Winfried Graninger - Academia.edu (original) (raw)

Papers by Winfried Graninger

Research paper thumbnail of Effects of ciprofloxacin and ofloxacin on adult human cartilage in vitro

Antimicrobial Agents and Chemotherapy, 1997

Chondrocyte toxicity and necrosis were seen with electron microscopy after incubation of human ad... more Chondrocyte toxicity and necrosis were seen with electron microscopy after incubation of human adult cartilage biopsy specimens in ciprofloxacin or ofloxacin. In vitro exposure of chondrocytes to fluoroquinolones did not affect apoptosis as determined by flow cytometry. While the immediate clinical significance of this finding remains unclear, the possibility of long-term cartilage damage after fluoroquinolone treatment cannot be excluded.

Research paper thumbnail of Enhanced transendothelial in vitro migration of scleroderma lymphocytes

Arthritis Research & Therapy, 2001

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with define... more We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2 The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients

Research paper thumbnail of Impaired T-cell rsponse to subsequent TCR-stimulation after anti-CD3 induced proliferation

Arthritis Research & Therapy, 2001

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with define... more We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2 The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients

Research paper thumbnail of 212 Increase in the Gene Expression of MMP 13 and BMP 2 in Human Arthrotic Cartilage Cells After the Treatment with 5 Aza Deoxy Cytidine

Osteoarthritis and Cartilage, 2007

while up-regulating all other genes, suggesting a possible protective effect. Caspase-3, a key si... more while up-regulating all other genes, suggesting a possible protective effect. Caspase-3, a key signaling molecule in apoptosis, was significantly up-regulated (12x FS) under Co alone. While previous studies showed increased apoptosis due to mechanical injury, we have now shown a direct link between chondrocyte apoptosis in normal cartilage due to co-culture with excised joint capsule.

Research paper thumbnail of 227 Decrease of Collagen Type II Expression in Human Cartilage Cells After the Treatment with 5 Aza Deoxy Cytidine

Osteoarthritis and Cartilage, 2007

Purpose: TGF-beta is known to play a pivotal role in the reparative process of osteoarthritic car... more Purpose: TGF-beta is known to play a pivotal role in the reparative process of osteoarthritic cartilage. We aimed to test if the wide use of "TGF-beta" to refer to the three isoforms TGF-beta1, TGF-beta2 and TGF-beta3, without distinction between them, can be assumed without risk in OA research. As a first step, we have explored if they are similarly expressed in human OA cartilage. Methods: A total of 24 patients that were undergoing total hip replacement were recruited. 12 of them had hip OA and 12 had hip fracture but without clinical, radiologic or macroscopic OA. Gene expression of the 3 TGF-beta isoforms, collagen type II and aggrecan was analyzed by RT-qPCR using RNA obtained from fresh femoral head cartilage. Immunohistochemistry was used to study the expression and distribution of TGF-beta isoforms in different stages of OA: normal, slightly and strongly altered hip cartilage. Results: Expression of the three TGF-beta isoforms was significantly increased in OA cartilage. The increase was more marked for the TGF-beta3 isoform than for TGF-beta1 and TGF-beta2. TGF-beta3 levels were independent from the levels of the other two isoforms suggesting differential regulation. The mRNA levels of TGF-beta1 and TGF-beta2 were strongly correlated in OA cartilage (Spearman's r=0.83, p=0.002) but not in control cartilage. Levels of TGF-beta2 were lower than those of TGF-beta1 and TGF-beta3. Immunohistochemistry was in agreement with the qPCR results and showed that the three isoforms were overexpressed in the superficial layer of OA cartilage and near the lesions. Conclusions: Assuming that TGF-beta isoforms act indistinctly in OA is unwarranted. TGF-beta3 showed differential regulation in relation with TGF-beta1 and TGF-beta2. This might be important for OA given recent evidence suggesting that TGF-beta isoforms may have differing biological activities 227

Research paper thumbnail of Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes

Journal of orthopaedic research : official publication of the Orthopaedic Research Society, Jan 21, 2015

Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is al... more Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Express...

Research paper thumbnail of T lymphocytes of patients with SLE lack Stat4

Arthritis Research & Therapy, 2001

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with define... more We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2 The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients

Research paper thumbnail of Treating rheumatoid arthritis to target: 2014 update of the recommendations of an international task force

Annals of the rheumatic diseases, Jan 12, 2015

Reaching the therapeutic target of remission or low-disease activity has improved outcomes in pat... more Reaching the therapeutic target of remission or low-disease activity has improved outcomes in patients with rheumatoid arthritis (RA) significantly. The treat-to-target recommendations, formulated in 2010, have provided a basis for implementation of a strategic approach towards this therapeutic goal in routine clinical practice, but these recommendations need to be re-evaluated for appropriateness and practicability in the light of new insights. To update the 2010 treat-to-target recommendations based on systematic literature reviews (SLR) and expert opinion. A task force of rheumatologists, patients and a nurse specialist assessed the SLR results and evaluated the individual items of the 2010 recommendations accordingly, reformulating many of the items. These were subsequently discussed, amended and voted upon by >40 experts, including 5 patients, from various regions of the world. Levels of evidence, strengths of recommendations and levels of agreement were derived. The update ...

Research paper thumbnail of Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary

Arthritis research & therapy, 2006

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissu... more The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [35S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1beta, were analyzed by RT-PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in...

Research paper thumbnail of Long term treatment of psoriatic arthritis with infliximab

Annals of the rheumatic diseases, 2004

Research paper thumbnail of Transcriptional overexpression of the proto-oncogene bcl-2 in patients with systemic Lupus erythematosus

Wiener klinische Wochenschrift, 1992

In systemic Lupus erythematosus (SLE) spontaneous hyperreactivity of the cells of the immune syst... more In systemic Lupus erythematosus (SLE) spontaneous hyperreactivity of the cells of the immune system leads to the production of pathogenic autoantibodies. A hyperproliferative state of lymphocytes is indicated by the increased expression of nuclear oncogenes. We investigated the expression of a putative proto-oncogene, bcl-2, which is responsible for prolonged survival of lymphocytes and protection from programmed cell death. In 19 of 24 patients with SLE an increased concentration of bcl-2 mRNA was found in unstimulated circulating blood lymphocytes. The overexpression of the bcl-2 gene was more pronounced in patients with active SLE. A pathogenic role of increased bcl-2 expression and prolonged survival of autoimmune memory cells in SLE can be hypothesized.

