culture proliferation assays and gene expression studies

... 10 donors cultured on different days and assayed by real-time PCR on different days show similar results when cultured with TGF-β and SF In addition, culture of cells from two patients and subsequent ... collagen type I and TLH expression are upregulated; after SF stimulation, α-sma and TLH expression are upregulated In the healthy FLSs, collagen I expression rather than α-sma expression was induced ... co-localisation of collagen type IV and CD55 expression (Figure 3e,f) In addition, expression of collagen type IV mRNA was found in cultured FLSs from both RA patients and healthy controls (Figure 3i),...

Ngày tải lên: 09/08/2014, 08:23

... control and antigenstimulated sample is or greater Depletion and purity were assessed by flow cytometry and found consistently to exceed 95% and 70% in CD4+CD25+ (containing also Treg) depleted and ... of patients with early RA and control DR4+ individuals PBMCs from patients and control individuals were prepared as described in the Materials and methods section and cultured at × 106 cells/ml ... study, and analysis and interpretation of the data He drafted the manuscript and gave final approval of the version to be published RP, MDS, CN, GDS, AF and AB conducted the research MO and DA...

Ngày tải lên: 09/08/2014, 13:22

... manuscript, and the statistical analysis MB and RDZ coordinated and organized the primary sample collection and critically evaluated both the study design and the manuscript VBK conceived and designed ... examination of deep and superficial cartilage and synovial fluid content of IsoAsp Isomerized aspartate varied by cartilage location and degeneration status We investigated IsoAsp, GAG, and protein content ... cartilage histological degeneration Materials and methods Cartilage and synovial fluid collection Waste articular cartilage and synovial fluid were obtained from 14 cases of randomly selected total...

Ngày tải lên: 09/08/2014, 14:20

... 5'-CGT-CAC-CGG-GTG-TAA-ATC-AGCTTG-3' TA Cloning of PCR products and generation of standard curve To verify the PCR products and to generate standard curves of miRNAs, thymine adenine (TA) cloning was ... IL-1β and TRAF6, which is a key molecule in the down stream of TNFα and IL-1β signaling [41] The expression of miR-155 is upregulated in RA FLS and has repressive effect to MMP-3 and [18] The expression ... miRNA concentrations in plasma and those in synovial fluid A and B, Plasma and synovial fluid concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in RA (A) and OA (B) The average concentrations...

Ngày tải lên: 12/08/2014, 12:20

... 2Immunology and Allergy Division, Geneva University Hospitals and University of Geneva, Rue Gabrielle Perret-Gentil 4, Geneva, CH-1211, Switzerland 3Department of Diagnostic Sciences and Special ... MCP-1/CCL2 production and fibroblast proliferation of RA patients [44] and by the promotion of osteoclastogenesis in collagen induced arthritis [45] Alterations in CD45 and CD3 ST expressions are in ... and 545.8 ± 253.2, P = 0.007) and CD3 (402.8 ± 203.0 and 224.8 ± 107.7, P = 0.039) ST expression Figure SF total white blood cells and knee clinical indexes before and after intra-articular TNF-a...

Ngày tải lên: 12/08/2014, 14:22

... available reagents and assays were performed as per the manufacturer’s instructions (details in Table 1) The remaining biomarker assays utilized methods that have been previously described and summarized ... on a shaker Samples were read at 595 nm and the GAG level determined from a linear standard curve (12.5 to 400 μg/ml) Table Summary and sources of the assays used in this study Analyte Details ... 0.012), COMP (P = 0.0001) and MMP-3 (P = 0.0001) The biggest differences between synovial fluid and serum were observed for MMP-3 and COMP with fold differences of 738.8 and 21.2 respectively Smaller...

Ngày tải lên: 12/08/2014, 15:22

... cluster in samples 1, 3, 4, 7, and (diagonal arrows pointing upward and to the left) (2) ~185-kDa (denoted by small brackets facing downward): OA samples and (b,d), and and (f,g) (blots/exposures ... 1, 3, 4, 7, and 8, as well as RA samples 11–15 (denoted by diagonal arrows pointing upward and to the left in Figs and 5; Table 1) In of the OA samples (1 and in Fig 4, also samples and in blots ... been observed to trigger gene expression for stromelysin, inducible nitric oxide synthetase, hyaluronan receptor proteins, and other biologically active molecules in cultured chondrocytes [20,21,23–26]...

