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Steffen Gay

Publications and source records attributed to Steffen Gay.

89 records · Page 5Linked to original sources
PubMed2003

Evaluation of differentially expressed genes by a combination of cDNA array and RAP-PCR using the AtlasImage 2.0 software.

Evaluation of genes regulated differentially is essential for the development of therapeutic approaches in multifactorial diseases. To characterize gene expression profiles in multifactorial inflammatory and malignant diseases such as rheumatoid arthritis (RA) or colon adenoma (CA), RNA arbitrarily primed PCR (RAP-PCR) combined with cDNA array hybridization were performed and evaluated using an array-specific software.RNA of synovial fibroblasts from patients with RA and osteoarthritis (OA), and laser microdissected normal and colon adenoma tissue was used. RAP-PCR reactions were hybridized to cDNA array membranes. Arrays were analyzed by phosphor imaging, and the AtlasImage 2.0 software with different normalization settings. The AtlasImage 2.0 software was a useful tool to evaluate differentially expressed genes. However, software settings were needed to be optimized for every experimental approach and should be used without changes for all experiments. To compare RA vs. OA synovial fibroblasts and normal vs. CA expression patterns, global normalization using the sum method is recommended.

Arthritis↗
PubMed2003

Apoptosis in rheumatoid arthritis.

Apoptosis is a key mechanism that regulates tissue composition and homeostasis. Alterations in the apoptosis of synovial cells have been described in residential synoviocytes as well as inflammatory cells and associated with the pathogenesis of rheumatoid arthritis. These changes constitute hallmarks of synovial cell activation and contribute to both chronic inflammation and hyperplasia. Rheumatoid arthritis synovial fibroblasts are affected most prominently, and their resistance to apoptosis has been linked closely to the progressive destruction of articular cartilage. Although a detailed understanding of mechanisms that prevent synovial fibroblasts from programmed cell death is lacking, several antiapoptotic molecules have been identified. Among them, downstream modulators of Fas-signaling, such as sentrin-1/small ubiquitin-like modifier (SUMO)-1 and Fas-associated death domain-like interleukin (IL)-1beta-converting enzyme-inhibitory protein (FLIP), as well as transcriptional regulators such as NFkappaB, Stat3, and p53, have been suggested to regulate apoptosis most prominently. Current efforts are aimed at elucidating the specific role of these molecules in regulating the apoptosis of rheumatoid fibroblasts and at identifying molecular targets to interfere with altered apoptosis.

Antirheumatic Agents↗
PubMed2003

Gene transfer as a future therapy for rheumatoid arthritis.

Inhibiting key pathogenic processes within the rheumatoid synovium is a most attractive goal to achieve, and the number of potential intra- and extracellular pathways operative in rheumatoid arthritis (RA) that could be used for a gene therapy strategy is increasing continuously. Gene transfer or gene therapy might also be one of the approaches to solve the problem of long-term expression of therapeutic genes, in order to replace the frequent application of recombinant proteins, in the future. However, at present, gene therapy has not reached a realistic clinical stage, which is mainly due to severe side effects in humans, the complexity of RA pathophysiology and the current state of available gene transfer techniques. On the other hand, novel gene delivery systems are not restricted to vectors or certain types of cells, as mobile cells including macrophages, dendritic cells, lymphocytes and multipotent stem cells can also be used as smart gene transfer vehicles. Moreover, the observation in animal models that application of viral vectors into a joint can exert additional therapeutic effects in nearby joints might also facilitate the transfer from animal to human gene therapy. Future strategies will also examine the potential of novel long-term expression vectors such as lentiviruses and cytomegalovirus (CMV)-based viruses as a basis for future clinical trials in RA.

Animals↗
PubMed2003

Detection of interleukin 1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha in skin of patients with fibromyalgia.

