PubMed Health⌕ Search

Biomedical subjects

S Gay

Publications and source records attributed to S Gay.

At least 37 records · Page 2Linked to original sources
PubMed2004

Galectin-3 is induced in rheumatoid arthritis synovial fibroblasts after adhesion to cartilage oligomeric matrix protein.

BACKGROUND: Galectin-3 is expressed in the synovial tissue of patients with rheumatoid arthritis (RA), particularly at sites of joint destruction. OBJECTIVE: To explore the possibilities that galectin-3 is induced either by proinflammatory cytokines or by adhesion to cartilage components. METHODS: Cell culture plates were coated with fibronectin, collagens I-VI, or cartilage oligomeric matrix protein (COMP), and the suspended cells were then added. The medium was changed after 1 hour at 37 degrees C. Adherent cells were further incubated for 18 hours in the presence or absence of tumour necrosis factor alpha (TNF alpha) or interleukin 1 beta. Cells were pretreated with murine IgG1, anti-CD29, -CD51, -CD61 (integrins), or -CD3 monoclonal antibodies and transferred to culture plates coated with COMP. Adherent cells were counted by light microscopy. The expression of intracellular galectin-3, or cell surface CD29, CD51, and CD61 was determined by flow cytometry before and after adhesion. RESULTS: Four times more RA synovial fibroblasts (SF) than osteoarthritis SF adhered to COMP. RA SF presented more cell surface integrins, and monoclonal antibodies against CD51 inhibited the adhesion to COMP by 80%. TNF alpha reduced the expression of CD61 and the adhesion to COMP, but did not reverse the adhesion once it had taken place. The adhesion of RA SF to COMP was found to increase the intracellular level of galectin-3. In contrast, intracellular galectin-3 decreased after exposure to TNF alpha. CONCLUSION: The increase of galectin-3 occurs after adhesion to COMP, and the alpha V beta 3 receptor (CD51/CD61) has a pivotal role in this process.

Arthritis, Rheumatoid↗
PubMed2004

Albumin-coupled methotrexate (MTX-HSA) is a new anti-arthritic drug which acts synergistically to MTX.

OBJECTIVE: To evaluate the anti-arthritic effects of the new inflammation-targeted drug MTX-HSA and to investigate whether peripheral blood mononuclear cells (PBMC) are potential target cells for albumin-mediated drug delivery. METHODS: The murine model of collagen-induced arthritis (CIA) was used to measure the anti-arthritic effect of MTX, MTX-HSA or a combination of both (n = 30 to 35 per group). In addition, the uptake of fluorescence-labelled albumin (AFLc-HSA) in PBMC of 14 patients with RA was measured by fluorescence-activated cell sorting (FACS). RESULTS: In equivalent doses of 7.5 mg/kg intravenously (IV) twice a week, MTX-HSA is significantly (P<0.02) superior to MTX in inhibiting the development of CIA and reducing the joint count as well as the number of affected paws. When given in lower doses as combination therapy, both drugs act synergistically (P<0.03). A mean of 96, 72 and 64% of the CD14-, CD16- and CD20-positive cells from peripheral blood of rheumatoid arthritis (RA) patients showed an uptake of albumin after incubation with AFLc-HSA in vitro. This finding was not significantly different in comparison to healthy controls. In contrast, the number of CD3-positive cells taking up albumin is increased significantly in RA patients in comparison to controls (26.3 +/- 12.9% s.d. vs 11.6 +/- 7.3% s.d.; P = 0.005). CONCLUSION: The data show that the effectiveness of MTX-HSA in CIA is superior to MTX and that both drugs act synergistically. In addition, albumin appears to be taken up by peripheral blood cells, suggesting that they might be one of the potential target cells of this novel anti-arthritic treatment approach.

Animals↗
PubMed2004

Role of specificity protein-1, PPARgamma, and pituitary protein transcription factor-1 in transcriptional regulation of the murine CORS-26 promoter.

