 phosphorylation, which might explain this inhibition. In contrast, we demonstrated that stimulation of ,5,1 integrin can induce BAFF synthesis and release per se and that stimulation of this pathway has no inhibitory effect on IFN,-induced BAFF synthesis. Conclusion Our findings indicate that BAFF secretion by resident cells in target organs of autoimmunity is tightly regulated by innate immunity, with positive and negative controls, depending on the receptors and the pathways triggered. [source] Sympathectomy suppresses tumor growth and alters gene-expression profiles in rat tongue cancerEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 4 2009Bina Raju Sympathetic nerves are known to affect carcinogenesis. Recently we found that sympathetic denervation decreases the size of rat tongue tumors. To identify genes involved in rat tongue carcinogenesis and to study the effect of sympathetic nerves on these genes, we compared gene-expression profiles in normal rat tongue (control) and in tumor-induced tongues with (SCGx) and without (Sham) bilateral sympathectomy. Significance analysis of microarrays revealed 280 genes (168 up-regulated, 112 down-regulated) that showed at least a twofold differential expression between Sham and SCGx tumors (false discovery rate < 5%). These included genes associated with cell adhesion, signaling, structure, proliferation, metabolism, angiogenesis, development, and immunity. Hierarchical clustering demonstrated that controls and sympathectomized tumors grouped together, while Sham tumors grouped separately. We identified 34 genes, known to be involved in carcinogenesis, that were not differentially expressed between sympathectomized tumors and control tongues, but which showed a significant change in expression in Sham tumors. Microarray results of 12 of these genes were confirmed by quantitative reverse transcription,polymerase chain reaction. In conclusion, sympathectomy significantly altered the gene-expression profile and inhibited tumor growth. The expression of several cancer genes were increased more than threefold in Sham tumors, but unaltered in the sympathectomized tumors when compared with controls, indicating that these genes may be of significance in rat tongue carcinogenesis. [source] The location and characteristics of two populations of dental pulp cells affect tooth developmentEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2009Yoshinori Sumita This study investigated the characteristics of two dental pulp cell populations during the early stages of crown formation in porcine teeth. A transplantation method was developed to reproduce epithelial cell,mesenchymal cell interactions during odontogenesis (tooth development). The technique allowed two types of cells/tissue to be combined in vivo. Populations of cells localized in the cervical loop epithelium region, dental pulp horn, and dental pulp core chambers were isolated and dissociated into single cells. Each population was examined for its gene-expression pattern using both semiquantitative and quantitative reverse transcription,polymerase chain reaction (RT-PCR) analyses, and for its tissue-formation capability by combining the cervical loop epithelial cells with either pulp horn cells or pulp core cells on biodegradable collagen scaffolds that were subsequently examined using histology and immunohistology. Gene-expression patterns showed that pulp horn cells were more mature than pulp core cells. Cervical loop epithelial cells combined with pulp horn cells mainly reconstituted dentin,cementum structures. By contrast, cervical loop epithelial cells combined with pulp core cells reconstituted enamel,dentin structures. These results suggest that mesenchymal cells residing in a specific location of the pulp possess a specific tissue-formation potential when combined with epithelial cells. [source] Role of transcription factors Ad4bp/SF-1 and DAX-1 in steroidogenesis and spermatogenesis in human testicular development and idiopathic azoospermiaINTERNATIONAL JOURNAL OF UROLOGY, Issue 6 2006YOSHIYUKI KOJIMA Background:, Ad4bp/SF-1 and DAX-1 are orphan members of the nuclear hormone receptor superfamily of transcription factors. In order to obtain better understandings of human testicular steroidogenesis and spermatogenesis, we examined the expression levels of both factors in human normal and idiopathic azoospermic testes and investigated their physical meaning. Methods:, First, we examined the expression level of Ad4bp/SF-1 and DAX-1 by quantitative reverse transcription,polymerase chain reaction (RT,PCR), immunohistochemistry and western blotting analysis using eight normal human testicular tissues