Article

Rituximab Treatment Induces the Expression of Genes Involved in Healing Processes in the Rheumatoid Arthritis Synovium

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Abstract

Rituximab displays therapeutic benefits in the treatment of patients with rheumatoid arthritis (RA) resistant to tumor necrosis factor (TNF) blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. We undertook this study to investigate the global molecular effects of rituximab in synovial biopsy samples obtained from anti-TNF-resistant RA patients before and after administration of the drug. Paired synovial biopsy samples were obtained from the affected knee of anti-TNF-resistant RA patients before (time 0) and 12 weeks after (time 12) initiation of rituximab therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction experiments were performed to confirm the differential expression of selected transcripts. According to Student's paired t-tests, 549 of 54,675 investigated probe sets were differentially expressed between time 0 and time 12. Pathway analysis revealed that genes down-regulated between time 0 and time 12 were significantly enriched in immunoglobulin genes and genes involved in chemotaxis, leukocyte activation, and immune responses (Gene Ontology annotations). In contrast, genes up-regulated between time 0 and time 12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (Gene Set Enrichment Analysis). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy. Rituximab displays unique effects on global gene expression profiles in the synovial tissue of RA patients. These observations open new perspectives in the understanding of the biologic effects of the drug and in the selection of patients likely to benefit from this therapy.

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... These clinical observations led to the hypothesis that DMARDs have convergent effects downstream of their immediate targets (the common pathway hypothesis). This is supported by a series of studies from our group showing that different DMARDs induce similar transcriptomic changes in paired (preversus posttreatment) RA synovial biopsies (6)(7)(8). ...
... We find abatacept mainly modulates lymphocyte-related transcripts (T Cellrelated genes and chemokines). By combining these data with those generated from four other DMARDs: methotrexate, tocilizumab, rituximab, and adalimumab (6)(7)(8), we provide compelling evidence, in a large series (50 pre-/post-treatment pairs) of synovial biopsies, for a shared set of highly inter-connected genes and pathways modulated downstream of RA therapies. ...
... Data from four other cohorts of RA patients with active disease, included in previous studies on pre/post treatment biopsies (6)(7)(8), were also analyzed: 2 × 8 patients treated with adalimumab (baseline cDMARD 8/8, 2 good responders (GR), 4 moderate responders (MR), and 2 non responders (MR); EULAR response criteria), 2 × 12 patients treated with rituximab (baseline cDMARD 12/12, 3 GR, 6 MR and 3 NR), 2 × 8 biopsies from patients treated with methotrexate (baseline cDMARD 0/8, 2 GR, 2 MR and 4 NR), and 2 × 12 patients treated with tocilizumab (baseline cDMARD 0/12, 7 GR, 4 MR and 1 NR). ...
Article
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Objectives Our goal was to assess for the histological and transcriptomic effects of abatacept on RA synovia, and to compare them with previously published data from four other DMARDs: tocilizumab, rituximab, methotrexate, and adalimumab. Methods Synovial tissue was obtained using ultrasound-guided biopsy from affected joints of 14 patients, before and 16 weeks after treatment with subcutaneous abatacept 125 mg weekly. Paraffin-sections were stained and scored for CD3 ⁺ , CD20 ⁺ , and CD68 ⁺ cell infiltration. Transcriptional profiling was performed using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix) and analyzed on Genespring GX (Agilent). Pathway analyses were performed on Genespring GX, Metascape, and EnrichR. Results Gene expression analysis identified 304 transcripts modulated by abatacept in synovial tissue. Downregulated genes were significantly enriched for immune processes, strongly overlapping with our findings on other therapies. Data were pooled across these studies, revealing that genes downregulated by DMARDs are significantly enriched for both T-cell and myeloid leukocyte activation pathways. Interestingly, DMARDs seem to have coordinate effects on the two pathways, with a stronger impact in good responders to therapy as compared to moderate and non-responders. Conclusion We provide evidence that the effects of five DMARDs on the RA synovium culminate in the same pathways. This confirms previous studies suggesting the existence of common mediators downstream of DMARDs, independent of their primary targets.