OBJECTIVE: To determine if abnormal collagen metabolism is correlated with neurogenic inflammation, a potential activator of collagen metabolism, in patients with fibromyalgia (FM). METHODS: The presence of inflammatory cytokines, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-a was investigated in skin tissues by using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Fifty-three skin biopsies from female patients with FM (30-65 years of age) were examined and compared to skin biopsies of 10 age and sex matched healthy controls. Biopsies were obtained from the left deltoid region. Rheumatoid arthritis synovial fibroblasts and tissues were used as positive controls for the expression of cytokines. Total RNA isolated from the tissue samples were reverse transcribed (RT) by random hexamers as the primer for RT followed by PCR amplification using specific primers for IL-1beta, IL-6 or TNF-a. Expression of IL-1beta, and TNF-a protein was investigated in the skin by immunohistochemistry using specific antibodies (avidin-biotin method). RESULTS: Positive signals (RT-PCR) were detected in skin tissues of 19/50 (38%) FM patients for IL-1beta, in 14/51 FM patients (27%) for IL-6, and in 17/53 patients (32%) for TNF-a. None of the cytokines could be detected in healthy control skin. Immunoreactivity for IL-1beta and TNF-a was demonstrated in certain skin tissues of our FM patients. CONCLUSION: The detection of cytokines in FM skin indicates the presence of inflammatory foci (neurogenic inflammation) in the skin of certain patients (about 30% of FM patients), suggesting an inflammatory component in the induction of pain. This may explain the response to nonsteroidal antiinflammatory therapy in a subset of FM patients.

Adult↗
PubMed2002

Anti-tumor necrosis factor-alpha treatment improves endothelial function in patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with accelerated atherosclerosis and increased cardiovascular morbidity and mortality. Striking similarities exist in the inflammatory and immunologic response in RA and atherosclerosis. Indeed, adhesion molecules and cytokines, tumor necrosis factor (TNF)-alpha in particular, are key mediators of joint inflammation and of vascular dysfunction and progression of atherosclerotic vascular disease. Hence, the aim of the present study was to assess the effect of chronic antiinflammatory treatment with the anti-TNF-alpha antibody infliximab on disease activity and endothelial function in patients with active RA. METHODS AND RESULTS: Eleven RA patients (mean age 46+/-5 years; disease duration 9+/-2 years) with high disease activity despite treatment with stable doses of methotrexate (<or=25 mg/wk) and prednisone (<or=10 mg/d) were investigated. Clinical status and endothelium-dependent and -independent vasodilation of the brachial artery as assessed by high-resolution ultrasound were measured before and after 12 weeks of infliximab therapy. Flow-mediated vasodilation improved from 3.2+/-0.4% to 4.1+/-0.5% (P=0.018), whereas endothelium-independent vasodilation with nitroglycerin and baseline diameter remained unchanged (13.6+/-1.2% versus 12.8+/-1.4%, P=0.98, and 3.74+/-0.15 versus 3.66+/-0.11 mm, P=0.54, respectively). Disease activity score (DAS28) was significantly reduced, from 5.6+/-0.3 to 3.5+/-0.6 (P=0.002). Erythrocyte sedimentation rate and C-reactive protein were lowered from 34+/-7 to 19+/-5 mm/h (P=0.04) and from 38+/-11 to 15+/-10 mg/L (P=0.08), respectively. CONCLUSIONS: This is the first study to show that anti-TNF-alpha treatment improves endothelial function in RA. The data suggest that in RA, endothelial dysfunction is part of the disease process and is mediated by TNF-alpha.

Antibodies, Monoclonal↗
PubMed2002

Expression of the apoptosis accelerator Bax in rheumatoid arthritis synovium.

