Background In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and reduc... more Background In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and reduction of inflammatory symptoms has been demonstrated and it is recommended for the treatment of arthritis, fibromyalgia and ankylosing spondylitis. The mode of action of this therapy, which consists in a brief exposure to temperatures between -110 and -160°C in special cryochambers, has not been fully elucidated. Objectives The aim of this study was to investigate the changes in the gene expression pattern in cells from peripheral blood of patients with fibromyalgia going through a series of three exposures to WBCT within three days. Methods Ten patients with fibromyalgia (9 female/1 male, mean age 48.7 (9.8) years) were included in the study and underwent 3 exposures to WBCT in a cryochamber system with 3 chambers (10 seconds at -10°C, 10 seconds at -60°C and for maximum 3 minutes at -110°C) at 3 consecutive days. The mean pain intensity (VAS in mm) before start of WBCT treatment was 61. Blood was collected immediately prior to (baseline) and directly after the first exposure to WBCT and after the third exposure using PAXgene™ RNA tubes. Total RNA was extracted with the PAXgene Blood RNA kit, no globin reduction protocol was applicated. Transcriptome analysis was performed using Affymetrix GeneChip(®) Human Gene 1.0 ST arrays. Data were analysed using the Partek Genomic Suite 6.6 Software. Results All 10 patients tolerated the application of the WBCT well and reported to benefit from this treatment. The mean pain intensity (VAS in mm) reduced from 61 at baseline to 36 directly after the third treatment in the cryochamber. Repeated exposure to WBCT caused statistically significant (p<0.05) greater than 1.2 fold up-regulation and <-1.2 fold down-regulation in expression (relative to baseline) of 90 out of 33,297 sequences.(72 down-regulated, 18 up-regulated). 34 of these transcripts were found significantly changed already early after a single exposure to WBCT. The majority of down-regulated transcripts belongs to the group of small nucleolar RNA (SNORD). An up-regulation was found amongst others for transcripts for PBX1, SFRP2, MAP2K3, and SLC25A39. Conclusions We observed significant changes in gene expression early in peripheral blood cells of fibromyalgia patients undergoing a WBCT. Small nucleolar RNAs represent the group with highest number of gene products that appear to be regulated by brief exposures to very cold temperatures. These molecules belong to so-called non-coding RNAs, which are considered as internal signals that control various levels of gene expression in physiology and development and are discussed as regulators of disease. Disclosure of Interest None Declared
Objective The cytokine IL-17 plays a crucial role in the development and promoting of inflammator... more Objective The cytokine IL-17 plays a crucial role in the development and promoting of inflammatory rheumatic diseases, such as psoriasis arthritis and ankylosing spondylitis. The influence of IL-17 on the osteoblast differentiation from mesenchymal stem cells has already been well studied. However, the effect of IL-17 on mature osteoblasts is not yet fully understood. Methods In this study, the influence of IL-17 on the expression of osteogenic markers and pro-inflammatory cytokines was analyzed on mRNA and protein level in an osteoblast cell culture model. Results Our data indicate that IL-17 alone has no significant influence on the expression of osteoblast-specific genes. However, a significant upregulation of pro-inflammatory cytokines at the transcriptional level by IL-17 was observed in primary osteoblasts. This effect on the regulation of pro-inflammatory cytokines was abolished completely by administration of a therapeutic anti-IL-17 antibody. Co-stimulation with TNF-α and IL-17 led to an upregulation of pro-inflammatory cytokines, which significantly exceeded the additive effect of both cytokines. In this co-stimulation, the anti-IL-17 antibody could not completely reverse the IL-17 effect. The same IL-17 and TNF-α effect was observed in osteoblast-like cells (MG63), whereas IL-17 alone did not induce the expression of pro-inflammatory cytokines. Conclusion The upregulation of the pro-inflammatory cytokines IL-1, IL-6, and IL-8 in primary osteoblasts by IL-17 indicates an indirect regulatory effect on osteoclastogenesis and activation of bone resorption. The therapeutic IL-17 antibody reduced the IL-17 induced release of pro-inflammatory cytokines by osteoblasts and this, in turn, could also reduce the effect on osteoclast differentiation and bone resorption. Our study underlines the important role of osteoblasts as major players in the osteoimmunologic network.
Background Interleukin (IL)-1 and metalloproteinases (MMP) are thought to play a crucial role in ... more Background Interleukin (IL)-1 and metalloproteinases (MMP) are thought to play a crucial role in the cartilage degradation of osteoarthritis (OA). Recently it has been proposed that CRP may be useful index of OA activity and severity. Objectives We studied the relationships between synovial fluid (SF) IL-1b, MMP1 and MMP3, and serum levels of CRP in patients with OA of the knee (KOA) to investigate the role of CRP in KOA. Methods In the SF from 25 patients with KOA (Altman criteria, Kellgren and Lawrence score of 2 or greater on X-ray plan films) we determined the levels of IL-1b, MMP-1 and MMP-3 (ELISA), along with WBC number. In the same patients serum concentration of CRP was analysed by ultrasensitive rate nephelometry. No patients were taking NSAIDs or other antirheumatic drugs. Results In SF, MMP1 was correlated with MMP3 (r = 0.61, p = 0.0009), and MMP3 with IL-1b (r = 0.67, p = 0.0002). In turn, serum CRP was correlated with MMP3 (r = 0.76, p < 0.0001) and, at lower level, with MMP1 (r = 0.49, p = 0.01) and IL-1b (r = 0.49, p = 0.01). Conclusion The correlations found between serum CRP and SF levels of substances believed to be crucial in OA cartilage degradation, such as MMPs and IL-1, confirm the role of marker of disease severity recently proposed for this acute phase protein.