Research paper thumbnail of Ultrasound composite scores for the assessment of inflammatory and structural pathologies in Psoriatic Arthritis (PsASon-Score)

Arthritis Research & Therapy, 2014

Introduction: This study was performed to develop ultrasound composite scores for the assessment ... more Introduction: This study was performed to develop ultrasound composite scores for the assessment of inflammatory and structural lesions in Psoriatic Arthritis (PsA). Methods: We performed a prospective study on 83 PsA patients undergoing two study visits scheduled 6 months apart. B-mode and Power Doppler (PD) findings were semi-quantitatively scored at 68 joints (evaluating synovia, perisynovial tissue, tendons and bone) and 14 entheses. We constructed bilateral and unilateral (focusing the dominant site) ultrasound composite scores selecting relevant sites by a hierarchical approach. We tested convergent construct validity, reliability and feasibility of inflammatory and structural elements of the scores as well as sensitivity to change for inflammatory items. Results: The bilateral score (termed PsASon22) included 22 joints (6 metacarpophalangeal joints (MCPs), 4 proximal interphalangeal joints (PIPs) of hands (H-PIPs), 2 metatarsophalangeal joints (MTPs), 4 distal interphalangeal joints (DIPs) of hands (H-DIPs), 2 DIPs of feet (F-DIPs), 4 large joints) and 4 entheses (bilateral assessment of lateral epicondyle and distal patellar tendon). The unilateral score (PsASon13) compromised 13 joints (2 MCPs, 3 H-PIPs, 1 PIP of feet (F-PIP), 2 MTPs, 1 H-DIP and 2 F-DIPs and 2 large joints) and 2 entheses (unilateral lateral epicondyle and distal patellar tendon). Both composite scores revealed a moderate to high sensitivity (bilateral composite score 43% to 100%, unilateral 36% to 100%) to detect inflammatory and structural lesions compared to the 68-joint/ 14-entheses score. The inflammatory and structural components of the composite scores correlated weakly with clinical markers of disease activity (corr coeffs 0 to 0.40) and the health assessment questionnaire (HAQ, corr coeffs 0 to 0.39), respectively. Patients with active disease achieving remission at follow-up yielded greater reductions of ultrasound inflammatory scores than those with stable clinical activity (Cohen's d effect size ranging from 0 to 0.79). Inter-rater reliability of bi-and unilateral composite scores was moderate to good with ICCs ranging from 0.42 to 0.96 and from 0.36 to 0.71, respectively for inflammatory and structural sub-scores. The PsASon22 and PsASon13 required 16 to 26 and 9 to 13 minutes, respectively to be completed. Conclusion: Both new PsA ultrasound composite scores (PsASon22 and PsASon13) revealed sufficient convergent construct validity, sensitivity to change, reliability and feasibility.

Research paper thumbnail of Clinical and immunological effects of anti-TNF therapy in systemic lupus erythematosus (SLE)

Arthritis Research & Therapy - ARTHRITIS RES THER, 2003

Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis ... more Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis of rheumatoid arthritis (RA). The only accepted serologic parameter (rheumatoid factor [RF]) is not disease specific, nor are any of several novel RA autoantibodies. We aimed at identifying profiles instead of individual autoreactivities allowing for unambiguous prediction of RA. Selected RA autoantigens were tested by ELISA (RF and anti-cyclic citrullinated peptide [anti-CCP]) or Western blot (heavy-chain-binding protein [BiP], heterogeneous ribonucleoprotein particle A2 [RA33/ hnRNP A2], calpastatin and calreticulin). Antibody reactivities were assayed from serum samples of 149 RA patients and 132 patients with other rheumatic diseases and from synovial fluids (SF) (58 RA, 65 non-RA). No single autoreactivity was sufficient for unambiguous prediction of RA. Frequencies of multiparameter profiles consisting of 3, 4, 5 and 6 autoreactivites were determined. Fifteen six-parameter serum profiles were exclusively expressed in RA patients, representing a cumulative sensitivity of 59%. Twelve SF profiles were exclusively expressed in 64% of RA patients. The self-learning classification algorithm CLASSIF1 was capable of accurately predicting RA when these profiles were present. Data profile analysis of RF/CCP/BiP/calpastatin/calreticulin/RA33 provided specific discrimination of 64% of RA. Most importantly, RA specific profiles were observed in 64% of patients with early disease (<12 months). For the first time, the accurate prediction of the class RA has been achieved by the use of multiparametric autoreactivity profiles. Because of early expression in disease, these profiles make it possible to start a disease-modifying therapy long before irreversible bone and joint destruction may develop. Additional RA-specific profiles are required to cover the entire group of RA patients. 2 Investigation of the reactivity patterns of antifilaggrin antibodies in sera and synovial fluids from patients with rheumatoid arthritis using citrullinated synthetic peptides

Research paper thumbnail of Poster Presentations (PP01-PP67)

Rheumatology, 2012

Background: Prophylaxis of tuberculosis in patients treated with anti-tumor necrosis factor (Anti... more Background: Prophylaxis of tuberculosis in patients treated with anti-tumor necrosis factor (Anti-TNF) prevents the latent tuberculosis infections (LTBI). But incidence rate of tuberculosis with anti-TNF therapy in tuberculosis-endemic area is higher than in other areas despite strict prophylaxis. We analyzed incidences and characteristics of patients with reactivation of LTBI and new tuberculosis infection. Method: We identified 549 patients treated with anti-TNF in tertiary care center from June 2003 through June 2010. The number of patients with ankylosing spondylitis, rheumatoid arthritis, psoariatic arthritis, spondyloarthropathy and behcet's disease were 317, 215, 11, 3 and 3 respectively. Their medical records including history of tuberculosis infection were reviewed. Result: Among 549 patients, 526 patients took tuberculosis screening test and 266 patients had LTBI prophylaxis. Nevertheless, we observed 11 cases of tuberculosis (2.0%) and the incidence rate of tuberculosis was 968 per 100,000 person-years. Patients infected by tuberculosis were 6 non-LTBI, 4 LTBI received prophylaxis and 1 without screening test. The mean duration of anti-TNF therapy was 21.73 months and patients of extrapulmonary tuberculosis were 9. Among 11 tuberculosis patients, 3 non-LTBI patients and 2 LTBI patients who received prophylaxis were infected by tuberculosis after anti-TNF therapy of 12 months and their mean duration of anti-TNF therapy was 40.8 months. In addition, three non-LTBI patients treated with anti-TNF therapy for less than 12 months were infected by new tuberculosis or reactivated LTBI with false-negative screening test. Conclusion: The incidence of tuberculosis in patients treated with anti-TNF was higher than other previous reports. This result show that the reactivation of LTBI and development of new tuberculosis infection as a long period of anti-TNF therapy for the endemic area. Screening and prophylaxis of LTBI are important but we also have to be concerned about new tuberculosis infection during anti-TNF therapy.