from infants to adults. Second, we performed quantitative RT,PCR using testicular biopsy samples obtained from 22 idiopathic azoospermic patients to examine the expression of Ad4bp/SF-1 and DAX-1, and analysed the correlation between the expression levels of both factors and the serum hormone levels or histological evaluation to study their potential correlation with steroidogenesis and spermatogenesis on idiopathic azoospermia. Results:, The expression levels of both factors in the normal testes increased with testicular development. Ad4bp/SF-1 was abundantly expressed in Leydig cell, whereas DAX-1 was expressed in Sertoli cells. The expression level of Ad4bp/SF-1 in idiopathic azoospermic patients testes positively correlated with serum testosterone (P < 0.05). The average expression levels of DAX-1 mRNA for patients with maturation arrest (0.39 ± 0.19) and Sertoli cell-only syndrome (0.13 ± 0.08) were lower than that with hypospermatogenesis (1.60 ± 1.32) and normal spermatogenesis (1.30 ± 1.41). Conclusion:, Ad4bp/SF-1 is important for the maintenance of steroidogenesis in the human testis. DAX-1 plays a critical role in spermatogenesis in the human testis, and Sertoli cell-only syndrome and maturation arrest may result from abnormal Sertoli cell function that disrupts the normal progression of spermatogenesis. [source] Reduced 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] Natural genetic variation in whole-genome expression in Arabidopsis thaliana: the impact of physiological QTL introgressionMOLECULAR ECOLOGY, Issue 5 2006THOMAS E. JUENGER Abstract A long-standing and fundamental question in biology is how genes influence complex phenotypes. Combining near-isogenic line mapping with genome expression profiling offers a unique opportunity for exploring the functional relationship between genotype and phenotype and for generating candidate genes for future study. We used a whole-genome microarray produced with ink-jet technology to measure the relative expression level of over 21 500 genes from an Arabidopsis thaliana near-isogenic line (NIL) and its recurrent parent. The NIL material contained two introgressions (bottom of chromosome II and top of chromosome III) of the Cvi-1 ecotype in a Ler -2 ecotype genome background. Each introgression ,captures' a Cvi allele of a physiological quantitative trait loci (QTL) that our previous studies have shown increases transpiration and reduces water-use efficiency at the whole-plant level. We used a mixed model anova framework for assessing sources of expression variability and for evaluating statistical significance in our array experiment. We discovered 25 differentially expressed genes in the introgression at a false-discovery rate (FDR) cut-off of 0.20 and identified new candidate genes for both QTL regions. Several differentially expressed genes were confirmed with QRT,PCR (quantitative reverse transcription,polymerase chain reaction) assays. In contrast, we found no statistically significant differentially expressed genes outside of the QTL introgressions after controlling for multiple tests. We discuss these results in the context of candidate genes, cloning QTL, and phenotypic evolution. [source] Wilms' tumor 1 message and protein expression in bone marrow failure syndrome and acute leukemiaPATHOLOGY INTERNATIONAL, Issue 10 2007Takashi Iwasaki Wilms' tumor 1 (WT1) is a useful marker for the diagnosis of acute leukemia and myelodysplastic syndromes (MDS). In the current study quantitative reverse transcription,polymerase chain reaction and immunostaining were used simultaneously to examine the relationship between WT1 RNA and protein level and also to evaluate WT1 as a tool to differentiate aplastic anemia (AA) and MDS refractory anemia (RA). Three types of WT1 messages (total, exon 5(+) and KTS(+)) and WT1 immunostaining of these diseases were analyzed. An increase of all three WT1 messages in high-grade MDS and acute leukemia was observed as compared with the normal control, whereas there was no significant difference in WT1 message between AA and RA, suggesting that WT1 message is not a good tool to discriminate AA and RA. No significant difference was observed between normal and RA, except for exon 5 message. Three WT1 message levels had a significant correlation, suggesting that the total WT1 message is sufficient for clinical practice. Positive immunostaining of WT1 was observed only in the portion of acute leukemia and overt leukemia (OL) transformed from MDS with a high WT1 message level, suggesting the relatively high detection threshold of WT1 protein with the immunostaining