Cre-directed S1P ablation (S1P Osx mice). S1P Osx mice and wild type (WT) littermates were studie... more Cre-directed S1P ablation (S1P Osx mice). S1P Osx mice and wild type (WT) littermates were studied by histology, in situ hybridization analysis (ISH), immunofluorescence (IF), real-time (RT) PCR and micro-computed tomography (mCT) techniques. Osteogenic differentiation assays analyzing for osteoblast differentiation in vitro were conducted with bone marrow harvested from 21 day old WT and mutant mice. Results: Mice with a homozygous deletion for S1P (S1P f/f ;Osx-Cre mice or S1P cko-Osx) suffer from severe chondrodysplasia; mice heterozygous for S1P deletion (S1P þ/f-Osx) have a size intermediate to S1P cko-Osx mice and WT littermates. Additionally, these mice are also kyphotic that gets more pronounced with age and was confirmed by mCT analysis. In situ
Background: Bone erosion in joint is the most deleterious effect induced by rheumatoid arthritis ... more Background: Bone erosion in joint is the most deleterious effect induced by rheumatoid arthritis (RA) and is the major cause of disability. New therapies are urgently required to prevent RA-associated bone destruction. The excessive bone resorption in RA was proved to be mediated by osteoclasts which are derived from the monocyte/macrophage lineage and induced by RANKL. Therefore, RANKL could be a potential target for the preservation of bone mass in RA. Objectives: Chrysotherapy showed potential effects in reducing of joint destruction but with serious side-effects in RA therapy. A rational designed gold nanoparticle named GN was prepared to provide a choice for suppressing bone loss in RA therapy with more safety. Methods: Mice macrophage cell line RAW264.7 was activated by RANKL to evaluate the potential osteoclastogenesis suppressing effects of GN. The differentiation, fusion and function of osteoclast were assessed by tartrateresistant acid (TRAP) staining, actin ring formation assay and osteologic discs detection respectively. Relative expressions of osteoclast-specific genes were evaluated by RT-PCR and the activity of NF-κB pathway as well as MAPK pathways were analyzed by immunoblotting. Results: Osteoclast differentiation, fusion and bone resorption were activated by RANKL in RAW264.7 cell, while GN significantly attenuates this process in a dose-dependent manner. The expressions of osteoclast-specific genes including TRAP and OSCAR were increased by RANKL stimulation but were effectively suppressed by GN. The activation of NF-kappa B pathway and MAPK pathways induced by RANKL was also suppressed by GN treatment. Conclusions: Our findings revealed that treatment with GN could prevent RANKLinduced osteoclastogenesis and reduce increased expression of osteoclastspecific genes which may through the suppression of NF-kB and MAPK activation in macrophage. GN may provide a basis for the design of chrysotherapy drugs that effect in progression of RA-associated articular erosions.
Outcome predictors of biologic therapeutic drugs like TNF inhibitors are of interest since side e... more Outcome predictors of biologic therapeutic drugs like TNF inhibitors are of interest since side effects like serious infections or malignancy cannot be completely ruled out. Response rates are heterogeneous. The present study addressed the question whether in patients with rheumatoid arthritis (RA) interleukin-10 (IL-10) promoter genotypes with potential relevance for IL-10 production capacity are associated with response to long-term treatment with etanercept. Caucasian RA patients that, according to the EULAR criteria, responded well (n = 25), moderately (n = 17) or not (n = 8) to etanercept therapy (median 36 months, range 4-52), and 160 matched controls were genotyped for the IL-10 promoter SNPs-2849 G>A (rs6703630),-1082 G>A (rs1800896),-819 C>T (rs1800871) and-592 C>A (rs1800872). Haplotypes were reconstructed via mathematic model and tested for associations with disease susceptibility and therapy response. We identified the four predominant haplotypes AGCC, GATA, GGCC, and GACC in almost equal distribution. Patients that responded well carried the putative IL-10 low producer allele-2849 A or the haplotypes AGCC and GATA (RR 2.1 and 4.0, respectively; 95% CI 1.1-4.0 and 1.1-14.8), whereas an unfavourable response was associated with carriage of the putative high producer haplotype GGCC (RR 1.9, 95% CI 1.1-3.3). No significant associations of alleles or haplotypes with disease susceptibility were observed. In RA, a low IL-10 production which is genetically determined rather by haplotypes than by SNPs may favour the response to etanercept treatment. Iatrogenic blockade of TNF may reveal proinflammatory effects of its endogeneous antagonist IL-10. Further studies are needed to correlate these genetic findings to direct cytokine measurements.
New experimental approaches are presented which are implemented in rheumatology research. DNA mic... more New experimental approaches are presented which are implemented in rheumatology research. DNA microarray technology and proteome analysis are two new methods which are applied to gain a global survey over the gene expression at the RNA and protein level under various conditions. Based on these methods of molecular medicine important functional proteins in the pathogenesis of rheumatoid arthritis (RA) as well as clinical relevant genetic polymorphisms shall be identified. New insights are expected which will help in the differentiation of clinical entities and in the search for new therapeutic strategies in the treatment of RA.