Research paper thumbnail of 239 SPHINGOSINE-1-PHOSPHATE INHIBITSII-1 and TNF-Α Induced Inos Expression via S1P1 and S1P2 in Human Articular Chondrocytes

Osteoarthritis and Cartilage, 2008

We have prior identified the bioactive Sphingolipid Sphingosine-1-Phosphate (S1P) as a potent inh... more We have prior identified the bioactive Sphingolipid Sphingosine-1-Phosphate (S1P) as a potent inhibitor of Interleukin-1 (Il-1) induced cartilage degradation in bovine cartilage. As micro-molar concentrations of S1P have been found in the synovial fluid of osteoarthritis and rheumatoid arthritis joints we investigated the role of S1P and its receptors in human cartilage. Methods: Human cartilage specimens were obtained from patients undergoing total knee joint replacement. Specimens were formalin fixed and paraffin embedded and S1P receptor iso-types S1P 1 , S1P 2 and S1P 3 were detected by immunohistochemistry. For cell culture human Chondrocytes were isolated using collagenase B. Cells grown in monolayer were cultured in Ham's F-12/DMEM (1:1) and 10% FCS over 3 passages. Cultured chondrocytes were serum starved for 24 hours and incubated with 10 ng/ml Il-1b or 100 ng/ml TNF-a alone or in combination with 0.1 mM up to 3 mM S1P. Expression of iNOS, ADAMTS-4 and MMP-13, was evaluated using real-time PCR. Results: All three investigated S1P-receptor iso-types were detected in human cartilage; however S1P 2 was the most prominent subtype. Interestingly, the expression of the receptors was confined to zones of cartilage damage and proliferating chondrocytes. Furthermore, expression of iNOS mRNA induced by Il-1b and TNF-a was dose dependently reduced by S1P by 47.0% and 56.2% respectively (P < 0.05). Cytokine induced ADAMTS-4 mRNA was diminished in the presence of S1P by 35.6% and 41.8% respectively (P < 0.05). In contrast to our previous observations in bovine cartilage MMP-13 mRNA expression was not affected by coincubation with S1P in human chondrocytes. Using specific inhibitors for S1P 1 (pertussis toxin) and S1P 2 (suramin) we found that inhibition of S1P 1 but not S1P 3 partly reversed iNOS and ADAMTS-4 inhibition. Conclusions: Our results suggest that S1P reduces Il-1 and TNF-a induced mRNA transcription of ADAMTS-4 and iNOS in human articular chondrocytes via S1P 1 and S1P 2. Therefore expression of these S1P receptor subtypes in damaged areas of human cartilage can be interpreted as a counter regulation of chondrocytes to inhibit further tissue degradation by Il-1b and TNF-a.

Research paper thumbnail of 234 Cartilage Calcification and BMP Signaling in Early Hand Osteoarthritis

Osteoarthritis and Cartilage, 2008

In contrast, cytomegalovirus promoter-driven Sp1 overexpression further enhanced Il-1-induced ADA... more In contrast, cytomegalovirus promoter-driven Sp1 overexpression further enhanced Il-1-induced ADAMTS-4 expression. Expression of constitutive ADAMTS-5 was not affected by any of the agents. Conclusions: These results provide pharmacological and genetic evidence for the importance of Sp1 in ADAMTS-4 gene regulation by Il-1.

Research paper thumbnail of 268 LAMA4 is Expressed by Clusters of Hypertrophic Chondrocytes in Osteoarthritic Cartilage and Regulated by Epigenetic Mechanims

Osteoarthritis and Cartilage, 2010

Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage ... more Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage whose expression is strongly decreased during osteoarthritis (OA) process. Specific proteins (Sp) and Sox proteins have been previously characterized to be major regulators of this gene. Besides, the existence of a link between estrogen deprivation and osteoarthritis in postmenopausal women suggests that 17β-estradiol (17β-E 2) may be a critical modulator of cartilaginous matrix homeostasis. The aim of present study was to investigate the molecular mechanisms regulating type II collagen gene expression under the effect of 17β-E 2 , and to characterize the genomic pathway via estrogen receptors (ER). Methods: Articular chondrocytes were isolated from 3-week old rabbits and incubated for 24 hours with increasing concentrations of 17β-E 2 (0 to 10 nM). Nuclear proteins were extracted and submitted to gel retardation assays in order to determine Sp and Sox proteins binding activities on COL2A1 gene. In some experiments, relative expression of Sp1/Sp3 or Sox-9 was inhibited by siRNAs strategies. Moreover, chromatin immunoprecipitation was used to study protein-protein interactions on COL2A1 promoter and enhancer sequences. Results: We have previously shown that 17β-E 2 could stimulate type II collagen neosynthesis, protein level and corresponding steady-state levels of COL2A1 mRNAs in primary and in vitro dedifferentiated rabbit articular chondrocytes. 17β-E 2 action was mediated, in part, by an ERα/Sp transactivating effect on the-266/-63 bp region of COL2A1 proximal promoter. Our new data showed that 17β-E 2 also increases the DNA-binding activities of Sp1/Sp3 and Soxs proteins to the first intron region including COL2A1 specific enhancer. In addition, Sp1, Sp3 and Sox-9 siRNAs prevented hERα66-induced transactivation of COL2A1, suggesting that these factors are required for COL2A1 up-regulation. Moreover, chromatin immunoprecipitation assays indicated that ERα physically interacts in vivo with Sp1, Sp3, Sox-9 and p300 transcription factors, on both COL2A1 promoter and enhancer sequences. Thus, considering that all these transcription factors are known to act in concert, we proposed that 17β-E 2 in vivo effect could be mediated by a bridging complex, composed of ERα, Sp1, Sp3, Sox-9 and p300 transcription factors, which could establish a physical link between the two main cisregulatory regions of the gene, and consequently, may facilitate COL2A1 transcription process. Besides, this transcriptional activation of COL2A1 gene requires the ERα transactivation domains, AF-1 and AF-2, as demonstrated by using ERα mutants lacking these functional domains. Conclusions: Understanding the molecular basis for 17β-E 2 /ERα induction of COL2A1 transcription provides new insights into molecular mechanisms of OA and could be therefore attractive for some applications in the tissue engineering of cartilage.