Ngày tải lên: 09/08/2014, 01:23

... (TNF) family and prevents apoptosis, increases survival, and stimulates cytokine production in dendritic cells [17,18] TRANCE and the ligand of RANK (RANKL) were originally cloned and sequenced ... streptavidinHRP conjugate and tetramethylbenzidine as a substrate for HRP Concentrations of RANK-L and OPG were obtained from a standard curve generated by known concentrations of human RANK-L and OPG The ... subjects expressed RANK-L and TRAF6, but not OPG and RANK (Figure 1a) PBMLs from healthy subjects expressed the four genes tested, and platelets expressed RANK-L, TRAF6, OPG, and not RANK (Figure...

Ngày tải lên: 09/08/2014, 10:20

... Tris-borate-EDTA gels (Labora AB, Sollentuna, Sweden) and the bands were revealed by silver staining The gels were scanned and the intensity of each band on the gel was analyzed with IPLab Gel software ... categories I and IV (P < 0.0001), and between the patients in categories II and III (P = 0.0036) Levels of 8-oxodG in the SF The mean level of 8-oxodG in the RA–SF samples was 3.2 ng/ml, and 41 of ... and 8-oxodG in the SF of patients with RA and the appearance of other laboratory parameters, such as RF, erosivity, and extra-articular manifestations The presence of both mtDNA (P = 0.043) and...

Ngày tải lên: 09/08/2014, 01:23

... chondrocyte hypertrophy and proliferation [10,11] Page of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol No Chubinskaya et al OP-1 gene expression and protein expression have ... diagnosed OA (n = 29) and RA (n = 25) enrolled in this study were of both sexes (13 males and 16 females for the OA group, and seven males and 18 females for the RA group) and represented similar ... donors, of OA patients, and of RA patients, and report statistical differences between the SF concentrations of this growth factor in OA patients and RA patients Patients and methods Subjects Synovial...

Ngày tải lên: 09/08/2014, 08:22

... have any effect on the expression of functional MMPs MAPK gene expression in synovial fibroblasts from patients with RA and OA Members of the MAPK gene family (such as ERK1/2 and p38MAPK) are involved ... induce multiple MMP expression in human dermal and synovial fibroblasts and that the response is similar to that induced by IL-1β/TNF-α The expression pattern of MAPK gene expression also indicates ... hours Cells were harvested and total cellular RNA was isolated and reverse-transcribed The MAPK family gene expression was analyzed using the Human MAPK Gene Family I Multigene-12 reverse transcription-polymerase...

Ngày tải lên: 09/08/2014, 08:23

... osteoclast gene expression, the mRNA expression of the genes encoding ctr, trap, and cathepsin K was also analysed with quantitative realtime PCR by means of seven SFs (10%) from patients with OA and ... dye VIC at the 5' end and quencher fluorescent dye TAMRA at the 3' end), and TaqMan Universal PCR Master Mix from Applied Biosystems; and the 1st Strand cDNA Synthesis Kit and PCR Core Kit from ... experiments Animal care and experiments were approved and conducted in accordance with accepted standards of humane animal care and use as deemed appropriate by the Animal Care and Use Committee of...

Ngày tải lên: 09/08/2014, 10:20

... and quantitative RT-PCR Changes in gene expression assessed by microarray analysis were confirmed by semiquantitative and quantitative RT-PCR for selected genes, namely CXCL10, CCL18, CCRL2 and ... the expression of CC chemokines (a) and CXC, C and CX3C chemokines (b) Data obtained by Gene Pix™ Analysis Software were normalized, transformed and denoted as x-fold regulation versus the expression ... A, Vaslin B, Le Grand R, Brew BJ, Dormont D: Expression of chemokines and their receptors in human and simian astrocytes: evidence for a central role of TNFα and IFNγ in CXCR4 and CCR5 modulation...