International Journal of Molecular Sciences, Oct 5, 2018
The balance of bone formation and resorption is the result of a regulated crosstalk between osteo... more The balance of bone formation and resorption is the result of a regulated crosstalk between osteoblasts, osteoclasts, and osteocytes. Inflammation, mechanical load, and external stimuli modulate this system. Exposure of bone cells to metal ions or wear particles are thought to cause osteolysis via activation of osteoclasts and inhibition of osteoblast activity. Co 2+ ions have been shown to impair osteoblast function and the expression of the three transforming growth factor (TGF)-β isoforms. The current study was performed to analyze how Co 2+ and Cr 3+ influence the expression, proliferation, and migration profile of osteoblast-like cells. The influence of Co 2+ , Cr 3+ , and CoCr particles on gene expression was analyzed using an osteogenesis PCR Array. The expression of different members of the TGF-β signaling cascade were down-regulated by Co 2+ , as well as several TGF-β regulated collagens, however, Cr 3+ had no effect. CoCr particles partially affected similar genes as the Co 2+ treatment. Total collagen production of Co 2+ treated osteoblasts was reduced, which can be explained by the reduced expression levels of various collagens. While proliferation of MG63 cells appears unaffected by Co 2+ , the migration capacity was impaired. Our data may improve the knowledge of changes in gene expression patterns, and the proliferation and migration effects caused by artificial materials.
AdA. Lesions developed more slowly in LA/CFA than in AdA. However, hock BMD as well as knee infla... more AdA. Lesions developed more slowly in LA/CFA than in AdA. However, hock BMD as well as knee inflammation and erosion scores found to be different (shown here at Onset + 5 days): Conclusion Inflammatory and skeletal changes for AdA and LA/ CFA were similar by clinical and histopathologic measures 10 days after onset, while inflammation receded by Onset + 25 days in LA/CFA more than it did in AdA. These data indicate that bone densitometric and morphologic findings can distinguish these two models of adjuvant arthritis despite their similar clinical presentations. This finding may be relevant in screening novel therapeutic candidates as these models may define different potential efficacious effects of a compound.
Background For about 15 years biological compounds belong to the therapeutic options for the trea... more Background For about 15 years biological compounds belong to the therapeutic options for the treatment of rheumatoid arthritis. By targeting of tumor necrosis factor alpha (TNFα), one of the key cytokines in the pathogenesis of rheumatoid arthritis (RA), patients benefit in terms of disease activity as well as radiologic progression. The application of biologic substances is subject to a risk to develop antibodies against these structures causing adverse events or loss of efficacy. Objectives It was the aim of the study to analyse the occurrence of anti-drug antibodies in the course of a long term treatment of patients with rheumatoid arthritis with etanercept (ETN). Methods In this retrospective analysis sera from 100 RA patients obtained during an up to 4 years treatment period with etanercept (after 6, 12, 24, 36 and 48 months) and 43 adalimumab (ADL)-treated patients were included for determination of anti-drug antibodies. If available the first serum sample obtained at least 2 weeks after discontinuation of ETN treatment (due to side effects, non-response, remission or surgery) was included for analysis to exclude any interference with high drug levels. For the determination of anti-ETN and anti-ADL levels Promonitor® -anti-ETN and –anti-ADL assays (Proteomika, Spain) were used according to the manufacturer's instructions. Both assays are designed as bridging assays with cut-off values of 142 AU/ml and 3.5 AU/ml for ETN and ADL, respectively. For calculation of results a data analysis software (http://www.myassays.com) was used. Results No anti-ETN antibodies were found in 100 RA patients, neither in the course of treatment nor in samples from 72 of these patients obtained after discontinuation of ETN treatment after mean 36.3 months (range 5-78 months). However, in the ADL-treated cohort anti-ADL antibodies were found in 51.2% of patients (22 out of 43) after discontinuation of therapy after mean 23.4 months (range 3-77 months). Conclusions Our study confirms the lack of anti-drug antibodies in a large cohort of RA patients in the course of a long-term treatment with ETN over up to 4 years. The absence of anti-ETN antibodies in serum samples after discontinuation of therapy confirms this result by exclusion of false negative results resulting from interference with high etanercept levels in the course of treatment. Acknowledgements Supported by Pfizer Pharma GbmH Disclosure of Interest S. Drynda Grant/research support from: Pfizer Pharma GmbH, R. Beuermann: None declared, J. Kekow: None declared
Background: In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and redu... more Background: In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and reduction of inflammatory symptoms has been demonstrated and it is recommended for the treatment of arthritis, fibromyalgia and ankylosing spondylitis. The mode of action of this therapy, which consists in a brief exposure to temperatures between-110 and-160°C in special cryochambers, has not been fully elucidated. Objectives: The aim of this study was to investigate the changes in the gene expression of selected genes (CCL4, TGFBR3, CD69 and MAP2K3) identified as significantly regulated in a small pilot study using Affymetrix GeneChip ® Human Gene 1.0 ST arrays in cells from peripheral blood of patients with fibromyalgia going through a series of three exposures to WBCT within three days. Methods: Twenty two patients with fibromyalgia (20 female/2 male, age 51.7±8.9 years (mean ± SD) were included in the study and underwent 3 exposures