Research paper thumbnail of Is pro-matrix metalloproteinase-3 a marker for posttraumatic cartilage degradation?

Osteoarthritis and Cartilage, 2003

Objective: Since the development of posttraumatic osteoarthritis (OA) is a relatively slow proces... more Objective: Since the development of posttraumatic osteoarthritis (OA) is a relatively slow process, estimation of OA risk would be of value with regard to chondroprotective measures and medication. In this study we investigated the significance of pro-matrixmetalloproteinase-3 (proMMP-3) for this purpose. Design: Synovial fluid (SF) and serum samples were collected from 259 patients of our trauma clinic at the time of arthroscopy. The extent of cartilage damage was assessed according to the Outerbridge-score. ProMMP-3 levels in SF and serum were determined by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody. Additionally we determined SF and serum levels of total MMP-3 and COMP levels as well as TIMP-1 and-2 concentrations in 40 randomly selected patients by ELISA. Results: Serum proMMP-3 levels of the total cohort were markedly increased compared to healthy controls (P<0.007). The comparison of serum and SF lavage proMMP-3 concentrations showed a significant correlation (r s ϭ0.41, P<0.0001), however, only 26% of the investigated samples were increased above normal ranges. The grade of cartilage damage did not correlate with enzyme concentration neither in patients' serum nor in SF samples. ProMMP-3 SF concentration was increased early after trauma. Furthermore, proMMP-3 correlated significantly with total MMP-3 serum and SF levels as well as COMP SF levels. Conclusions: The measurement of proMMP-3 in serum or SF did not reflect the present cartilage damage and thus appears to have only minor potential for clinical use, but it should be considered for longitudinal studies, since it may reflect a risk for cartilage degradation in a subset of patients.

Research paper thumbnail of P177 Crosstalk Between INTERLEUKIN-1 and Sphingolipids: SPHINGOSINE-1-PHOSPHATE Abrogates Nitric Oxide Production and Loss of Glycosaminoglycans in Bovine Chondrocytes

Osteoarthritis and Cartilage, 2006

Purpose: Osteoarthritis is a complicated process comprising osteoclast, osteoblast and chondrocyt... more Purpose: Osteoarthritis is a complicated process comprising osteoclast, osteoblast and chondrocyte pathologies. These include osteoclastic bone resorption, osteoblastic bone formation, chondrocyte hypertrophy and subsequent cartilage degradation. At present, there are no ex vivo models which allow for investigation of the combined metabolism of all the implicated cell types in one single simple ex vivo model. The remodelling in the growth plates may partly resemble the cartilage pathology during OA progression. Therefore we developed a growth plate model in which parameters of the OA pathology involving both bone and cartilage parameters can be investigated. We developed a new ELISA assay and techniques to assess MMP derived cartilage degradation, cartilage formation as well as osteoclastic bone resorption. Methods: Tibiae were dissected from embryonic NMRi mice at day 17 post vaginal plug, as previously described. The experiment was designed with a paired control. One tibia was stimulated with 100nM salmon calcitonin (sCT) and compared to vehicle treatment. The experiment was performed in 4 replicates. The ex vivo tissue was cultured in 0.1% BSA in alpha-MEM for 7 days with refreshment of medium other day. We assessed parameters of tissue turnover in the conditioned medium by an array of ELISA assays 1) bone resorption by CTX-I 2) MMP mediated collagen type II degradation by CTX-II 3) cartilage formation by pro-collagen type II, PIIb. For clarification, conditioned medium was measured at all days, and data are accumulated over the entire culture period. Results: Calcitonin inhibited bone resorption (CTX-I) (P<0.01) by 50%, and collagen type II degradation (CTX-II) by 50%. Collagen type II formation (PIIb) was stimulated by 75% (P<0.01). Conclusions: By combining a novel ex vivo tissue culture with biochemical markers that are specific products of enzyme activity, we have developed a biological model in which most of the parameters in the progression of OA can be investigated. This model system allows for accurate profiling of hormones and other molecules that positively could affect the disease mechanisms of OA and therefore progression of OA. Calcitonin has been suggested to be a possible treatment for OA. Calcitonin inhibited cartilage and bone degradation, and stimulated cartilage formation. Calcitonin affects both osteoclast and chondrocyte metabolism, and seems to shift chondrocyte activity toward a cartilage formation rather than a cartilage degradation phenotype.

Research paper thumbnail of Listeria-Associated Arthritis in a Patient Undergoing Etanercept Therapy: Case Report and Review of the Literature

Journal of Clinical Microbiology, 2005

Listeriosis can be a cause of infectious arthritis. Here, we present a case of articular listerio... more Listeriosis can be a cause of infectious arthritis. Here, we present a case of articular listeriosis in a patient with rheumatoid arthritis receiving treatment with etanercept, a tumor necrosis factor antagonist. We review the literature of articular listeriosis and discuss the role of tumor necrosis factor blockade in precipitating listeriosis.

Research paper thumbnail of Effects of ciprofloxacin and ofloxacin on adult human cartilage in vitro

Antimicrobial Agents and Chemotherapy, 1997

Chondrocyte toxicity and necrosis were seen with electron microscopy after incubation of human ad... more Chondrocyte toxicity and necrosis were seen with electron microscopy after incubation of human adult cartilage biopsy specimens in ciprofloxacin or ofloxacin. In vitro exposure of chondrocytes to fluoroquinolones did not affect apoptosis as determined by flow cytometry. While the immediate clinical significance of this finding remains unclear, the possibility of long-term cartilage damage after fluoroquinolone treatment cannot be excluded.