... In parallel, synovial biopsy samples were harvested from DMARD-naive RA patients before and 12 weeks after the initiation of MTX therapy. We previously identified specific molecular pathways that were associated with response to treatment with adalimumab (ADA) and rituximab (RTX) in RA patients (4,5). In the present study, we sought to determine whether the molecular changes induced in the synovium by treatment with TCZ might overlap with the molecular effects of these other treatments. ...
... The following parameters were evaluated: synovial hyperplasia, lymphoplasmacytic cell infiltrates, fibrinoid necrosis, and vascular hyperplasia. Immunolabeling experiments were performed using a standard protocol, as previously described (5). The following antibodies were used: anti-CD3 (Neomarkers), anti-CD20 (Biocare Medical), anti-CD68, and anti-CD138 (both from DakoCytomation). ...
... T cell activation and immune response pathways were not differentially affected in the TCZ-and MTX-treated patients, regardless of whether any patients achieved clinical remission. In patients who achieved remission at 6 months after the start of TCZ therapy, genes involved in, for example, induction of apoptosis and myeloid cell differentiation were more down-regulated, and genes involved in regulation of Ras protein signal transduction and ubiquitin-dependent protein catabolic processes were more up-regulated (see Supplementary Table 4 Previous studies by our group have demonstrated that ADA therapy and RTX therapy each display distinct molecular effects on global gene expression profiles in the RA synovium (4,5). In particular, ADA induces a significant down-regulation of genes involved in cell proliferation (4), whereas RTX down-regulates transcripts that are significantly enriched in immunoglobulin and T cell activation genes (5). ...
Article
Objective: To investigate the global molecular effects of tocilizumab (TCZ) in comparison with methotrexate (MTX) treatment in synovial biopsy tissue obtained from patients with previously untreated rheumatoid arthritis (RA) before therapy (T0) and 12 weeks after the initiation of therapy (T12), and to compare the results with previous gene expression data obtained in synovial biopsy tissue from adalimumab (ADA)- and rituximab (RTX)-treated patients with RA. Methods: Paired synovial biopsy samples were obtained at T0 and T12 from the affected knee of TCZ-treated RA patients and MTX-treated RA patients. Gene expression studies were performed using GeneChip Human Genome U133 Plus 2.0 microarrays, and confirmatory quantitative real-time reverse transcription-polymerase chain reaction experiments were performed on selected transcripts. The effects of TCZ and MTX on synovial cell populations and histologic characteristics were assessed by immunohistochemistry. Results: Gene expression studies showed that blockade of the interleukin-6 receptor (IL-6R) gene (IL6R) using TCZ induced a significant decrease in the expression of numerous chemokine and T cell activation genes in the RA synovium. These effects strongly correlated with the molecular effects of MTX and RTX therapy on RA synovial tissue, but differed from the molecular changes induced by ADA (decreased expression of genes involved in cell proliferation). Conclusion: The molecular similarities between the effects of TCZ, RTX, and MTX therapies in the RA synovium indicate that B cell- and IL-6-dependent pathways play synergistic roles in the pathogenesis of the disease, in particular through activation of T cell responses. Moreover, these results open perspectives for the individualization of therapeutic decisions, based on a better knowledge of the synovial molecular effects of each type of RA therapy.
... Recent microarray studies have focused on gene expression in synovial tissue and/or blood in an attempt to identify transcriptional profi les, better categorise patients and predict responsiveness to treatment. [2][3][4][5][6][7][8] These studies have yielded valuable information on molecular pathways involved in the disease pathogenesis and allowed identifi cation of transcriptional infl ammatory and repair/remodelling profi les in groups of patients. 4 8 Comprehensive quantitative PCR (qPCR) provides greater sensitivity and dynamic range for gene expression analysis, and might reveal additional biology. ...