to WBCT in a cryochamber system with 3 chambers (10 seconds at-10°C, 10 seconds at-60°C and for maximum 3 minutes at-110°C) at 3 consecutive days. During the study patients did not change their medication. Blood was collected immediately prior to (baseline) and directly after the first exposure to WBCT and after the third exposure using PAXgene™ RNA tubes. Total RNA was extracted with the PAXgene Blood RNA kit. Gene expression levels of MAP2K3, CCL4, TGFBR3, and CD69 were analysed by real-time PCR using sequence-specific primers and probes (TaqMan). Results: All 22 patients tolerated the application of the WBCT well and reported to benefit from this treatment. The expression levels of CCL4 in cells from peripheral blood reduced significantly to mean 67% in 19 of 22 patients after the third exposure compared to baseline. The expression level of CD69 was also reduced in the majority of patients (16 out of 22) significantly to 59%. In contrast, the expression of MAP2K3 was found to be up-regulated in 13 patients to mean 180%, while the expression levels in the other nine patients remained almost unchanged. The changes of gene expression levels observed after the third exposure compared to baseline became apparent already after the first cold exposure, without reaching statistical significance. The down-regulation of TGFBR3 observed in the pilot study could not be confirmed in the larger cohort. Conclusions: The results of our study indicate, that the whole-body cryotherapy may cause significant changes in gene expression levels of CCL4, CD69 and MAP2K3. The MAP2K3 expression is known to be regulated by environmental stress, which is in accordance with the observed up-regulation of MAP2K3 expression. The down-regulation of the CD69, a marker for T-cell activation and the chemokine CCL4 that is produced by T-cells indicate that the exposure to WBCT has an effect on peripheral T-cells. Whether this influence on T-cells contributes to the therapeutic effect needs to be elucidated.
Background: Remission is the current aim of treatment in rheumatoid arthritis (RA). Remission is ... more Background: Remission is the current aim of treatment in rheumatoid arthritis (RA). Remission is ideally the absence of detectable disease and absence of structural and functional worsening over the time. Infra-clinical synovitis, detected by ultrasound, may persist in patients with remission leading to further structural damage. Objectives: The aim of this study was to assess the frequency of relapse after clinical remission and the value of US to predict this relapse. Methods: A prospective study of 30 patients followed-up for RA in remission was conducted. The remission was defined by a Disease Activity Score 28 joints (DAS28) ≤2,6 for at least 3 months under the same therapy. A B-mode and Power Doppler (PD) US examination was assessed by an experienced rheumatologist blinded to clinical data for 22 joints (wrists, 10 metacarpo-phalangeals, 10 proximal interphalangeals joints). A semi-quantitative scale running from 0 to 3 depending on US activity was attributed for each joint for PD. Patients were clinically controlled 3 to 6 months after performing the US. The variability of DAS28 was noted, it was considered significant if the p value <0,05. Results: The mean age of the thirty patients was 48 years-old (25 females, 5 males). All of them were in remission according to DAS28 with an average of 2,03 [1,13-2,6]. US examination showed synovial hypertrophy in B-mode in 80% of patients and PD signals in 56,7% of patients. The follow-up control was performed on average after 4 months for 27 patients. The variation of DAS28 from baseline was significant (p<0,005). In fact the DAS28 increased of 0,75 [0,10-3,1] for 20 patients, among them 14 were no longer in remission. The DAS28 remained stable for 2 patients and decreased for 5 others. For patients who have reactivation of RA, the mean US PD scale was 4,4, while it was 1,1 for patients who remained in remission. However, the difference wasn't significant (p=0,12). Conclusions: For patients in remission, persistent active synovitis in US is a risk factor for futur reactivation of the disease. PD monitoring in daily practice might lead to reduce disease relapses and structural damages.
European journal of mass spectrometry, Feb 1, 2010
As the potential of epitope chips for routine application in diagnostics relies on the careful se... more As the potential of epitope chips for routine application in diagnostics relies on the careful selection of peptides, reliable epitope mapping results are of utmost interest to the medical community. Mass spectrometric epitope mapping, in combination with peptide chip analysis, showed that autoantibodies from patients who suffered from rheumatoid arthritis (RA) were directed against distinct surface structures on the full-length human autoantigen RA33 as well as against partial sequences. Using the combined mass spectrometric epitope extraction and peptide chip analysis approach, four sequence motifs on RA33 emerged as immuno-positive, showing that epitopes were not randomly distributed on the entire RA33 amino acid sequence. A sequential epitope motif (245GYGGG249) was determined on the C-terminal part of RA33 which matched with the Western blot patient screening results using the full-length protein and, thus, was regarded as a disease-associated epitope. Other epitope motifs were found on N-terminal partial sequences (59RSRGFGF65, 111KKLFVG116) and again on the C-terminal part (266NQQPSNYG273) of RA33. As recognition of these latter three motifs was also recorded by peptide chip analysis using control samples which were negative in the Western blot screening, these latter motifs were regarded as “cryptic epitopes”. Knowledge of disease-associated epitopes is crucial for improving the design of a customized epitope peptide chip for RA and mass spectrometric epitope mapping pivotally assisted with selecting the most informative peptide(s) to be used for future diagnostic purposes.