The collagenous repeat-containing sequence of 26-kDa protein (CORS-26) was recently described as a new gene that is induced during adipocyte differentiation. Since the transcription factors specificity protein-1 (SP-1) and PPARgamma have been demonstrated to modulate transcriptional activation of adipocytic genes, we investigated the putative role of SP-1 and PPARgamma in the regulation of the murine CORS-26 promoter. Computer-based sequence analysis revealed two putative SP-1 binding sites and binding sites for PPARgamma and Pit-1 within the TATA-box containing promoter. Electrophoretic mobility shift assays (EMSA) with nuclear extracts from 3T3-L1 adipocytes and appropriate promoter fragments demonstrated that SP-1 binds specifically to both SP-1 binding sites. Specificity was demonstrated by (i) the appearance of supershift bands, (ii) competition experiments and, (iii) by using oligonucleotides carrying mutated SP-1 binding sites. Functional promoter activity was analyzed by Luciferase reporter gene assays and SP-1 was shown to exert inhibitory effects on the transcriptional activation of the murine CORS-26 gene. Additionally, specific binding activity of PPARgamma and Pit-1 to the CORS-26 promoter was demonstrated. Taken together, the present data demonstrate the functionality of the proximal murine CORS-26 promoter, which is regulated specifically by two SP-1 binding sites via SP-3-independent repressive effects of SP-1 on transcriptional activation. Pit-1 and PPARgamma can bind specifically to the promoter and might play an additive functional role in gene regulation of murine CORS-26.

3T3-L1 Cells↗
PubMed2004

In-phantom imaging of all dose components in boron neutron capture therapy by means of gel dosimeters.

The experimental method for in-phantom imaging and profiling the absorbed dose in neutron capture therapy has been improved. The method separates the contributions of the various secondary radiation components and is based on suitably designed gel dosimeters in the form of layers. The discrimination of the dose components is achieved by means of pixel-to-pixel manipulations of images obtained with gel dosimeters having different isotopic composition. Large dose images are obtainable with this method, because the layer geometry of dosimeters avoids sensible variation of neutron transport due to the isotopic composition of gel. Operation modalities aimed at attaining more reliable results have been studied. Some results, together with the results of punctual measurements performed with conventional dosimeters and with MC calculations, are here reported.

Boron Neutron Capture Therapy↗
PubMed2004

Targeting cathepsin L (CL) by specific ribozymes decreases CL protein synthesis and cartilage destruction in rheumatoid arthritis.

The present study was undertaken to examine whether ribozymes cleaving specifically cathepsin L (CL) mRNA are able to decrease the synthesis of CL protease in rheumatoid arthritis synovial fibroblasts (RA-SF) and thereby reduce the invasiveness into cartilage both in vitro and in the SCID mouse coimplantation model of RA. Two different ribozymes that cleave CL mRNA specifically at positions 533 (RzCL533) and 790 (RzCL790) were generated. Using retroviral gene transfer, RA-SF were transduced with the ribozyme constructs or the empty vector. To examine the effect of the ribozymes on the mRNA level, quantitative analysis for CL mRNA was performed using real-time PCR. For evaluation on the protein level, ELISA using specific anti-CL antibodies was performed. In addition, transduced RA-SF were examined in vitro in a three-dimensional destruction assay evaluating their ability to degrade extracellular matrix produced by human chondrocytes. Matrix destruction was monitored by the release of soluble glycosaminoglycans (sGAG). Using the in vivo SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF and control cells were coimplanted with human cartilage for 60 days. After being killed, invasion of RA-SF into the cartilage was evaluated by using a semiquantitative score. Transduction of RA-SF with RzCL533 and RzCL790 ribozymes decreased significantly the expression of CL mRNA to 44% (range 25-62%) and 20% (range 1-43%), respectively, when compared to mock-transduced cells. The protein concentration of CL in the cell culture supernatants of transduced RA-SF was decreased from 16.0 ng/ml in the mock constructs to 4.1 and 8.2 ng/ml (mean), respectively. Using the in vitro cartilage destruction assay, the release of sGAG decreased to 46 and 60%, respectively, after 14 days when compared to mock-transduced cells. In the SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF revealed a significant lower cartilage invasion when compared to mock and untransduced cells. Using retroviral gene transfer, ribozymes cleaving CL mRNA inhibit specifically the synthesis of this matrix-degrading enzyme and reduce cartilage destruction in in vitro and in vivo models. Our study therefore suggests that ribozymes targeting CL could be a novel and efficient tool to inhibit joint destruction in RA.

Animals↗
PubMed2004

Study of a method based on TLD detectors for in-phantom dosimetry in BNCT.

A method has been developed, based on thermoluminescent dosemeters (TLD), aimed at measuring the absorbed dose in tissue-equivalent phantoms exposed to thermal or epithermal neutrons, separating the contributions of various secondary radiation generated by neutrons. The proposed method takes advantage of the very low sensitivity of CaF2:Tm (TLD-300) to low energy neutrons and to the different responses to thermal neutrons of LiF:Mg,Ti dosemeters with different 6Li percentage (TLD-100, TLD-700, TLD-600). The comparison of the results with those obtained by means of gel dosemeters and activation foils has confirmed the reliability of the method. The experimental modalities allowing reliable results have been studied. The glow curves of TLD-300 after gamma or neutron irradiation have been compared; moreover, both internal irradiation effect and energy dependence have been investigated. For TLD-600, TLD-100 and TLD-700, the suitable fluence limits have been determined in order to avoid radiation damage and loss of linearity.