... In line with our fi ndings, an infl ammatory expression pattern has previously been associated with response, but this study concluded that baseline serum IgG concentration and synovial immunoglobulin light chain were better discriminators between responders and non-responders than synovial gene expression. 2 To identify which baseline genes contributed most to response, we created a GS using genes that best correlated with ΔDAS BL-M. This baseline GS also correlated with response at months 9, 15 and 21. ...
Article
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Personalised healthcare is contingent on the identification of biomarkers that represent disease relevant pathways and predict drug response. The authors aimed to develop a gene expression signature in synovial tissue that could enrich clinical response of rheumatoid arthritis (RA) patients to rituximab. The authors studied synovial gene expression using high-throughput quantitative real-time-PCR in 20 RA patients who underwent arthroscopy before and after treatment with rituximab. Several objective approaches were used to explore patterns in the data and to find genes associated with changes in disease activity due to treatment. This analysis revealed two patient populations associated with distinct clinical, laboratory and histological features and, importantly, showed enrichment for response (60% non-responders vs 90% responders). A composite baseline gene score (GS) correlated with change in disease activity score (ΔDAS) between baseline and month 3 (r=0.74, p=0.0002), but also with ΔDAS at later time-points (month 9, r=0.54, p=0.016; month 15, r=0.45, p=0.06; month 21, r=0.72, p=0.003). Notably, the GS significantly correlated with baseline erythrocyte sedimentation rate (r=0.69, p=0.0008), but not with other DAS components. The GS genes represented T cell, macrophage, remodelling and interferon-α biology. Responders demonstrated higher expression of macrophage and T cell genes, while non-responders showed higher expression of interferon-α and remodelling genes. This study reveals a baseline synovial GS that correlates with early and late clinical responses to rituximab. The GS biology suggests that T cells and macrophages are important for response to B cell depleting therapy, while expression of remodelling and interferon-α genes correlates with poor response.
... Recent microarray studies have focused on gene expression in synovial tissue and/or blood in an attempt to identify transcriptional profi les, better categorise patients and predict responsiveness to treatment. [2][3][4][5][6][7][8] These studies have yielded valuable information on molecular pathways involved in the disease pathogenesis and allowed identifi cation of transcriptional infl ammatory and repair/remodelling profi les in groups of patients. 4 8 Comprehensive quantitative PCR (qPCR) provides greater sensitivity and dynamic range for gene expression analysis, and might reveal additional biology. ...
... In line with our fi ndings, an infl ammatory expression pattern has previously been associated with response, but this study concluded that baseline serum IgG concentration and synovial immunoglobulin light chain were better discriminators between responders and non-responders than synovial gene expression. 2 To identify which baseline genes contributed most to response, we created a GS using genes that best correlated with ΔDAS BL-M. This baseline GS also correlated with response at months 9, 15 and 21. ...
Article
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Objective To determine the effects of the B cell depleting drug rituximab on gene expression in synovial tissue from patients with refractory rheumatoid arthritis (RA) who have been on rituximab for 2 years.Methods Synovial tissue biopsies were collected from 19 patients with refractory RA at baseline, and 3 months after the first infusion of rituximab. 117 genes of interest were selected for analysis, including immune cell genes and fibrosis genes. Analysis was carried out using microfluidic real-time q-PCR and CT values were then obtained using the fluidigm gene expression data analysis software (version 2.1.1 Fluidigm).ResultsB cell depletion was evident in both responders and non-responders of rituximab between baseline and 3 months as assessed by CD20 gene expression. An unsupervised hierarchical cluster analysis revealed the presence of two groups of RA patients. One with high inflammation at baseline and one with low inflammation at baseline. The high inflammation group had significantly higher Disease Activity Score using 28 joint counts (DAS28), higher C reactive protein levels, higher erythrocyte sedimentation rate and a greater change in DAS28 over 3 months.Conclusion These results show the existence of two distinct RA patient subgroups, where high inflammation tissue is associated with more severe disease and a better response to rituximab.