Background In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and reduc... more Background In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and reduction of inflammatory symptoms has been demonstrated and it is recommended for the treatment of arthritis, fibromyalgia and ankylosing spondylitis. The mode of action of this therapy, which consists in a brief exposure to temperatures between -110 and -160°C in special cryochambers, has not been fully elucidated. Objectives The aim of this study was to investigate the changes in the gene expression pattern in cells from peripheral blood of patients with fibromyalgia going through a series of three exposures to WBCT within three days. Methods Ten patients with fibromyalgia (9 female/1 male, mean age 48.7 (9.8) years) were included in the study and underwent 3 exposures to WBCT in a cryochamber system with 3 chambers (10 seconds at -10°C, 10 seconds at -60°C and for maximum 3 minutes at -110°C) at 3 consecutive days. The mean pain intensity (VAS in mm) before start of WBCT treatment was 61. Blood was collected immediately prior to (baseline) and directly after the first exposure to WBCT and after the third exposure using PAXgene™ RNA tubes. Total RNA was extracted with the PAXgene Blood RNA kit, no globin reduction protocol was applicated. Transcriptome analysis was performed using Affymetrix GeneChip(®) Human Gene 1.0 ST arrays. Data were analysed using the Partek Genomic Suite 6.6 Software. Results All 10 patients tolerated the application of the WBCT well and reported to benefit from this treatment. The mean pain intensity (VAS in mm) reduced from 61 at baseline to 36 directly after the third treatment in the cryochamber. Repeated exposure to WBCT caused statistically significant (p<0.05) greater than 1.2 fold up-regulation and <-1.2 fold down-regulation in expression (relative to baseline) of 90 out of 33,297 sequences.(72 down-regulated, 18 up-regulated). 34 of these transcripts were found significantly changed already early after a single exposure to WBCT. The majority of down-regulated transcripts belongs to the group of small nucleolar RNA (SNORD). An up-regulation was found amongst others for transcripts for PBX1, SFRP2, MAP2K3, and SLC25A39. Conclusions We observed significant changes in gene expression early in peripheral blood cells of fibromyalgia patients undergoing a WBCT. Small nucleolar RNAs represent the group with highest number of gene products that appear to be regulated by brief exposures to very cold temperatures. These molecules belong to so-called non-coding RNAs, which are considered as internal signals that control various levels of gene expression in physiology and development and are discussed as regulators of disease. Disclosure of Interest None Declared
Objective The cytokine IL-17 plays a crucial role in the development and promoting of inflammator... more Objective The cytokine IL-17 plays a crucial role in the development and promoting of inflammatory rheumatic diseases, such as psoriasis arthritis and ankylosing spondylitis. The influence of IL-17 on the osteoblast differentiation from mesenchymal stem cells has already been well studied. However, the effect of IL-17 on mature osteoblasts is not yet fully understood. Methods In this study, the influence of IL-17 on the expression of osteogenic markers and pro-inflammatory cytokines was analyzed on mRNA and protein level in an osteoblast cell culture model. Results Our data indicate that IL-17 alone has no significant influence on the expression of osteoblast-specific genes. However, a significant upregulation of pro-inflammatory cytokines at the transcriptional level by IL-17 was observed in primary osteoblasts. This effect on the regulation of pro-inflammatory cytokines was abolished completely by administration of a therapeutic anti-IL-17 antibody. Co-stimulation with TNF-α and IL-17 led to an upregulation of pro-inflammatory cytokines, which significantly exceeded the additive effect of both cytokines. In this co-stimulation, the anti-IL-17 antibody could not completely reverse the IL-17 effect. The same IL-17 and TNF-α effect was observed in osteoblast-like cells (MG63), whereas IL-17 alone did not induce the expression of pro-inflammatory cytokines. Conclusion The upregulation of the pro-inflammatory cytokines IL-1, IL-6, and IL-8 in primary osteoblasts by IL-17 indicates an indirect regulatory effect on osteoclastogenesis and activation of bone resorption. The therapeutic IL-17 antibody reduced the IL-17 induced release of pro-inflammatory cytokines by osteoblasts and this, in turn, could also reduce the effect on osteoclast differentiation and bone resorption. Our study underlines the important role of osteoblasts as major players in the osteoimmunologic network.
Background Interleukin (IL)-1 and metalloproteinases (MMP) are thought to play a crucial role in ... more Background Interleukin (IL)-1 and metalloproteinases (MMP) are thought to play a crucial role in the cartilage degradation of osteoarthritis (OA). Recently it has been proposed that CRP may be useful index of OA activity and severity. Objectives We studied the relationships between synovial fluid (SF) IL-1b, MMP1 and MMP3, and serum levels of CRP in patients with OA of the knee (KOA) to investigate the role of CRP in KOA. Methods In the SF from 25 patients with KOA (Altman criteria, Kellgren and Lawrence score of 2 or greater on X-ray plan films) we determined the levels of IL-1b, MMP-1 and MMP-3 (ELISA), along with WBC number. In the same patients serum concentration of CRP was analysed by ultrasensitive rate nephelometry. No patients were taking NSAIDs or other antirheumatic drugs. Results In SF, MMP1 was correlated with MMP3 (r = 0.61, p = 0.0009), and MMP3 with IL-1b (r = 0.67, p = 0.0002). In turn, serum CRP was correlated with MMP3 (r = 0.76, p < 0.0001) and, at lower level, with MMP1 (r = 0.49, p = 0.01) and IL-1b (r = 0.49, p = 0.01). Conclusion The correlations found between serum CRP and SF levels of substances believed to be crucial in OA cartilage degradation, such as MMPs and IL-1, confirm the role of marker of disease severity recently proposed for this acute phase protein.