method. [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] DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 11 2009Ko Hashimoto Objective To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1, (IL-1,) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. Methods IL-1,, tumor necrosis factor , (TNF,) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4,5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green,based quantitative reverse transcription,polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B -expressing and IL1B -nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (,299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1, into the culture media was assessed by enzyme-linked immunosorbent assay. Results Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100,1,000-fold by treatment with TNF,/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1, and was almost abolished by TNF,/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. Conclusion These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes. [source] Profiling microRNA expression in bovine articular cartilage and implications for mechanotransductionARTHRITIS & RHEUMATISM, Issue 8 2009Walter Dunn Objective Articular cartilage is an avascular tissue with precise polarity and organization comprising 3 distinct functional zones: the surface, middle, and deep zones. Each zone has a different gene expression pattern that plays a specific role in articular cartilage development and maintenance. MicroRNA (miRNA) are small noncoding gene products that play an important regulatory role in determining cell differentiation and function. The purpose of this study was to test our hypothesis that miRNA expression profiles in the different articular cartilage zones as well as between regions subjected to different levels of weight-bearing stresses are unique. Methods Using an miRNA microarray approach in conjunction with quantitative reverse transcription,polymerase chain reaction, we identified miRNA in bovine articular cartilage that were differentially expressed in the different functional zones and in the anterior weight-bearing and posterior non,weight-bearing regions of the medial femoral condyle (M1 and M4, respectively). Results We identified miRNA-221 and miR-222 as part of a subset of differentially expressed miRNA that were up-regulated in articular cartilage in the anterior, M1, greater weight-bearing location. Additionally, miR-126, miR-145, and miR-335 were down-regulated in monolayers of tissue-cultured chondrocytes as compared with levels determined directly from intact native cartilage. Conclusion In conclusion, miR-222 expression patterns in articular cartilage are higher in the weight-bearing anterior medial condyle as compared with the posterior non,weight-bearing medial condyle. Thus, miR-222 might be a potential regulator of an articular cartilage mechanotransduction pathway. These data implicate miRNA in the maintenance of articular cartilage homeostasis and are therefore targets for articular cartilage tissue engineering and regenerative medicine. [source] Familial mediterranean fever with a single MEFV mutation: Where is the second hit?ARTHRITIS & RHEUMATISM, Issue 6 2009Matthew G. Booty Objective Familial Mediterranean fever (FMF) has traditionally been considered an autosomal-recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only 1 demonstrable MEFV mutation. The purpose of this study was to perform an extensive search for a second MEFV mutation in 46 patients diagnosed clinically as having FMF and carrying only 1 high-penetrance FMF mutation. Methods MEFV and other candidate genes were sequenced by standard capillary electrophoresis. In 10 patients, the entire 15-kb MEFV genomic region was resequenced using hybridization-based chip technology. MEFV gene expression levels were determined by quantitative reverse transcription,polymerase chain reaction. Pyrin protein levels were examined by Western blotting. Results A second MEFV mutation was not identified in any of the patients who were screened. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation; however, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance. Conclusion Our data underscore the existence of a significant subset of FMF patients who are carriers of only 1 MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of the most common mutations and detection of a single mutation appears to be sufficient in the presence of clinical symptoms for the diagnosis of FMF and the initiation of a trial of colchicine. [source] Indoleamine 2,3 dioxygenase,mediated tryptophan catabolism regulates accumulation of Th1/Th17 cells in the joint in collagen-induced arthritisARTHRITIS & RHEUMATISM, Issue 5 2009Gabriel Criado Objective Indoleamine 2,3 dioxygenase (IDO) is a catabolic enzyme that initiates the kynurenine pathway of tryptophan degradation and has immunomodulatory properties. The aim of this study was to investigate the regulation of collagen-induced arthritis by tryptophan catabolism mediated by IDO. Methods Arthritis was induced by immunization with type II collagen. After induction of arthritis, the expression of IDO was analyzed by quantitative reverse transcription,polymerase chain reaction. The effect of IDO deficiency on collagen-induced arthritis was assessed in vivo by administration of 1-methyltryptophan and clinical and histologic evaluation of IDO-deficient mice. The requirement for IDO activation was bypassed by administration of L -kynurenine. Results IDO was induced in lymph node dendritic cells after collagen immunization. Systemic inhibition of tryptophan catabolism during active arthritis increased disease severity. Conversely, bypassing the requirement for tryptophan degradation by the administration of L -kynurenine resulted in amelioration of arthritis. Furthermore, IDO-deficient mice showed a higher incidence of arthritis and exacerbated disease severity compared with IDO-competent mice. Such increased disease activity in IDO-deficient mice correlated early with increased production of the proinflammatory cytokines interferon-, and interleukin-17 by lymph node T cells and later with increased infiltration of Th1 and Th17 cells in the inflamed joints. Conclusion Our data indicate that the induction of IDO controls the accumulation of Th1 and Th17 pathogenic T cells at the site of inflammation during collagen-induced arthritis. Therefore, manipulation of the kynurenine pathway of tryptophan degradation provides the potential for therapeutic intervention in rheumatoid arthritis. [source] Abnormal basement membrane type IV collagen ,-chain composition in labial salivary glands in Sjögren's syndromeARTHRITIS & RHEUMATISM, Issue 4 2009P. Poduval Objective Sjögren's syndrome (SS) is characterized by atrophy and malfunction of the acinar cells. The aim of this study was to investigate whether type IV collagen ,-chain composition of acinar cell compartments could be abnormal in diseased glands. Methods Messenger RNA (mRNA) from human submandibular gland (HSG) cells, cultured with or without growth factor,depleted Matrigel, was analyzed using quantitative reverse transcription,polymerase chain reaction (RT-PCR). Labial salivary glands were analyzed using quantitative RT-PCR and immunohistochemistry. Results HSG cells of both the ductal and acinar phenotypes synthesized all ,-chain mRNA, in particular those of the ,1 and ,2 chains. Labial salivary glands (LSGs) contained ,1/2 chains but also contained mRNA of all the other ,-chains, although the mRNA copy numbers for the ,3 and ,4 chains were low, and the corresponding proteins were absent. Type IV collagen ,1/2-chains were observed in all tubuloalveolar basement membranes. In healthy glands, ,5 and ,6 chains were continuous around ducts but discontinuous around acini. In SS glands, these chains were absent or patchy around the ducts and absent around the acini. Conclusion Ductal and acinar epithelial cells are able to locally produce mRNA for all 6 different ,-chains. Type IV collagen ,1/2-chains seem to form the backbone in the tubuloalveolar basement membrane in salivary glands. Type IV collagen ,3 and ,4 chain mRNA were found in cultured salivary epithelial cells and LSG explants but were not translated to the corresponding ,-chains in LSGs. Both ,5 and ,6 mRNA were observed in salivary epithelial cells and glands. In healthy glands, immunolabeling always disclosed corresponding ,-chains around ducts, but their synthesis and/or degradation seemed to be locally regulated around acinar cells. [source] Expression of MicroRNA-146a in osteoarthritis cartilageARTHRITIS & RHEUMATISM, Issue 4 2009Keiichiro Yamasaki Objective A role of microRNA, which are ,22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). Methods The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription,polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1, (IL-1,) was examined by quantitative RT-PCR. Results All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0,5, grade II = moderate OA scored 6,10, and grade III = severe OA scored 11,14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1, stimulation in human chondrocytes in vitro. Conclusion This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1,. Thus, miR-146 might play a role in OA cartilage pathogenesis. [source] Interleukin-1, and tumor necrosis factor , inhibit chondrogenesis