... In other studies, we evaluated the effects of therapies on global gene expression patterns in prospective synovial biopsy samples obtained prior to and 3 months after initiation of therapy with methotrexate, tocilizumab, rituximab or adalimumab. We showed that methotrexate, tocilizumab and rituximab display very similar molecular effects in RA synovitis, characterized by a decrease in T cell activation genes [5,6]. By contrast, TNF blockade resulted in a decrease in the expression of transcripts involved in cell proliferation and inflammation. ...
... Transcriptomic data (GeneChip Human Genome U133 Plus2.0.CEL files, Affymetrix) from 65 samples obtained by needle-arthroscopic knee synovial biopsy were used in the present analyses. These samples were obtained in untreated RA patients (<1 year disease duration for the majority of them), prior to and 3 months after initiation of tocilizumab (n = 13 and 12 samples, respectively) or methotrexate (n = 2 × 8 samples) therapy [GEO:GSE45867] (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/geo), and in RA patients resistant to TNF blockade, prior to and 3 months after administration of rituximab (n = 2 × 12 samples, GSE24742) therapy [5,6]. All patients met the ACR/European League Against Rheumatism (EULAR) 2010 RA classification criteria, and the ACR 1987 revised classification for RA. ...
Article
Background Gene expression profiling studies indicate that IL6-related T cell activation, and TNFα-dependent cell proliferation are major targets of therapy in the RA synovium1. We investigated whether expression of these pathways in early RA synovial biopsies is associated with clinically relevant information. Methods We performed global transcriptomic studies (HGU133 Plus2.0) on early RA synovial biopsies, (GSE45867) and in TNFα-stimulated synovial fibroblasts (GSE15615). Immunostaining experiments (GADD45B, PDE4D) were performed on independent sets of early untreated RA samples, obtained by needle-arthroscopy (n=46), or by US-guided biopsies (n=35), next quantitated digitally. Results In an initial set of 20 untreated early RA patients, 1,431 transcripts displayed at least a moderate correlation (r >0.4) and 77 of them displayed a good correlation (r >0.6) with DAS28-CRP. T cell associated genes were enriched in the 0.4 – 0.6 correlation range, while 38 out of the 77 transcripts with a correlation >0.6, were found to be induced by TNFα in cultured synovial fibroblasts (GADD45B, PDE4D, and CXCL14 were represented several times, by different probe sets). Immunostaining experiments on 46 independent synovial biopsy samples confirmed a higher PDE4D (median score 3.23 vs. 0.57, p =0.04) and GADD45B (median 0.60 vs. 0.31, p =0.09) staining in patients with DAS28-CRP >5.1. Higher synovial expression of TNFα-induced genes predicts absence of response to TNF blockade in MTX-resistant RA patients2. We therefore wondered whether higher expression of these genes at baseline also predicts absence of response to first line therapy in early RA. In the microarray data, expression of 6 (GADD45B (x2), PDE4D, ADAMTS1, WWP2, MPPED1) out of 38 TNFα-dependent probe sets was significantly higher in patients who did not reach SDAI remission at month 6 in response to MTX therapy (all patients were DAS responders). In an independent group of patients, immunostaining of GADD45B (median score 2.39 vs. 0.29, p =0.002) and PDE4D (median score 5.47 vs. 0.48, p =0.002) produced a higher signal in baseline synovial biopsies of 14 EULAR non-responders (at 3 months) out of 46 early RA patients who received first line therapy, and in 8 non-responders out of the 16 who received methotrexate as a first line agent (GADD45B: 2.87 vs. 0.25, p =0.01; PDE4D: 7.74 vs. 0.48, p =0.07). Similarly, GADD45B immunostaining in US-guided biopsies was significantly higher at baseline in another set of 15 non-responders out of 35 early RA patients treated with MTX (median 0.76 vs. 0.22, p =0.03); no significant difference in PDE4D staining was observed in this set of samples. Disease activity at baseline itself did not predict response to therapy. Conclusions Higher expression of TNFα-induced transcripts in early RA synovitis drives disease activity, and predicts poor response to first-line therapy. These results are important for patients' stratification in clinical trials, and open perspectives in terms of personalized medicine approaches in clinical practice. References Disclosure of Interest A. De Groof: None declared, F. Humby: None declared, J. Ducreux: None declared, S. Kelly: None declared, A. Nzeusseu Toukap: None declared, C. Pitzalis: None declared, P. Durez: None declared, B. Lauwerys Shareholder of: DNAlytics