Cre-directed S1P ablation (S1P Osx mice). S1P Osx mice and wild type (WT) littermates were studie... more Cre-directed S1P ablation (S1P Osx mice). S1P Osx mice and wild type (WT) littermates were studied by histology, in situ hybridization analysis (ISH), immunofluorescence (IF), real-time (RT) PCR and micro-computed tomography (mCT) techniques. Osteogenic differentiation assays analyzing for osteoblast differentiation in vitro were conducted with bone marrow harvested from 21 day old WT and mutant mice. Results: Mice with a homozygous deletion for S1P (S1P f/f ;Osx-Cre mice or S1P cko-Osx) suffer from severe chondrodysplasia; mice heterozygous for S1P deletion (S1P þ/f-Osx) have a size intermediate to S1P cko-Osx mice and WT littermates. Additionally, these mice are also kyphotic that gets more pronounced with age and was confirmed by mCT analysis. In situ
Background: Bone erosion in joint is the most deleterious effect induced by rheumatoid arthritis ... more Background: Bone erosion in joint is the most deleterious effect induced by rheumatoid arthritis (RA) and is the major cause of disability. New therapies are urgently required to prevent RA-associated bone destruction. The excessive bone resorption in RA was proved to be mediated by osteoclasts which are derived from the monocyte/macrophage lineage and induced by RANKL. Therefore, RANKL could be a potential target for the preservation of bone mass in RA. Objectives: Chrysotherapy showed potential effects in reducing of joint destruction but with serious side-effects in RA therapy. A rational designed gold nanoparticle named GN was prepared to provide a choice for suppressing bone loss in RA therapy with more safety. Methods: Mice macrophage cell line RAW264.7 was activated by RANKL to evaluate the potential osteoclastogenesis suppressing effects of GN. The differentiation, fusion and function of osteoclast were assessed by tartrateresistant acid (TRAP) staining, actin ring formation assay and osteologic discs detection respectively. Relative expressions of osteoclast-specific genes were evaluated by RT-PCR and the activity of NF-κB pathway as well as MAPK pathways were analyzed by immunoblotting. Results: Osteoclast differentiation, fusion and bone resorption were activated by RANKL in RAW264.7 cell, while GN significantly attenuates this process in a dose-dependent manner. The expressions of osteoclast-specific genes including TRAP and OSCAR were increased by RANKL stimulation but were effectively suppressed by GN. The activation of NF-kappa B pathway and MAPK pathways induced by RANKL was also suppressed by GN treatment. Conclusions: Our findings revealed that treatment with GN could prevent RANKLinduced osteoclastogenesis and reduce increased expression of osteoclastspecific genes which may through the suppression of NF-kB and MAPK activation in macrophage. GN may provide a basis for the design of chrysotherapy drugs that effect in progression of RA-associated articular erosions.
Outcome predictors of biologic therapeutic drugs like TNF inhibitors are of interest since side e... more Outcome predictors of biologic therapeutic drugs like TNF inhibitors are of interest since side effects like serious infections or malignancy cannot be completely ruled out. Response rates are heterogeneous. The present study addressed the question whether in patients with rheumatoid arthritis (RA) interleukin-10 (IL-10) promoter genotypes with potential relevance for IL-10 production capacity are associated with response to long-term treatment with etanercept. Caucasian RA patients that, according to the EULAR criteria, responded well (n = 25), moderately (n = 17) or not (n = 8) to etanercept therapy (median 36 months, range 4-52), and 160 matched controls were genotyped for the IL-10 promoter SNPs-2849 G>A (rs6703630),-1082 G>A (rs1800896),-819 C>T (rs1800871) and-592 C>A (rs1800872). Haplotypes were reconstructed via mathematic model and tested for associations with disease susceptibility and therapy response. We identified the four predominant haplotypes AGCC, GATA, GGCC, and GACC in almost equal distribution. Patients that responded well carried the putative IL-10 low producer allele-2849 A or the haplotypes AGCC and GATA (RR 2.1 and 4.0, respectively; 95% CI 1.1-4.0 and 1.1-14.8), whereas an unfavourable response was associated with carriage of the putative high producer haplotype GGCC (RR 1.9, 95% CI 1.1-3.3). No significant associations of alleles or haplotypes with disease susceptibility were observed. In RA, a low IL-10 production which is genetically determined rather by haplotypes than by SNPs may favour the response to etanercept treatment. Iatrogenic blockade of TNF may reveal proinflammatory effects of its endogeneous antagonist IL-10. Further studies are needed to correlate these genetic findings to direct cytokine measurements.
New experimental approaches are presented which are implemented in rheumatology research. DNA mic... more New experimental approaches are presented which are implemented in rheumatology research. DNA microarray technology and proteome analysis are two new methods which are applied to gain a global survey over the gene expression at the RNA and protein level under various conditions. Based on these methods of molecular medicine important functional proteins in the pathogenesis of rheumatoid arthritis (RA) as well as clinical relevant genetic polymorphisms shall be identified. New insights are expected which will help in the differentiation of clinical entities and in the search for new therapeutic strategies in the treatment of RA.