OBJECTIVE: The aim of this study was to analyze the expression of apoptosis-related molecules in rheumatoid arthritis (RA) synovium, with special emphasis on the apoptosis accelerator Bax. METHODS: Immunohistochemical analysis of Bax, Bcl-2, and Bcl-x(L) was performed in tissue specimens of patients with RA and compared to normal synovial tissue. Expression of Bax was additionally determined by double labeling with CD68, p53, and Ki-67 (clone MIB-1). Apoptotic cells were further identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In RA, expression of Bax was higher than in healthy controls and occurred in CD68-positive and -negative synoviocytes. Strong Bax staining was also found in chondrocytes at sites of cartilage degradation. Bax-positive synoviocytes could be detected with p53 and also with Ki-67. Bax and Bcl-x(L) were markedly colocalized in synovium. The TUNEL method revealed only few positive synoviocytes. CONCLUSIONS: The marked colocalization of Bax and antiapoptotic Bcl-x(L) as well as the low frequency of TUNEL-positive cells in RA synovium suggest that Bax activity is not sufficient to decrease synovial hyperplasia in RA. Apoptotic mechanisms in RA chondrocytes might also be important for the pathogenesis of joint damage.

Adult↗
PubMed2002

Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers.

To examine whether the lack of sufficient neoangiogenesis in systemic sclerosis (SSc) is caused by a decrease in angiogenic factors and/or an increase in angiostatic factors, the potent proangiogenic molecules vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, and the angiostatic factor endostatin were determined in patients with SSc and in healthy controls. Forty-three patients with established SSc and nine patients with pre-SSc were included in the study. Serum levels of VEGF, basic fibroblast growth factor and endostatin were measured by ELISA. Age-matched and sex-matched healthy volunteers were used as controls. Highly significant differences were found in serum levels of VEGF between SSc patients and healthy controls, whereas no differences could be detected for endostatin and basic fibroblast growth factor. Significantly higher levels of VEGF were detected in patients with Scl-70 autoantibodies and in patients with diffuse SSc. Patients with pre-SSc and short disease duration showed significant higher levels of VEGF than healthy controls, indicating that elevated serum levels of VEGF are a feature of the earliest disease stages. Patients without fingertip ulcers were found to have higher levels of VEGF than patients with fingertip ulcers. Levels of endostatin were associated with the presence of giant capillaries in nailfold capillaroscopy, but not with any other clinical parameter. The results show that the concentration of VEGF is already increased in the serum of SSc patients at the earliest stages of the disease. VEGF appears to be protective against ischemic manifestations when concentrations of VEGF exceed a certain threshold level.

Adult↗
PubMed2002

Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation.

The aim of this study was to explore the molecular profile of proliferating rheumatoid arthritis synovial fibroblasts (RA-SF). Total RNA was extracted from two cultures of RA-SF (low-density [LD] proliferating cells and high-density [HD] nonproliferating cells) and suppression subtractive hybridization was performed to compare differential gene expression of these two cultures. Subtracted cDNA was subcloned, and nucleotide sequences were analyzed to identify each clone. Differential expression of distinct clones was confirmed by semiquantitative RT-PCR. The expression of certain genes in synovial tissues was examined by in situ hybridization. In both LD and HD cells, 44 clones were upregulated. Of the 88 total clones, 46 were identical to sequences that have previously been characterized. Twenty-nine clones were identical to cDNAs that have been identified, but with unknown functions so far, and 13 clones did not show any significant homology to sequences in GenBank (NCBI). Differential expression of distinct clones was confirmed by RT-PCR. In situ hybridization showed that certain genes, such as S100A4, NFAT5, unr and Fbx3, were also expressed predominantly in synovial tissues from patients with RA but not from normal individuals. The expression of distinct genes in proliferating RA-SF could also be found in RA synovium, suggesting that these molecules are involved in synovial activation in RA. Most importantly, the data indicate that the expression of certain genes in RA-SF depends on the stage of proliferation; therefore, the stage needs to be considered in any analysis of differential gene expression in SF.

Arthritis, Rheumatoid↗
PubMed2002

Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three-dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice.

OBJECTIVE: To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP-13]) and collagenase 1 (MMP-1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3-dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice. METHODS: Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell-cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts. RESULTS: Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3-dimensional collagen gels was associated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2-4-fold increase upon treatment with interleukin-1beta or tumor necrosis factor a. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3-dimensional collagen or by proinflammatory cytokines. CONCLUSION: The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3-dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell-matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.