by human mesenchymal stem cells through NF-,B,dependent pathways,ARTHRITIS & RHEUMATISM, Issue 3 2009N. Wehling Objective The differentiation of mesenchymal stem cells (MSCs) into chondrocytes provides an attractive basis for the repair and regeneration of articular cartilage. Under clinical conditions, chondrogenesis will often need to occur in the presence of mediators of inflammation produced in response to injury or disease. The purpose of this study was to examine the effects of 2 important inflammatory cytokines, interleukin-1, (IL-1,) and tumor necrosis factor , (TNF,), on the chondrogenic behavior of human MSCs. Methods Aggregate cultures of MSCs recovered from the femoral intermedullary canal were used. Chondrogenesis was assessed by the expression of relevant transcripts by quantitative reverse transcription,polymerase chain reaction analysis and examination of aggregates by histologic and immunohistochemical analyses. The possible involvement of NF-,B in mediating the effects of IL-1, was examined by delivering a luciferase reporter construct and a dominant-negative inhibitor of NF-,B (suppressor-repressor form of I,B [srI,B]) with adenovirus vectors. Results Both IL-1, and TNF, inhibited chondrogenesis in a dose-dependent manner. This was associated with a marked activation of NF-,B. Delivery of srI,B abrogated the activation of NF-,B and rescued the chondrogenic response. Although expression of type X collagen followed this pattern, other markers of hypertrophic differentiation responded differently. Matrix metalloproteinase 13 was induced by IL-1, in a NF-,B,dependent manner. Alkaline phosphatase activity, in contrast, was inhibited by IL-1, regardless of srI,B delivery. Conclusion Cell-based repair of lesions in articular cartilage will be compromised in inflamed joints. Strategies for enabling repair under these conditions include the use of specific antagonists of individual pyrogens, such as IL-1, and TNF,, or the targeting of important intracellular mediators, such as NF-,B. [source] Involvement of the notch pathway in the regulation of matrix metalloproteinase 13 and the dedifferentiation of articular chondrocytes in murine cartilageARTHRITIS & RHEUMATISM, Issue 2 2009Régis Blaise Objective To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. Methods Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch -expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription,polymerase chain reaction, immunoblotting, and immunocytochemistry. Results Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. Conclusion The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch. [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] Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cellsARTHRITIS & RHEUMATISM, Issue 5 2008Michael B. Mueller Objective Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy-associated markers. Methods Human MSC pellet cultures were predifferentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor , (TGF,), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription,polymerase chain reaction. Results The combination of TGF, withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1,3, parathyroid hormone,related protein receptor, retinoic acid receptor ,, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed. Conclusion Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC-derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy-inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering. [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] Expression of the stem cell marker nestin in peripheral blood of patients with melanomaBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2010A. Fusi Summary Background, There is continued interest in markers indicative of circulating melanoma cells. Nestin is a neuroepithelial intermediate filament protein that was found to be expressed in melanoma and in various cancer stem cells. Objectives, We investigated expression of nestin in peripheral blood of patients with melanoma. Methods, We analysed nestin expression by flow cytometry and by quantitative reverse transcription,polymerase chain reaction both in tissues (n = 23) and in blood samples (n = 102) from patients with American Joint Committee on Cancer stage III,IV melanoma. Forty-six negative controls were also added. Results, Flow cytometry did not reveal nestin-expressing cells in peripheral blood of healthy volunteers. In patients with melanoma, however, nestin protein was expressed in a proportion of melanoma cells enriched from peripheral blood by immunomagnetic sorting. In melanoma tissue samples a significant correlation was found between mRNAs