... By contrast, adalimumab (a TNFα-blocking antibody) displayed relatively stronger effects on proportions of CD68 positive cells compared to other synovial cell populations, 3 months after administration of the drug to methotrexate-resistant RA patients (45). We also investigated the effects of rituximab (anti-CD20 antibody) therapy on synovial cell populations before and 3 months after therapy (46). We found that B cells were depleted in the majority (18/20) of the samples, but the drug also displayed a significant effect on IL17 producing T cells (47), thereby supporting the hypothesis that B cells also play a role as antigenpresenting cells in RA synovitis. ...
... The results of single cell RNA sequencing studies will make it easier to understand how specific drugs interfere with dysregulated cellular pathways in the disease. This is for example the case regarding the induction of transcripts involved in wound healing pathways in response to several drugs (23,46), an effect in which increased representation of resident cells (following a decrease in the presence of inflammatory cells) could play a role. Nevertheless, such description of the global effects of these drugs in the synovium provided clinicians and researchers with unique tools, leading to new research hypotheses on e.g., potential drug interactions in the treatment of RA (drugs that do not share the same molecular effects might have additive or synergistic effects) and response to therapy. ...
Article
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Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease targeting the joints. Current treatment strategies are based on clinical, biological and radiological features, yet still fail to reach the goal of early low disease activity in a significant number of cases. Hence, there is a need for refining current treatment algorithms, using accurate markers of response to therapy. Because RA induces histological and molecular alterations in the synovium even before apparition of clinical symptoms, synovial biopsies are a promising tool in the search of such new biomarkers. Histological and molecular characteristics of RA synovitis are heterogeneous. Variations in synovial lining layer hyperplasia, in cellular infiltration of the sublining by immune cells of myeloid and lymphoid lineages, and in molecular triggers of these features are currently categorized using well-defined pathotypes: myeloid, lymphoid, fibroid and pauci-immune. Here, we first bring the plasticity of RA synovitis under scrutiny, i.e., how variations in synovial characteristics are associated with relevant clinical features (disease duration, disease activity, effects of therapies, disease severity). Primary response to a specific drug could be, at least theoretically, related to the representation of the molecular pathway targeted by the drug in the synovium. Alternatively, absence of primary response to a specific agent could be due to disease severity, i.e., overrepresentation of all synovial molecular pathways driving disease activity overwhelming the capacity of any drug to block them. Using this theoretical frame, we will highlight how the findings of previous studies trying to link response to therapy with synovial changes provide promising perspectives on bridging the gap to personalized medicine in RA.
... In other studies, we evaluated the effects of therapies on global gene expression patterns in prospective synovial biopsy samples obtained prior to and 3 months after initiation of therapy with methotrexate, tocilizumab, rituximab or adalimumab. We showed that methotrexate, tocilizumab and rituximab display very similar molecular effects in RA synovitis, characterized by a decrease in T cell activation genes [5,6]. By contrast, TNF blockade resulted in a decrease in the expression of transcripts involved in cell proliferation and inflammation. ...