Research paper thumbnail of Enhanced transendothelial in vitro migration of scleroderma lymphocytes

Arthritis Research & Therapy, 2001

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with define... more We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2 The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients

Research paper thumbnail of Impaired T-cell rsponse to subsequent TCR-stimulation after anti-CD3 induced proliferation

Arthritis Research & Therapy, 2001

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with define... more We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2 The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients

Research paper thumbnail of 212 Increase in the Gene Expression of MMP 13 and BMP 2 in Human Arthrotic Cartilage Cells After the Treatment with 5 Aza Deoxy Cytidine

Osteoarthritis and Cartilage, 2007

while up-regulating all other genes, suggesting a possible protective effect. Caspase-3, a key si... more while up-regulating all other genes, suggesting a possible protective effect. Caspase-3, a key signaling molecule in apoptosis, was significantly up-regulated (12x FS) under Co alone. While previous studies showed increased apoptosis due to mechanical injury, we have now shown a direct link between chondrocyte apoptosis in normal cartilage due to co-culture with excised joint capsule.

Research paper thumbnail of 227 Decrease of Collagen Type II Expression in Human Cartilage Cells After the Treatment with 5 Aza Deoxy Cytidine

Osteoarthritis and Cartilage, 2007

Purpose: TGF-beta is known to play a pivotal role in the reparative process of osteoarthritic car... more Purpose: TGF-beta is known to play a pivotal role in the reparative process of osteoarthritic cartilage. We aimed to test if the wide use of "TGF-beta" to refer to the three isoforms TGF-beta1, TGF-beta2 and TGF-beta3, without distinction between them, can be assumed without risk in OA research. As a first step, we have explored if they are similarly expressed in human OA cartilage. Methods: A total of 24 patients that were undergoing total hip replacement were recruited. 12 of them had hip OA and 12 had hip fracture but without clinical, radiologic or macroscopic OA. Gene expression of the 3 TGF-beta isoforms, collagen type II and aggrecan was analyzed by RT-qPCR using RNA obtained from fresh femoral head cartilage. Immunohistochemistry was used to study the expression and distribution of TGF-beta isoforms in different stages of OA: normal, slightly and strongly altered hip cartilage. Results: Expression of the three TGF-beta isoforms was significantly increased in OA cartilage. The increase was more marked for the TGF-beta3 isoform than for TGF-beta1 and TGF-beta2. TGF-beta3 levels were independent from the levels of the other two isoforms suggesting differential regulation. The mRNA levels of TGF-beta1 and TGF-beta2 were strongly correlated in OA cartilage (Spearman's r=0.83, p=0.002) but not in control cartilage. Levels of TGF-beta2 were lower than those of TGF-beta1 and TGF-beta3. Immunohistochemistry was in agreement with the qPCR results and showed that the three isoforms were overexpressed in the superficial layer of OA cartilage and near the lesions. Conclusions: Assuming that TGF-beta isoforms act indistinctly in OA is unwarranted. TGF-beta3 showed differential regulation in relation with TGF-beta1 and TGF-beta2. This might be important for OA given recent evidence suggesting that TGF-beta isoforms may have differing biological activities 227

Research paper thumbnail of Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes

Journal of orthopaedic research : official publication of the Orthopaedic Research Society, Jan 21, 2015

Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is al... more Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Express...

Research paper thumbnail of T lymphocytes of patients with SLE lack Stat4

Arthritis Research & Therapy, 2001

We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with define... more We discuss the presence of anti-keratin antibodies (AKA) of the IgG class in patients with defined juvenile idiopathic arthritis (JIA). An indirect immunofluorescence test and rat oesophagus substrate was used for the detection and quantification of AKA antibodies in patients´ sera. Overall 33/60 patients with JIA had sera positive for AKA (55 %, P = 0,0001) ranging from 1:10 to 1:160 dilutions. Following idiopathic arthritis of childhood classification criteria AKA occurred in 2/7 patients with systemic disease (28,6 %), in 13/30 patients with RF negative polyarthritis (43,3 %, P = 0,008) and in 15/18 RF positive polyarthritis (83,3 %, P = 0,000002). AKA were also found in a small cohort of patients with oligoarthritis (1/3) and psoriatic arthritis (2/2). AKA positivity occurred in 3/26 healthy controls at a 1:20 dilution. The presence of AKA was correlated as well as with the severity of the disease. Our study revealed that AKA was present overall in 18/29 patients (62%) with severe JIA and in 12/26 patients (46,2 %) with non-severe disease, however this did not reach statistical significance (P = 0,18). We also observed that AKA remained positive regardless of disease activity. AKA were detectable in 55,6 % patients with active JIA and in 48,6 % patients in the complete or near remission. Acknowledgement: This research was supported by a European Commission (Acronym: EUROBANK, contract no: QOL-2000-14.1), web site http://www.ncl.ac.uk and by grant of 2nd Medical Faculty, Charles University in Prague, VZ no. 111300003. P2 The significance of antibodies to cyclic citrullinated peptide, antikeratin antibodies, antiperinuclear factor, rheumatoid factor isotypes and HLA shared epitope in prediction of erosive disease in early rheumatoid arthritis patients

Research paper thumbnail of Treating rheumatoid arthritis to target: 2014 update of the recommendations of an international task force

Annals of the rheumatic diseases, Jan 12, 2015

Reaching the therapeutic target of remission or low-disease activity has improved outcomes in pat... more Reaching the therapeutic target of remission or low-disease activity has improved outcomes in patients with rheumatoid arthritis (RA) significantly. The treat-to-target recommendations, formulated in 2010, have provided a basis for implementation of a strategic approach towards this therapeutic goal in routine clinical practice, but these recommendations need to be re-evaluated for appropriateness and practicability in the light of new insights. To update the 2010 treat-to-target recommendations based on systematic literature reviews (SLR) and expert opinion. A task force of rheumatologists, patients and a nurse specialist assessed the SLR results and evaluated the individual items of the 2010 recommendations accordingly, reformulating many of the items. These were subsequently discussed, amended and voted upon by >40 experts, including 5 patients, from various regions of the world. Levels of evidence, strengths of recommendations and levels of agreement were derived. The update ...

Research paper thumbnail of Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary

Arthritis research & therapy, 2006

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissu... more The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [35S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1beta, were analyzed by RT-PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in...