Body Burden↗
PubMed2004

Characterisation of the TAPIRO BNCT thermal facility.

Dosimetry and spectrometry measurements have been carried out in the thermal column of the research fast reactor RSV-TAPIRO (ENEA-Casaccia, Rome) in order to investigate its suitability for irradiation of cells or mice, with a view to research in the interests of boron neutron capture therapy (BNCT). The thermal column consists of a graphite moderator (40 cm thick) containing a lead shield (13 cm thick) in order to shield reactor background. The irradiation volume, inside this structure, has cubic shape (18 x 18 x 18 cm3). Besides measurements of fluence and dose rates in air or in phantom performed with thermoluminescence dosemeters (TLDs) and using the activation technique, dose and fluence profiles have been generated using a method based on gel dosemeters analysed with optical imaging. To check the consistency of the results, spectrometry measurements in the same irradiation volume have been performed by means of bubble detectors.

Animals↗
PubMed2004

Increased DNA fragmentation and ultrastructural changes in fibromyalgic muscle fibres.

OBJECTIVE: To determine whether there is evidence of increased DNA fragmentation and ultrastructural changes in muscle tissue of patients with fibromyalgia (FM) compared with healthy controls. METHODS: Muscle tissues from 10 community residents with FM and 10 age and sex matched healthy controls were examined "blindly" for the presence of DNA fragmentation by two different methods: terminal deoxynucleotidyl transferase (TdT) staining (TUNEL) and the FragEL-Klenow DNA fragmentation detection kit. Ultrastructural analysis of tissue was performed by electron microscopy. RESULTS: DNA fragmentation was detected by both methods in 55.4 (SEM 2.5)% of the nuclei in muscle tissue of patients with FM compared with 16.1 (4.1)% (p<0.001) of the nuclei in healthy controls. Contrary to expectation, no typical features of apoptosis could be detected by electron microscopy. The myofibres and actin filaments were disorganised and lipofuscin bodies were seen; glycogen and lipid accumulation were also found. The number of mitochondria was significantly lower in patients with FM than in controls and seemed to be morphologically altered. CONCLUSION: The ultrastructural changes described suggest that patients with FM are characterised by abnormalities in muscle tissue that include increased DNA fragmentation and changes in the number and size of mitochondria. These cellular changes are not signs of apoptosis. Persistent focal contractions in muscle may contribute to ultrastructural tissue abnormalities as well as to the induction and/or chronicity of nociceptive transmission from muscle to the central nervous system.

Adult↗
PubMed2004

Discrepancy between mRNA and protein expression of tumour suppressor maspin in synovial tissue may contribute to synovial hyperplasia in rheumatoid arthritis.

OBJECTIVE: To investigate the expression of maspin in RA synovial tissue and compare it with the expression in osteoarthritis (OA) and normal synovial tissue (NS). METHODS: Using specific primers for maspin, a 237 bp fragment was amplified from cDNA obtained from cultured RA, OA, and normal synovial fibroblasts (SF) by RT-PCR. Additionally, mRNA expression levels were determined quantitatively by real time PCR. mRNA expression of maspin was investigated on snap frozen and paraffin embedded synovial tissue sections by in situ hybridisation. Immunohistochemistry was used to identify the cell type expressing maspin. SDS-PAGE and western blotting were performed to evaluate the protein expression in cultured SF. To confirm protein synthesis in situ, immunohistochemistry with specific anti-maspin antibodies was performed in synovial tissue sections of patients with RA. RESULTS: RT-PCR showed expression of maspin in all cDNA samples from cultured SF. Maspin mRNA was found to be decreased in RA SF twofold and 70-fold compared with OA SF and NS SF, respectively. Maspin mRNA was expressed in RA, OA, and normal synovial tissue. Importantly, maspin transcripts were also found at sites of invasion into cartilage and bone. At the protein level, maspin could be detected in RA and, less prominently, OA SF. In RA synovial tissue, maspin protein was detected in only a few synovial lining cells. CONCLUSION: Maspin is expressed intensively in RA SF at the mRNA level, but only slightly at the protein level, possibly owing to down regulation of maspin; this may contribute to the hyperplasia of synovial tissue in RA.