International Journal of Molecular Sciences, Oct 5, 2018
The balance of bone formation and resorption is the result of a regulated crosstalk between osteo... more The balance of bone formation and resorption is the result of a regulated crosstalk between osteoblasts, osteoclasts, and osteocytes. Inflammation, mechanical load, and external stimuli modulate this system. Exposure of bone cells to metal ions or wear particles are thought to cause osteolysis via activation of osteoclasts and inhibition of osteoblast activity. Co 2+ ions have been shown to impair osteoblast function and the expression of the three transforming growth factor (TGF)-β isoforms. The current study was performed to analyze how Co 2+ and Cr 3+ influence the expression, proliferation, and migration profile of osteoblast-like cells. The influence of Co 2+ , Cr 3+ , and CoCr particles on gene expression was analyzed using an osteogenesis PCR Array. The expression of different members of the TGF-β signaling cascade were down-regulated by Co 2+ , as well as several TGF-β regulated collagens, however, Cr 3+ had no effect. CoCr particles partially affected similar genes as the Co 2+ treatment. Total collagen production of Co 2+ treated osteoblasts was reduced, which can be explained by the reduced expression levels of various collagens. While proliferation of MG63 cells appears unaffected by Co 2+ , the migration capacity was impaired. Our data may improve the knowledge of changes in gene expression patterns, and the proliferation and migration effects caused by artificial materials.
AdA. Lesions developed more slowly in LA/CFA than in AdA. However, hock BMD as well as knee infla... more AdA. Lesions developed more slowly in LA/CFA than in AdA. However, hock BMD as well as knee inflammation and erosion scores found to be different (shown here at Onset + 5 days): Conclusion Inflammatory and skeletal changes for AdA and LA/ CFA were similar by clinical and histopathologic measures 10 days after onset, while inflammation receded by Onset + 25 days in LA/CFA more than it did in AdA. These data indicate that bone densitometric and morphologic findings can distinguish these two models of adjuvant arthritis despite their similar clinical presentations. This finding may be relevant in screening novel therapeutic candidates as these models may define different potential efficacious effects of a compound.
Background For about 15 years biological compounds belong to the therapeutic options for the trea... more Background For about 15 years biological compounds belong to the therapeutic options for the treatment of rheumatoid arthritis. By targeting of tumor necrosis factor alpha (TNFα), one of the key cytokines in the pathogenesis of rheumatoid arthritis (RA), patients benefit in terms of disease activity as well as radiologic progression. The application of biologic substances is subject to a risk to develop antibodies against these structures causing adverse events or loss of efficacy. Objectives It was the aim of the study to analyse the occurrence of anti-drug antibodies in the course of a long term treatment of patients with rheumatoid arthritis with etanercept (ETN). Methods In this retrospective analysis sera from 100 RA patients obtained during an up to 4 years treatment period with etanercept (after 6, 12, 24, 36 and 48 months) and 43 adalimumab (ADL)-treated patients were included for determination of anti-drug antibodies. If available the first serum sample obtained at least 2 weeks after discontinuation of ETN treatment (due to side effects, non-response, remission or surgery) was included for analysis to exclude any interference with high drug levels. For the determination of anti-ETN and anti-ADL levels Promonitor® -anti-ETN and –anti-ADL assays (Proteomika, Spain) were used according to the manufacturer's instructions. Both assays are designed as bridging assays with cut-off values of 142 AU/ml and 3.5 AU/ml for ETN and ADL, respectively. For calculation of results a data analysis software (http://www.myassays.com) was used. Results No anti-ETN antibodies were found in 100 RA patients, neither in the course of treatment nor in samples from 72 of these patients obtained after discontinuation of ETN treatment after mean 36.3 months (range 5-78 months). However, in the ADL-treated cohort anti-ADL antibodies were found in 51.2% of patients (22 out of 43) after discontinuation of therapy after mean 23.4 months (range 3-77 months). Conclusions Our study confirms the lack of anti-drug antibodies in a large cohort of RA patients in the course of a long-term treatment with ETN over up to 4 years. The absence of anti-ETN antibodies in serum samples after discontinuation of therapy confirms this result by exclusion of false negative results resulting from interference with high etanercept levels in the course of treatment. Acknowledgements Supported by Pfizer Pharma GbmH Disclosure of Interest S. Drynda Grant/research support from: Pfizer Pharma GmbH, R. Beuermann: None declared, J. Kekow: None declared
Background: In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and redu... more Background: In several studies an effect of whole-body cryotherapy (WBCT) on pain relief and reduction of inflammatory symptoms has been demonstrated and it is recommended for the treatment of arthritis, fibromyalgia and ankylosing spondylitis. The mode of action of this therapy, which consists in a brief exposure to temperatures between-110 and-160°C in special cryochambers, has not been fully elucidated. Objectives: The aim of this study was to investigate the changes in the gene expression of selected genes (CCL4, TGFBR3, CD69 and MAP2K3) identified as significantly regulated in a small pilot study using Affymetrix GeneChip ® Human Gene 1.0 ST arrays in cells from peripheral blood of patients with fibromyalgia going through a series of three exposures to WBCT within three days. Methods: Twenty two patients with fibromyalgia (20 female/2 male, age 51.7±8.9 years (mean ± SD) were included in the study and underwent 3 exposures to WBCT in a cryochamber