Research paper thumbnail of Long term treatment of psoriatic arthritis with infliximab

Annals of the rheumatic diseases, 2004

Research paper thumbnail of Transcriptional overexpression of the proto-oncogene bcl-2 in patients with systemic Lupus erythematosus

Wiener klinische Wochenschrift, 1992

In systemic Lupus erythematosus (SLE) spontaneous hyperreactivity of the cells of the immune syst... more In systemic Lupus erythematosus (SLE) spontaneous hyperreactivity of the cells of the immune system leads to the production of pathogenic autoantibodies. A hyperproliferative state of lymphocytes is indicated by the increased expression of nuclear oncogenes. We investigated the expression of a putative proto-oncogene, bcl-2, which is responsible for prolonged survival of lymphocytes and protection from programmed cell death. In 19 of 24 patients with SLE an increased concentration of bcl-2 mRNA was found in unstimulated circulating blood lymphocytes. The overexpression of the bcl-2 gene was more pronounced in patients with active SLE. A pathogenic role of increased bcl-2 expression and prolonged survival of autoimmune memory cells in SLE can be hypothesized.

Research paper thumbnail of Ultrasound composite scores for the assessment of inflammatory and structural pathologies in Psoriatic Arthritis (PsASon-Score)

Arthritis Research & Therapy, 2014

Introduction: This study was performed to develop ultrasound composite scores for the assessment ... more Introduction: This study was performed to develop ultrasound composite scores for the assessment of inflammatory and structural lesions in Psoriatic Arthritis (PsA). Methods: We performed a prospective study on 83 PsA patients undergoing two study visits scheduled 6 months apart. B-mode and Power Doppler (PD) findings were semi-quantitatively scored at 68 joints (evaluating synovia, perisynovial tissue, tendons and bone) and 14 entheses. We constructed bilateral and unilateral (focusing the dominant site) ultrasound composite scores selecting relevant sites by a hierarchical approach. We tested convergent construct validity, reliability and feasibility of inflammatory and structural elements of the scores as well as sensitivity to change for inflammatory items. Results: The bilateral score (termed PsASon22) included 22 joints (6 metacarpophalangeal joints (MCPs), 4 proximal interphalangeal joints (PIPs) of hands (H-PIPs), 2 metatarsophalangeal joints (MTPs), 4 distal interphalangeal joints (DIPs) of hands (H-DIPs), 2 DIPs of feet (F-DIPs), 4 large joints) and 4 entheses (bilateral assessment of lateral epicondyle and distal patellar tendon). The unilateral score (PsASon13) compromised 13 joints (2 MCPs, 3 H-PIPs, 1 PIP of feet (F-PIP), 2 MTPs, 1 H-DIP and 2 F-DIPs and 2 large joints) and 2 entheses (unilateral lateral epicondyle and distal patellar tendon). Both composite scores revealed a moderate to high sensitivity (bilateral composite score 43% to 100%, unilateral 36% to 100%) to detect inflammatory and structural lesions compared to the 68-joint/ 14-entheses score. The inflammatory and structural components of the composite scores correlated weakly with clinical markers of disease activity (corr coeffs 0 to 0.40) and the health assessment questionnaire (HAQ, corr coeffs 0 to 0.39), respectively. Patients with active disease achieving remission at follow-up yielded greater reductions of ultrasound inflammatory scores than those with stable clinical activity (Cohen's d effect size ranging from 0 to 0.79). Inter-rater reliability of bi-and unilateral composite scores was moderate to good with ICCs ranging from 0.42 to 0.96 and from 0.36 to 0.71, respectively for inflammatory and structural sub-scores. The PsASon22 and PsASon13 required 16 to 26 and 9 to 13 minutes, respectively to be completed. Conclusion: Both new PsA ultrasound composite scores (PsASon22 and PsASon13) revealed sufficient convergent construct validity, sensitivity to change, reliability and feasibility.

Research paper thumbnail of Clinical and immunological effects of anti-TNF therapy in systemic lupus erythematosus (SLE)

Arthritis Research & Therapy - ARTHRITIS RES THER, 2003

Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis ... more Heterogeneity and multifactoriality complicate diagnostics and our understanding of pathogenesis of rheumatoid arthritis (RA). The only accepted serologic parameter (rheumatoid factor [RF]) is not disease specific, nor are any of several novel RA autoantibodies. We aimed at identifying profiles instead of individual autoreactivities allowing for unambiguous prediction of RA. Selected RA autoantigens were tested by ELISA (RF and anti-cyclic citrullinated peptide [anti-CCP]) or Western blot (heavy-chain-binding protein [BiP], heterogeneous ribonucleoprotein particle A2 [RA33/ hnRNP A2], calpastatin and calreticulin). Antibody reactivities were assayed from serum samples of 149 RA patients and 132 patients with other rheumatic diseases and from synovial fluids (SF) (58 RA, 65 non-RA). No single autoreactivity was sufficient for unambiguous prediction of RA. Frequencies of multiparameter profiles consisting of 3, 4, 5 and 6 autoreactivites were determined. Fifteen six-parameter serum profiles were exclusively expressed in RA patients, representing a cumulative sensitivity of 59%. Twelve SF profiles were exclusively expressed in 64% of RA patients. The self-learning classification algorithm CLASSIF1 was capable of accurately predicting RA when these profiles were present. Data profile analysis of RF/CCP/BiP/calpastatin/calreticulin/RA33 provided specific discrimination of 64% of RA. Most importantly, RA specific profiles were observed in 64% of patients with early disease (<12 months). For the first time, the accurate prediction of the class RA has been achieved by the use of multiparametric autoreactivity profiles. Because of early expression in disease, these profiles make it possible to start a disease-modifying therapy long before irreversible bone and joint destruction may develop. Additional RA-specific profiles are required to cover the entire group of RA patients. 2 Investigation of the reactivity patterns of antifilaggrin antibodies in sera and synovial fluids from patients with rheumatoid arthritis using citrullinated synthetic peptides

Research paper thumbnail of Poster Presentations (PP01-PP67)