Adult↗
PubMed2004

Methotrexate (MTX) and albumin coupled with MTX (MTX-HSA) suppress synovial fibroblast invasion and cartilage degradation in vivo.

OBJECTIVE: To investigate the effect of methotrexate (MTX) and albumin coupled with methotrexate (MTX-HSA) on cartilage invasion and induction of perichondrocytic degradation by rheumatoid arthritis synovial fibroblasts (RA SF) in vivo. METHODS: Human cartilage and RA SF were co-transplanted in three groups of severe combined immunodeficient mice (SCID), which received 1 mg/kg free MTX (n = 9), 1 mg/kg MTX-HSA (n = 6), or 0.9% NaCl (n = 5), respectively, intraperitoneally twice a week. After 4 weeks' treatment, the mice were killed and the implants analysed histologically. RESULTS: The control group had a mean (SEM) score for cartilage invasion of RA SF of 2.0 (0.26) and for perichondrocytic cartilage degradation of 1.5 (0.34). In contrast, mice which received MTX showed a significantly reduced invasion (0.78 (0.14), p<0.01) and a reduction in perichondrocytic cartilage degradation scores (0.69 (0.2), p<0.05) in comparison with the control group. Mice treated with MTX-HSA also had significantly reduced scores for RA SF invasion into the cartilage (0.92 (0.41), p<0.05) and for cartilage degradation (0.83 (0.44), p<0.05) compared with controls. The effects of MTX and MTX-HSA were not significantly different between these two groups. CONCLUSION: Treatment with MTX or MTX-HSA significantly ameliorates cartilage destruction in the SCID mouse model for human RA.

Albumins↗
PubMed2003

Genomic organization, promoter, amino acid sequence, chromosomal localization, and expression of the human gene for CORS-26 (collagenous repeat-containing sequence of 26-kDa protein).

The murine gene for CORS-26 is located on mouse chromosome 15A2 and its expression has been reported to be restricted to fibroblasts, cartilage and kidney. Here, the complete genomic organization of the corresponding human CORS-26 gene with exon/intron boundaries and exon-specific primer combinations is presented. Additionally, a 1.2 kb fragment of the TATA box-containing promoter region was cloned and analyzed for putative transcription factor binding sites. The deduced amino acid sequence is presented completely. Northern blot analysis using a human multiple-tissue cDNA panel demonstrated expression of human CORS-26 mRNA in colon and small intestine. Additionally, RT-PCR analysis revealed expression of CORS-26 mRNA in placenta, fibroblasts and white adipose tissue. The chromosomal localization of the human CORS-26 gene was mapped to human chromosome 5p by fluorescence in situ hybridization (FISH). In humans, chromosomal imbalances on chromosome 5p were reported to be involved in the pathogenesis of osteosarcoma. Therefore, a human bone tumor cDNA panel was investigated and a strong CORS-26 mRNA expression was found in osteosarcoma, chondroblastoma and giant cell tumor. The present data provide the basis for further investigation of CORS-26 gene regulation in the context of mesenchymal tissue development and in the pathogenesis of bone or skeletal disease.

Adipokines↗
PubMed2003

Genomic organization, chromosomal localization and adipocytic expression of the murine gene for CORS-26 (collagenous repeat-containing sequence of 26 kDa protein).

The murine gene for CORS-26 shows striking homologies to the adipocyte-specific secretory protein adiponectin (belonging to the newly discovered C1q/TNF molecular superfamily) and its expression has been reported to be restricted to fibroblasts, cartilage and kidney. However, the present data demonstrate specific induction of CORS-26 mRNA expression in hormonally differentiated 3T3-L1 adipocytes, but not in preadipocytes. Furthermore, CORS-26 mRNA expression could be demonstrated in human synovial adipocytes of the knee by in situ hybridization. Since the genes for CORS-26 and adiponectin are homologous for their COOH-terminal globular domain and of their N-terminal collagenous domain, they might have originated by divergence from an innate mesenchymal precursor molecule directing the development of myocytes, adipocytes and chondrocytes from a mesenchymal stem cell. Here, the complete genomic organization with exon/intron boundaries together with exon-specific primer combinations are presented. Additionally, approximately 1 kb of the TATA-box-containing promoter region was cloned and analyzed for putative transcription factor binding sites. The chromosomal localization of the murine CORS-26 gene was mapped to mouse chromosome 15 A2 by fluorescence in situ hybridization (FISH). Since the linkage loci for proteoglycan-induced arthritis and MRL/lpr arthritis in mice have been mapped to that chromosomal region, CORS-26 might represent the underlying mechanism of disease. The present data provide the basis for further investigation of the CORS-26 gene regulation in the context of mesenchymal tissue development, chondrocyte/adipocyte function and bone or skeletal disease.