... Transcriptomic data (GeneChip Human Genome U133 Plus2.0.CEL files, Affymetrix) from 65 samples obtained by needle-arthroscopic knee synovial biopsy were used in the present analyses. These samples were obtained in untreated RA patients (<1 year disease duration for the majority of them), prior to and 3 months after initiation of tocilizumab (n = 13 and 12 samples, respectively) or methotrexate (n = 2 × 8 samples) therapy [GEO:GSE45867] (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/geo), and in RA patients resistant to TNF blockade, prior to and 3 months after administration of rituximab (n = 2 × 12 samples, GSE24742) therapy [5,6]. All patients met the ACR/European League Against Rheumatism (EULAR) 2010 RA classification criteria, and the ACR 1987 revised classification for RA. ...
Article
Full-text available
Background: IL6-related T cell activation and TNFα-dependent cell proliferation are major targets of therapy in the RA synovium. We investigated whether expression of these pathways in RA synovial biopsies is associated with disease activity and response to therapy. Method: Correlation and gene set enrichment studies were performed using gene expression profiles from RA synovial biopsies. Immunostaining experiments of GADD45B and PDE4D were performed on independent additional sets of early untreated RA samples, obtained in two different centers by needle-arthroscopy or US-guided biopsies. Results: In 65 RA synovial biopsies, transcripts correlating with disease activity were strongly enriched in TNFα-induced genes. Out of the individual variables used in disease-activity scores, tender joint count, swollen joint count and physician's global assessment, but not CRP or patient's global assessment displayed a similar correlation with the expression of TNFα-dependent genes. In addition, TNFα-induced genes were also significantly enriched in transcripts over-expressed in synovial biopsy samples obtained from poor-responders to methotrexate or tocilizumab, prior to initiation of therapy. GADD45B (induced by TNFα in monocytes) and PDE4D (induced by TNFα in FLS) immunostaining was significantly higher in overall poor-responders to therapy in 46 independent baseline samples obtained from early untreated RA patients prior to initiation of therapy. GADD45B (but not PDE4D) immunostaining was significantly higher in the sub-group of patients with poor-response to methotrexate therapy, and this was confirmed in another population of methotrexate-treated patients. Conclusion: Higher expression of TNFα-induced transcripts in early RA synovitis is associated with higher disease activity, and predicts poor response to first-line therapy. That over-expression of TNFα-induced genes predicts poor-response to therapy regardless of the drug administered, indicates that this molecular signature is associated with disease severity, rather than with specific pathways of escape to therapy.
... Needle arthroscopy using 2-mm grasping forceps and obtaining 6 biopsy specimens provided at least 15-50 mg for further RNA analysis [49]. Other studies using needle arthroscopy approach showed that at least 4 biopsy samples provided 1-2 μg of total RNA [50,51]. Yield from ultrasound guided synovial tissue biopsy taking 6 samples with 16/14G quick core needles provided at least 10 mg of synovial tissue for RNA isolation, with a median RNA yield 0.54-0.89 ...
... Examples of comprehensive gene expression analysis aiming for individualized therapy and understanding of mode of action of novel therapies are summarized in Table 2. Importantly, the differences between study designs and selection of patients should be considered. For example, treatment with methotrexate, tocilizumab and rituximab had similar molecular effects on transcriptomic changes (albeit of different magnitudes) in the RA synovium, which were distinct from the molecular changes induced by adalimumab [50,51,73]. It is important to notice that patients included in these trials were at different stages of the disease, since patients on tocilizumab or methotrexate were treatment naive, patients on adalimumab had failed DMARD therapy and patients commencing rituximab were both DMARDs anti-TNF failures. ...