system with 3 chambers (10 seconds at-10°C, 10 seconds at-60°C and for maximum 3 minutes at-110°C) at 3 consecutive days. During the study patients did not change their medication. Blood was collected immediately prior to (baseline) and directly after the first exposure to WBCT and after the third exposure using PAXgene™ RNA tubes. Total RNA was extracted with the PAXgene Blood RNA kit. Gene expression levels of MAP2K3, CCL4, TGFBR3, and CD69 were analysed by real-time PCR using sequence-specific primers and probes (TaqMan). Results: All 22 patients tolerated the application of the WBCT well and reported to benefit from this treatment. The expression levels of CCL4 in cells from peripheral blood reduced significantly to mean 67% in 19 of 22 patients after the third exposure compared to baseline. The expression level of CD69 was also reduced in the majority of patients (16 out of 22) significantly to 59%. In contrast, the expression of MAP2K3 was found to be up-regulated in 13 patients to mean 180%, while the expression levels in the other nine patients remained almost unchanged. The changes of gene expression levels observed after the third exposure compared to baseline became apparent already after the first cold exposure, without reaching statistical significance. The down-regulation of TGFBR3 observed in the pilot study could not be confirmed in the larger cohort. Conclusions: The results of our study indicate, that the whole-body cryotherapy may cause significant changes in gene expression levels of CCL4, CD69 and MAP2K3. The MAP2K3 expression is known to be regulated by environmental stress, which is in accordance with the observed up-regulation of MAP2K3 expression. The down-regulation of the CD69, a marker for T-cell activation and the chemokine CCL4 that is produced by T-cells indicate that the exposure to WBCT has an effect on peripheral T-cells. Whether this influence on T-cells contributes to the therapeutic effect needs to be elucidated.
Background: Remission is the current aim of treatment in rheumatoid arthritis (RA). Remission is ... more Background: Remission is the current aim of treatment in rheumatoid arthritis (RA). Remission is ideally the absence of detectable disease and absence of structural and functional worsening over the time. Infra-clinical synovitis, detected by ultrasound, may persist in patients with remission leading to further structural damage. Objectives: The aim of this study was to assess the frequency of relapse after clinical remission and the value of US to predict this relapse. Methods: A prospective study of 30 patients followed-up for RA in remission was conducted. The remission was defined by a Disease Activity Score 28 joints (DAS28) ≤2,6 for at least 3 months under the same therapy. A B-mode and Power Doppler (PD) US examination was assessed by an experienced rheumatologist blinded to clinical data for 22 joints (wrists, 10 metacarpo-phalangeals, 10 proximal interphalangeals joints). A semi-quantitative scale running from 0 to 3 depending on US activity was attributed for each joint for PD. Patients were clinically controlled 3 to 6 months after performing the US. The variability of DAS28 was noted, it was considered significant if the p value <0,05. Results: The mean age of the thirty patients was 48 years-old (25 females, 5 males). All of them were in remission according to DAS28 with an average of 2,03 [1,13-2,6]. US examination showed synovial hypertrophy in B-mode in 80% of patients and PD signals in 56,7% of patients. The follow-up control was performed on average after 4 months for 27 patients. The variation of DAS28 from baseline was significant (p<0,005). In fact the DAS28 increased of 0,75 [0,10-3,1] for 20 patients, among them 14 were no longer in remission. The DAS28 remained stable for 2 patients and decreased for 5 others. For patients who have reactivation of RA, the mean US PD scale was 4,4, while it was 1,1 for patients who remained in remission. However, the difference wasn't significant (p=0,12). Conclusions: For patients in remission, persistent active synovitis in US is a risk factor for futur reactivation of the disease. PD monitoring in daily practice might lead to reduce disease relapses and structural damages.
European journal of mass spectrometry, Feb 1, 2010
As the potential of epitope chips for routine application in diagnostics relies on the careful se... more As the potential of epitope chips for routine application in diagnostics relies on the careful selection of peptides, reliable epitope mapping results are of utmost interest to the medical community. Mass spectrometric epitope mapping, in combination with peptide chip analysis, showed that autoantibodies from patients who suffered from rheumatoid arthritis (RA) were directed against distinct surface structures on the full-length human autoantigen RA33 as well as against partial sequences. Using the combined mass spectrometric epitope extraction and peptide chip analysis approach, four sequence motifs on RA33 emerged as immuno-positive, showing that epitopes were not randomly distributed on the entire RA33 amino acid sequence. A sequential epitope motif (245GYGGG249) was determined on the C-terminal part of RA33 which matched with the Western blot patient screening results using the full-length protein and, thus, was regarded as a disease-associated epitope. Other epitope motifs were found on N-terminal partial sequences (59RSRGFGF65, 111KKLFVG116) and again on the C-terminal part (266NQQPSNYG273) of RA33. As recognition of these latter three motifs was also recorded by peptide chip analysis using control samples which were negative in the Western blot screening, these latter motifs were regarded as “cryptic epitopes”. Knowledge of disease-associated epitopes is crucial for improving the design of a customized epitope peptide chip for RA and mass spectrometric epitope mapping pivotally assisted with selecting the most informative peptide(s) to be used for future diagnostic purposes.
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Papers by Susanne Drynda