3T3 Cells↗
PubMed2003

Expression of fibronectin splice variants and oncofetal glycosylated fibronectin in the synovial membranes of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: The aim of this study was to define and compare the expression of fibronectin (Fn) isoforms in synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Using monoclonal antibodies specific for total Fn, extra domain (ED)-A Fn, ED-B Fn, and oncofetal glycosylated Fn, we studied the expression of the Fn isoforms in synovium. Furthermore, in situ hybridization for the detection of ED-B Fn mRNA including a double labeling technique for the detection of cell type was applied. RESULTS: Strong expression of total Fn, ED-A Fn, oncofetal glycosylated Fn and, to a lesser extent, ED-B Fn could be demonstrated in the synovial lining layer in both RA and OA. Stromal and vessel expression of Fn isoforms was more prominent in RA tissue. Pannus tissue showed strong labeling with ED-B Fn. CONCLUSION: The expression of alternatively spliced isoforms of Fn is associated with tissue remodeling and, as a partial process of this phenomenon, with neovascularization rather than underlying disease, X-ray status, or parameters of acute inflammation. In the lining layer, Fn expression correlates with hyperplasia associated with cell recruitment but not with proliferative status. Most remarkably, the expression of ED-B Fn in pannus tissue seems to be associated with the invasive phenotype described in RA tissue.

Alternative Splicing↗
PubMed2003

Laser-mediated microdissection facilitates analysis of area-specific gene expression in rheumatoid synovium.

OBJECTIVE: Current approaches to analyzing gene expression in rheumatoid arthritis (RA) synovium are based on RNA isolated either from cultured synovial cells or from synovial biopsy specimens. This strategy does not, in general, allow distinction of specific gene expression between cells originating from different synovial areas, due to potential mixture of expression profiles. Therefore, we established the combination of laser-mediated microdissection (LMM) and differential display to analyze profiles of gene expression in histologically defined areas of rheumatoid synovium. The present study was undertaken to establish parameters for this technique and assess its usefulness for gene expression analysis. METHODS: Cryosections derived from RA synovial tissues were used to obtain cell samples from synovial lining versus sublining, using a microbeam laser microscope. RNA was isolated and analyzed by nested RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) for differential display fingerprinting. Differentially expressed bands were cut out, and PCR products were eluted, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry analysis. RESULTS: Microdissected sections of RA synovial tissue containing approximately 600 cells yielded enough RNA to produce a reproducible RNA fingerprint pattern. Several genes could be identified as being expressed differentially between the synovial lining and the sublining, and their expression could be confirmed at the messenger RNA and protein levels. CONCLUSION: The combination of LMM and RAP-PCR presents a valuable tool to obtain novel insights into the area-dependent differential regulation of gene expression in RA synovium. Both known and previously unknown genes were revealed with this technique. This study is the first to demonstrate the potential of this analytic strategy in the investigation of a nonmalignant, multifactorial, inflammatory disease.

Arthritis, Rheumatoid↗
PubMed2003

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.

Adenoviridae↗
PubMed2003

p53 in rheumatoid arthritis synovial fibroblasts at sites of invasion.

OBJECTIVE: To analyse the functional response of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 modulates the destructive process of RASF. METHODS: RASF and controls grown on chamber slides were either directly examined with DO7 anti-p53 antibodies by immunofluorescence or irradiated with 10 Gy x rays and analysed time dependently for the expression of p53. The percentage of positive cells was evaluated by a quantitative scoring system. RASF and normal (N) SF cultured in vitro were co-implanted with human cartilage in SCID mice for 60 days. Consecutively, the invasion score was evaluated, and the number of p53 positive cells was determined at the sites of invasion by immunohistochemistry. In addition, synovial tissues from RA, osteoarthritis, and normal synovia were stained with DO7 antibodies. RESULTS: In vitro the rate of expression of p53 in RASF was low (<5%), but transiently inducible by ionising irradiation (50%). In vitro low p53 expressing RASF disclosed, when invading articular cartilage, a nuclear p53 signal in 20% of the cells, indicating the induction of p53 in a distinct population of RASF during the invasive process. CONCLUSIONS: These data suggest an inductive p53 response at sites of cartilage invasion during the destructive process driven by activated RASF.

Animals↗