Animals↗
PubMed2002

Local production of complement proteins in rheumatoid arthritis synovium.

OBJECTIVE: Complement has been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA) based on studies showing reduced levels of native complement components and increased levels of complement metabolites in plasma, synovial fluid (SF), and synovial tissue (ST) of RA patients. However, there is limited information on local production and activation of key factors of the complement cascade in RA synovium and their potential modulation by novel anticytokine therapies. This study was undertaken to characterize the expression of complement proteins and receptors in RA SF and ST. METHODS: Using in situ hybridization, immunohistochemistry, and Western blot techniques, we assessed the presence of complement proteins C3, factor B (FB), and C5b-9, as well as the expression of complement receptors C3aR and C5aR in rheumatoid synovium. C3 and FB levels in SF were determined by enzyme-linked immunosorbent assay. Functional assessment was performed by examining the effects of soluble tumor necrosis factor receptor (sTNFR) p55 gene transfer in the SCID mouse model of RA. RESULTS: Complement proteins and receptors could be localized in all RA synovial specimens, whereas in osteoarthritis (OA) synovium, only a few, single cells expressed complement proteins and receptors. No differences were noted in the concentration of C3 between RA and OA in SF; however, FB levels were markedly reduced in RA versus OA SF. In RA synovium, in contrast to OA synovium, local expression of complement factor and complement receptor messenger RNA was found throughout the various ST compartments, suggesting that activation of the complement cascade occurs in all parts of the rheumatoid synovium. Moreover, C5aR expression was up-regulated following overexpression of sTNFR p55 by adenovirus-based gene transfer. CONCLUSION: In summary, local complement production and activation may play an important role in RA, and specific modulation and inhibition of local complement production could be an attractive therapeutic target for RA.

Aged↗
PubMed2002

FLICE-inhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by a hyperplastic synovial tissue, inflammatory infiltrates, and a progressive destruction of cartilage and bone. FLICE-inhibitory protein (FLIP) prevents the association of caspase 8 with FADD and thus exerts an antiapoptotic effect through inhibition of Fas-mediated apoptosis. We undertook this study to examine the expression of FLIP in RA, osteoarthritic (OA), and normal synovial tissues. METHODS: We investigated the expression of FLIP (long form) in 5 RA, 2 OA, and 2 normal synovial tissue samples. A 393-bp fragment was amplified from complementary DNA obtained from cultured RA synovial fibroblasts (RASF) by reverse transcription-polymerase chain reaction (RT-PCR). Using in situ hybridization, the expression of FLIP messenger RNA (mRNA) in paraffin-embedded synovial tissue sections was investigated semiquantitatively by analyzing the lining layer, the sublining, and sites of invasion. Immunohistochemistry with anti-CD68 antibodies was performed on serial tissue sections to further characterize the cell types expressing FLIP. In addition, quantitative expression of FLIP was measured by real-time PCR. RESULTS: RT-PCR revealed the expression of FLIP mRNA in all RA and OA samples tested. Using in situ hybridization in synovial tissue, FLIP was detected in all 5 RA samples and in 1 of 2 OA samples, but in neither of the 2 normal control samples. In RA, FLIP expression could be found in both the lining and sublining layers; most importantly, it could also be identified at sites of cartilage invasion and bone destruction. Moreover, quantitative PCR analysis showed 50% higher FLIP expression in RASF than in OASF. CONCLUSION: The expression of antiapoptotic FLIP in RA synovial tissue and in synovial fibroblasts suggests the idea of a novel pathway in RA that potentially extends the lifespan of cartilage- and bone-degrading synovial cells, thus contributing to the progression of joint destruction.

Arthritis, Rheumatoid↗
PubMed2002

Role of nuclear factor kappaB in synovial inflammation.