coding for nestin and tyrosinase (P = 0·001) and melan-A (P = 0·002), whereas in blood a significant correlation was observed only for tyrosinase (P = 0·015), but not for melan-A (P = 0·53). Nestin expression was higher in stage IV patients compared with stage III/IV with no evidence of disease, in patients with high tumour burden, and was positively correlated to expression of tyrosinase and melan-A. Conclusions, Nestin was found to be an additional marker of interest for circulating melanoma cells. Prospective studies should investigate its potential added informative value in comparison with markers already in use for melanoma cell detection. [source] Adalimumab therapy rapidly inhibits p38 mitogen-activated protein kinase activity in lesional psoriatic skin preceding clinical improvementBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2010L. Soegaard-Madsen Summary Background, The pathogenesis of psoriasis and the mechanisms of action of antitumour necrosis factor (TNF)-, therapies are incompletely understood. Objectives, To investigate the early molecular effects of adalimumab in psoriatic skin. Methods, Biopsies taken from patients with psoriasis were examined before and after the onset of adalimumab therapy. TNF-, protein level and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative reverse transcription,polymerase chain reaction, respectively. The activities of p38 mitogen-activated protein kinase (MAPK) and extracellular regulated kinase 1 and 2 (ERK1/2) as well as the downstream kinases MAPK-activated protein kinase 2 (MK2) and mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2) were measured by Western blot analyses. Results, We demonstrated that clinical and histological improvements were detected from day 14. The increased activity of p38 MAPK in lesional psoriatic skin was significantly inhibited by adalimumab already at day 4. The activities of ERK1/2, MSK1/2 and MK2 were reduced at the end of study (day 84) when the level of TNF-, in lesional psoriatic skin reached the nonlesional level, and the Psoriasis Area and Severity Index score was reduced. Conclusions, The rapid inhibition of p38 MAPK by adalimumab in lesional psoriatic skin preceded clinical and histological improvements, demonstrating an association between TNF-, neutralization and p38 MAPK inhibition. Thus, inhibition of p38 MAPK may be a novel mechanism by which adalimumab mediates its antipsoriatic effect. [source] In vitro model for penetration of sensory nerve fibres on a Matrigel basement membrane: implications for possible application to intractable pruritusBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2009M. Tominaga Summary Background, Epidermal hyperinnervation occurs in dermatoses with intractable pruritus, such as atopic dermatitis, suggesting that the hyperinnervation is partly responsible for abnormal itch perception. Objectives, To investigate the mechanisms of penetration of sensory nerve fibres into the basement membrane of the skin. Methods, A rat dorsal root ganglion neurone culture system consisting of Matrigel and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. In some experiments, matrix metalloproteinase (MMP) blockers and semaphorin 3A (Sema3A) were added to the culture system. Matrigel-coated membranes were stained with anti-Tau antibody, and the number of nerve fibres that crossed the membrane was counted. Expression of MMPs in the cultured neurones was examined at mRNA and protein levels by quantitative reverse transcription,polymerase chain reaction and immunocytochemistry, respectively. The activity was also examined by zymography. Results, Nerve fibres penetrated into Matrigel in the presence of an NGF concentration gradient, which was dose-dependently inhibited by GM6001, a broad-spectrum MMP inhibitor. Transcripts for MMP2, but not MMP9, were increased in the cultured neurones, and the penetration was dose-dependently inhibited by MMP-2 blockers. MMP-2 and its activity were partially localized on the NGF-responsive growth cones. NGF also upregulated pro-MMP-2 activation molecules in the cultured neurones. Sema3A stimulation showed the opposite effects on these NGF-dependent events. Interestingly, MMP2 expression was modulated by extracellular matrix (ECM) substrates for this enzyme. Conclusions, Membrane-associated MMP-2 contributes to penetration of nerve fibres into Matrigel through modulation by axonal guidance molecules and/or ECM. These findings provide insight for understanding the development of intractable pruritus involving epidermal nerve density. [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. 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