Ngày tải lên: 09/08/2014, 10:21

... aminovorans, B niacini, Diaphorobacter species, and uncultured candidate division OP10 bacterium Several studies have also detected unsuspected uncultured and/ or cultured bacteria not considered as human ... × uncultured Sphingobacterium species, × uncultured Flavobacterium species, × uncultured β proteobacterium, × uncultured Firmicutes bacterium 15 24 10 × Stenotrophomonas maltophilia, × uncultured ... × uncultured β proteobacterium, × uncultured Flavobacterium species, × uncultured Sphingobacterium species, × Comamonas testosterone, × uncultured bacterium 17 × Escherichia species, × uncultured...

Ngày tải lên: 09/08/2014, 14:22

... structural gene displacement, allowing generation of His+MutS phenotype [47] PCR-positive His+Mut+ and His+MutS clones were cultured in buffered glycerol-complex medium, and for expression induction, cultures ... curves and data were normalized to the copy number of 18S RNA Expression of ACMSD isoforms in E coli pGEM-ACMSD I and pGEM-ACMSD II were digested with XhoI and Bpu1102I and cloned into pET-15b and ... it is present only in kidney, brain and liver Several studies have demonstrated that in rats, nutritional factors and hormones affect both gene expression and enzymatic activity In particular,...

Ngày tải lên: 19/02/2014, 02:20

... ligand affinity between Y7 and Y2 may prove very useful for studies of ligand–receptor interactions and 3D modeling, and we have previously been able to utilize differences between chicken and ... Experimental procedures Isolation and sequencing of the chicken Y6 gene and cloning into an expression vector Degenerate PCR primers, based on several mammalian and the nontetrapod Y1 subfamily, ... radioligand This radioligand had iodinated tyrosines at positions 21 and 27 and a specific activity of 4000 CiÆmmol)1 Saturation experiments were carried out with serial dilutions of radioligand, and...

Ngày tải lên: 19/02/2014, 07:20

... (A) and mouse (B) PNPO gene Exons are designated by closed boxes, and introns by bold lines The ORF is marked black, and grey boxes denote the 5¢- and 3¢-UTR sequences The locations of CpG islands ... PNPO gene contains CpG islands with a CGobs/CGexp ratio in excess of 0.6 and a G + C content of 62% spanning two regions from )377 to )158 and from )137 to +136 of the start codon Such a CpG island ... gel, and the product band was extracted from the gel, purified, and ligated into the pGEM vector Then a BamHI digested fragment was subcloned into pET28a expression vector (pET28a/PNPOx) and used...

Ngày tải lên: 23/03/2014, 12:20

... 11 cultures were shown to be viable and uncontaminated To assure viability and sterility, a vial was recovered from the freezer and passaged times and then tested for mycoplasmal, bacterial and ... Authors' contributions MTC and HB conceived of the study and MTC and RD devised the experimental design for the scratch assays HB harvested the wound tissue in the OR and HE helped in logging de-identified ... logging de-identified clinical data and delivering the specimens to MTC MTC supervised OS and SV to carry out the culture the cells in-vitro and perform the scratch assays A portion of the biopsies...

Ngày tải lên: 18/06/2014, 15:20

... assay included a standard curve derived from positive and negative controls Negative controls consisted of 2% BSA and human serum, free of sHLA-I and sHLA-II Positive control standards were prepared ... sHLA-I standards (100, 50, 25, 12.5, 6.25, 3.1 and ng/ml) of pure sHLA-I protein The test values were calculated from the curve described by these standards All sHLA-I assays were standardized ... interpreting the neuro-radiology studies and generating neuro-radiology data 16 17 18 19 20 Dr Featherston has contributed to this manuscript by doing statistical analysis and generating the figures 21...

Ngày tải lên: 19/06/2014, 22:20

... understand these apparent differences Figure Expression of TLR by HFF and Ectocervical tissue Expression of TLR by HFF and Ectocervical tissue Expression of TLR2, TLR3, TLR4 and TLR9 in cells and ... sodium pyruvate, and 10% FBS TLR ligands were added to wells and cultured for 24 hours Culture supernatants were removed and assayed for cytokines Tissue pieces were washed and infected with ... of the cultures experiments and participated in writing of the manuscript NL obtained and processed cervical tissue and provided direction to the studies MRZ performed the TLR expression studies...

Ngày tải lên: 20/06/2014, 01:20