The evaluation of molecular pathways has revealed novel insights into the pathophysiology of rheumatoid arthritis in the last several years. Gene transcription factors such as nuclear factor kB (NFkB) are activated by extracellular signals or cell-to-cell interactions that are converted into intracellular activation signals through receptor molecules located in the cell membrane. The number of known genes being translated after NFkB activation is increasing steadily. These genes includes cytokines, chemokines, growth factors, cellular ligands, and adhesion molecules. Because many of these genes are part of the pathogenesis of RA, there is considerable interest in the evaluation of the synovium-specific effects of NFkB to unveil its potential for future therapeutic strategies. The goal is to evolve these strategies from the therapies that have a wide spectrum of effects and side effects into rheumatoid arthritis-specific therapies designed to inhibit distinct molecular pathways within the synovium.

Arthritis, Rheumatoid↗
PubMed2002

Transcription factor Egr-1 activates collagen expression in immortalized fibroblasts or fibrosarcoma cells.

Synovial fibroblasts from rheumatoid arthritis patients express elevated levels of the transcription factor Egr-1. The metabolic consequences of Egr-1 overexpression in fibroblasts are not known in detail. Therefore we searched for gene products that are differentially expressed in Egr-1(high) versus Egr-1(low) fibroblasts. Immortalized synovial fibroblasts were transfected with two different Egr-1 expression vectors. Expression of recombinant Egr-1 was confirmed by RT-PCR and immunoblots. Random arbitrarily primed PCR revealed that Egr-1 induces enhanced transcription levels of the alpha1 chain of type I collagen. Increased expression of the alpha2 (I) chain could also be observed. We found enhanced levels of type I collagen propeptide in supernatants and stronger signals of alpha2 (I) protein in extracts of the Egr-1(high) expressing clone versus controls. Additionally, Egr-1 was transiently expressed in fibrosarcoma cells. These cells showed a pronounced elevation of type I collagen (alpha1) transcripts as well. Moreover, we could demonstrate that Egr-1 induces transcription of other genes including type II collagen (alpha1) and plateled-derived growth factor beta1. These data suggest that upregulation of Egr-1 might contribute tofibrosis observed in rheumatoid arthritis synovium by activation of genes encoding the alpha1 and alpha2 chains of type I collagen.

Cloning, Molecular↗
PubMed2001

Ex vivo gene transfer in the years to come.

Synovial fibroblasts (SFs) have become a major target for ex vivo gene transfer in rheumatoid arthritis (RA), but efficient transduction of RA-SFs still is a major problem. The low proliferation rate and heterogeneity of RA-SFs, together with their lack of highly specific surface receptors, have hampered a more extensive application of this technique. Improving transduction protocols with conventional viral vectors, therefore, as well as developing novel strategies, such as alternative target cells, and novel delivery systems constitute a major challenge. Recent progress in this field will lead to the achievement of high transgene expression, and will facilitate the use of gene transfer in human trials.

Arthritis, Rheumatoid↗
PubMedDate not supplied

The molecular control of angiogenesis.

Angiogenesis is a key event in a broad range of pathological conditions including both diseases with an enhanced and insufficient angiogenesis. Angiogenesis is often initiated with vasodilation accompanied by an increase in vascular permeability. After destabilization of the vessel wall and degradation of the surrounding extracellular matrix, extravasation of plasma proteins provides a provisional scaffold for the migration of endothelial cells. Endothelial cell proliferation and migration themselves are under tight control by a balance of angiogenesis inducers and inhibitors. A large number of angiogenic factors work together in a highly coordinated manner to induce endothelial cell outgrowth and the formation of functional vessels. On the other hand, angiostatic factors may play a critical role in the pathogenesis of ischemic diseases and contribute to the termination of physiological angiogenesis. Angiogenesis ends with the recruitment of pericytes and smooth muscle cells, which stabilize the newly formed vessel. The rapid increase in the knowledge about the molecular mechanisms of angiogenesis has led to first treatment trials in diseases with both enhanced and reduced angiogenesis. Although initial results are promising, much more work has to be done to consider anti-angiogenic or pro-angiogenic approaches as reliable therapeutic tools.

Angiostatins↗