Rheumatology, 2012

Background: Prophylaxis of tuberculosis in patients treated with anti-tumor necrosis factor (Anti... more Background: Prophylaxis of tuberculosis in patients treated with anti-tumor necrosis factor (Anti-TNF) prevents the latent tuberculosis infections (LTBI). But incidence rate of tuberculosis with anti-TNF therapy in tuberculosis-endemic area is higher than in other areas despite strict prophylaxis. We analyzed incidences and characteristics of patients with reactivation of LTBI and new tuberculosis infection. Method: We identified 549 patients treated with anti-TNF in tertiary care center from June 2003 through June 2010. The number of patients with ankylosing spondylitis, rheumatoid arthritis, psoariatic arthritis, spondyloarthropathy and behcet's disease were 317, 215, 11, 3 and 3 respectively. Their medical records including history of tuberculosis infection were reviewed. Result: Among 549 patients, 526 patients took tuberculosis screening test and 266 patients had LTBI prophylaxis. Nevertheless, we observed 11 cases of tuberculosis (2.0%) and the incidence rate of tuberculosis was 968 per 100,000 person-years. Patients infected by tuberculosis were 6 non-LTBI, 4 LTBI received prophylaxis and 1 without screening test. The mean duration of anti-TNF therapy was 21.73 months and patients of extrapulmonary tuberculosis were 9. Among 11 tuberculosis patients, 3 non-LTBI patients and 2 LTBI patients who received prophylaxis were infected by tuberculosis after anti-TNF therapy of 12 months and their mean duration of anti-TNF therapy was 40.8 months. In addition, three non-LTBI patients treated with anti-TNF therapy for less than 12 months were infected by new tuberculosis or reactivated LTBI with false-negative screening test. Conclusion: The incidence of tuberculosis in patients treated with anti-TNF was higher than other previous reports. This result show that the reactivation of LTBI and development of new tuberculosis infection as a long period of anti-TNF therapy for the endemic area. Screening and prophylaxis of LTBI are important but we also have to be concerned about new tuberculosis infection during anti-TNF therapy.

Research paper thumbnail of 239 SPHINGOSINE-1-PHOSPHATE INHIBITSII-1 and TNF-Α Induced Inos Expression via S1P1 and S1P2 in Human Articular Chondrocytes

Osteoarthritis and Cartilage, 2008

We have prior identified the bioactive Sphingolipid Sphingosine-1-Phosphate (S1P) as a potent inh... more We have prior identified the bioactive Sphingolipid Sphingosine-1-Phosphate (S1P) as a potent inhibitor of Interleukin-1 (Il-1) induced cartilage degradation in bovine cartilage. As micro-molar concentrations of S1P have been found in the synovial fluid of osteoarthritis and rheumatoid arthritis joints we investigated the role of S1P and its receptors in human cartilage. Methods: Human cartilage specimens were obtained from patients undergoing total knee joint replacement. Specimens were formalin fixed and paraffin embedded and S1P receptor iso-types S1P 1 , S1P 2 and S1P 3 were detected by immunohistochemistry. For cell culture human Chondrocytes were isolated using collagenase B. Cells grown in monolayer were cultured in Ham's F-12/DMEM (1:1) and 10% FCS over 3 passages. Cultured chondrocytes were serum starved for 24 hours and incubated with 10 ng/ml Il-1b or 100 ng/ml TNF-a alone or in combination with 0.1 mM up to 3 mM S1P. Expression of iNOS, ADAMTS-4 and MMP-13, was evaluated using real-time PCR. Results: All three investigated S1P-receptor iso-types were detected in human cartilage; however S1P 2 was the most prominent subtype. Interestingly, the expression of the receptors was confined to zones of cartilage damage and proliferating chondrocytes. Furthermore, expression of iNOS mRNA induced by Il-1b and TNF-a was dose dependently reduced by S1P by 47.0% and 56.2% respectively (P < 0.05). Cytokine induced ADAMTS-4 mRNA was diminished in the presence of S1P by 35.6% and 41.8% respectively (P < 0.05). In contrast to our previous observations in bovine cartilage MMP-13 mRNA expression was not affected by coincubation with S1P in human chondrocytes. Using specific inhibitors for S1P 1 (pertussis toxin) and S1P 2 (suramin) we found that inhibition of S1P 1 but not S1P 3 partly reversed iNOS and ADAMTS-4 inhibition. Conclusions: Our results suggest that S1P reduces Il-1 and TNF-a induced mRNA transcription of ADAMTS-4 and iNOS in human articular chondrocytes via S1P 1 and S1P 2. Therefore expression of these S1P receptor subtypes in damaged areas of human cartilage can be interpreted as a counter regulation of chondrocytes to inhibit further tissue degradation by Il-1b and TNF-a.

Research paper thumbnail of 234 Cartilage Calcification and BMP Signaling in Early Hand Osteoarthritis

Osteoarthritis and Cartilage, 2008

In contrast, cytomegalovirus promoter-driven Sp1 overexpression further enhanced Il-1-induced ADA... more In contrast, cytomegalovirus promoter-driven Sp1 overexpression further enhanced Il-1-induced ADAMTS-4 expression. Expression of constitutive ADAMTS-5 was not affected by any of the agents. Conclusions: These results provide pharmacological and genetic evidence for the importance of Sp1 in ADAMTS-4 gene regulation by Il-1.

Research paper thumbnail of 268 LAMA4 is Expressed by Clusters of Hypertrophic Chondrocytes in Osteoarthritic Cartilage and Regulated by Epigenetic Mechanims

Osteoarthritis and Cartilage, 2010

Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage ... more Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage whose expression is strongly decreased during osteoarthritis (OA) process. Specific proteins (Sp) and Sox proteins have been previously characterized to be major regulators of this gene. Besides, the existence of a link between estrogen deprivation and osteoarthritis in postmenopausal women suggests that 17β-estradiol (17β-E 2) may be a critical modulator of cartilaginous matrix homeostasis. The aim of present study was to investigate the molecular mechanisms regulating type II collagen gene expression under the effect of 17β-E 2 , and to characterize the genomic pathway via estrogen receptors (ER). Methods: Articular chondrocytes were isolated from 3-week old rabbits and incubated for 24 hours with increasing concentrations of 17β-E 2 (0 to 10 nM). Nuclear proteins were extracted and submitted to gel retardation assays in order to determine Sp and Sox proteins binding activities on COL2A1 gene. In some experiments, relative expression of Sp1/Sp3 or Sox-9 was inhibited by siRNAs strategies. Moreover, chromatin immunoprecipitation was used to study protein-protein interactions on COL2A1 promoter and enhancer sequences. Results: We have previously shown that 17β-E 2 could stimulate type II collagen neosynthesis, protein level and corresponding steady-state levels of COL2A1 mRNAs in primary and in vitro dedifferentiated rabbit articular chondrocytes. 17β-E 2 action was mediated, in part, by an ERα/Sp transactivating effect on the-266/-63 bp region of COL2A1 proximal promoter. Our new data showed that 17β-E 2 also increases the DNA-binding activities of Sp1/Sp3 and Soxs proteins to the first intron region including COL2A1 specific enhancer. In addition, Sp1, Sp3 and Sox-9 siRNAs prevented hERα66-induced transactivation of COL2A1, suggesting that these factors are required for COL2A1 up-regulation. Moreover, chromatin immunoprecipitation assays indicated that ERα physically interacts in vivo with Sp1, Sp3, Sox-9 and p300 transcription factors, on both COL2A1 promoter and enhancer sequences. Thus, considering that all these transcription factors are known to act in concert, we proposed that 17β-E 2 in vivo effect could be mediated by a bridging complex, composed of ERα, Sp1, Sp3, Sox-9 and p300 transcription factors, which could establish a physical link between the two main cisregulatory regions of the gene, and consequently, may facilitate COL2A1 transcription process. Besides, this transcriptional activation of COL2A1 gene requires the ERα transactivation domains, AF-1 and AF-2, as demonstrated by using ERα mutants lacking these functional domains. Conclusions: Understanding the molecular basis for 17β-E 2 /ERα induction of COL2A1 transcription provides new insights into molecular mechanisms of OA and could be therefore attractive for some applications in the tissue engineering of cartilage.

Research paper thumbnail of Is pro-matrix metalloproteinase-3 a marker for posttraumatic cartilage degradation?

Osteoarthritis and Cartilage, 2003

Objective: Since the development of posttraumatic osteoarthritis (OA) is a relatively slow proces... more Objective: Since the development of posttraumatic osteoarthritis (OA) is a relatively slow process, estimation of OA risk would be of value with regard to chondroprotective measures and medication. In this study we investigated the significance of pro-matrixmetalloproteinase-3 (proMMP-3) for this purpose. Design: Synovial fluid (SF) and serum samples were collected from 259 patients of our trauma clinic at the time of arthroscopy. The extent of cartilage damage was assessed according to the Outerbridge-score. ProMMP-3 levels in SF and serum were determined by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody. Additionally we determined SF and serum levels of total MMP-3 and COMP levels as well as TIMP-1 and-2 concentrations in 40 randomly selected patients by ELISA. Results: Serum proMMP-3 levels of the total cohort were markedly increased compared to healthy controls (P<0.007). The comparison of serum and SF lavage proMMP-3 concentrations showed a significant correlation (r s ϭ0.41, P<0.0001), however, only 26% of the investigated samples were increased above normal ranges. The grade of cartilage damage did not correlate with enzyme concentration neither in patients' serum nor in SF samples. ProMMP-3 SF concentration was increased early after trauma. Furthermore, proMMP-3 correlated significantly with total MMP-3 serum and SF levels as well as COMP SF levels. Conclusions: The measurement of proMMP-3 in serum or SF did not reflect the present cartilage damage and thus appears to have only minor potential for clinical use, but it should be considered for longitudinal studies, since it may reflect a risk for cartilage degradation in a subset of patients.

Research paper thumbnail of P177 Crosstalk Between INTERLEUKIN-1 and Sphingolipids: SPHINGOSINE-1-PHOSPHATE Abrogates Nitric Oxide Production and Loss of Glycosaminoglycans in Bovine Chondrocytes

Osteoarthritis and Cartilage, 2006

Purpose: Osteoarthritis is a complicated process comprising osteoclast, osteoblast and chondrocyt... more Purpose: Osteoarthritis is a complicated process comprising osteoclast, osteoblast and chondrocyte pathologies. These include osteoclastic bone resorption, osteoblastic bone formation, chondrocyte hypertrophy and subsequent cartilage degradation. At present, there are no ex vivo models which allow for investigation of the combined metabolism of all the implicated cell types in one single simple ex vivo model. The remodelling in the growth plates may partly resemble the cartilage pathology during OA progression. Therefore we developed a growth plate model in which parameters of the OA pathology involving both bone and cartilage parameters can be investigated. We developed a new ELISA assay and techniques to assess MMP derived cartilage degradation, cartilage formation as well as osteoclastic bone resorption. Methods: Tibiae were dissected from embryonic NMRi mice at day 17 post vaginal plug, as previously described. The experiment was designed with a paired control. One tibia was stimulated with 100nM salmon calcitonin (sCT) and compared to vehicle treatment. The experiment was performed in 4 replicates. The ex vivo tissue was cultured in 0.1% BSA in alpha-MEM for 7 days with refreshment of medium other day. We assessed parameters of tissue turnover in the conditioned medium by an array of ELISA assays 1) bone resorption by CTX-I 2) MMP mediated collagen type II degradation by CTX-II 3) cartilage formation by pro-collagen type II, PIIb. For clarification, conditioned medium was measured at all days, and data are accumulated over the entire culture period. Results: Calcitonin inhibited bone resorption (CTX-I) (P<0.01) by 50%, and collagen type II degradation (CTX-II) by 50%. Collagen type II formation (PIIb) was stimulated by 75% (P<0.01). Conclusions: By combining a novel ex vivo tissue culture with biochemical markers that are specific products of enzyme activity, we have developed a biological model in which most of the parameters in the progression of OA can be investigated. This model system allows for accurate profiling of hormones and other molecules that positively could affect the disease mechanisms of OA and therefore progression of OA. Calcitonin has been suggested to be a possible treatment for OA. Calcitonin inhibited cartilage and bone degradation, and stimulated cartilage formation. Calcitonin affects both osteoclast and chondrocyte metabolism, and seems to shift chondrocyte activity toward a cartilage formation rather than a cartilage degradation phenotype.

Research paper thumbnail of Listeria-Associated Arthritis in a Patient Undergoing Etanercept Therapy: Case Report and Review of the Literature

Journal of Clinical Microbiology, 2005

Listeriosis can be a cause of infectious arthritis. Here, we present a case of articular listerio... more Listeriosis can be a cause of infectious arthritis. Here, we present a case of articular listeriosis in a patient with rheumatoid arthritis receiving treatment with etanercept, a tumor necrosis factor antagonist. We review the literature of articular listeriosis and discuss the role of tumor necrosis factor blockade in precipitating listeriosis.