Article
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Rheumatoid arthritis (RA) is an autoimmune disease which causes significant pain, joint deformity, functional disability. The pathological hallmark of RA is inflammation of the synovium characterized by involvement of inflammatory and resident stromal cells, soluble mediators and signalling pathways leading to irreversible joint destruction. The treatment goal in RA has evolved over the last decade towards a target of disease remission that is achieved in less than a third of patients in clinical trials. The lack of therapeutic response to current treatments is suggestive of alternative drivers of RA pathogenesis that might serve as promising therapeutic targets. There are data to justify the use of synovial tissue in early drug development. Synovial tissue represents an appropriate compartment to be studied in patients with inflammatory arthritis and provides information that is distinct from peripheral blood. Modern techniques have made the procedure much more accessible and ultrasound guided biopsies represent a safe and acceptable option. Advances in analytic technologies allowing transcriptomic level of analysis can provide unique inside to target organ/tissue following the exposure to investigational medicinal product. However, there are still caveats with regard to both the choice of technique and analytical methods. Therefore the significance of synovial biopsy remains to be determined in future clinical trials. The aim of the current debate is to explore the potential for accessing and evaluating synovial tissue in early drug development, to summarize lessons we have learned from clinical trials and to discuss the challenges that have arisen so far.
... The biopsy-driven observational studies that enrolled RA patients have suggested that certain synovial tissue signatures are associated with treatment response to TNF-i, IL-6 and Bcell depletion therapy (53)(54)(55)(56)(57). However, autoimmune diseases such as RA are usually treated without biopsy (joint biopsy). ...
Article
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The establishment of precision medicine is considered particularly important in heterogeneous autoimmune diseases (e.g., psoriatic arthritis, systemic lupus erythematosus), which reveal clinical and molecular heterogeneity. The selection of optimal treatment strategies for individual patients may be more important and complex in autoimmune diseases than in other diseases. Two factors are important in precision medicine: patient stratification and use of targeted. When both factors work, patients are likely to have good outcomes. However, research into precision medicine and its practice in systemic autoimmune diseases is lacking. In contrast, the usefulness of peripheral immune cell phenotyping in the evaluation of immunological characteristics and stratification into subgroups of individual patients with systemic autoimmune diseases such as immunoglobulin 4-related disease, systemic lupus erythematosus, and anti-neutrophil cytoplasmic antibody-related vasculitis was reported. Furthermore, the potential of precision medicine using biological disease-modifying antirheumatic drugs based on peripheral immune cell phenotyping was recently demonstrated for psoriatic arthritis in the clinical setting. Precision medicine has not yet been sufficiently investigated in real world clinical settings. However, a dawn of precision medicine has emerged. We should shed further light on precision medicine in PsA and other autoimmune diseases. Here, we first review the usefulness of peripheral immune cell phenotyping in systemic autoimmune diseases and the potential of precision medicine in PsA based on this method.
... Clinical improvement may be associated with IL-15/memory T-cell-related mechanisms. 61 (IL-15 is known to expand and activate NK cells.) 7. Decreased IL-2, IL-6, IL-7, and IL-10 serum levels 62 8. Decreased production of IFN-γ and IL-1β by T cells 55 9. Downregulation of genes involved in immunoglobulins, chemotaxis, leukocyte activation, and immune responses with upregulation of genes involved in TGF-β pathway in synovial tissue 63 1. Although anti-T-cell therapies (eg, anti-CD3 antibody) are effective in treatment of DM1, 85 RTX was also shown to be helpful in new-onset DM1 86 2. Reduction in autoantibody production or autoreactive B-cell counts 87 3. Increased Treg cells in short term 86 4. Modification of the B7-2/CD28 co-stimulation pathway that plays a critical role in priming islet-reactive Th cells 88 Note. ...
Article
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Treatment with rituximab, a monoclonal antibody against the B-lymphocyte surface protein CD20, leads to the depletion of B cells. Recently, rituximab was reported to effectively prevent relapses of glucocorticoid-dependent or frequently relapsing minimal change disease (MCD). MCD is thought to be T-cell mediated; how rituximab controls MCD is not understood. In this review, we summarize key clinical studies demonstrating the efficacy of rituximab in idiopathic nephrotic syndrome, mainly MCD. We then discuss immunological features of this disease and potential mechanisms of action of rituximab in its treatment based on what is known about the therapeutic action of rituximab in other immune-mediated disorders. We believe that studies aimed at understanding the mechanisms of action of rituximab in MCD will provide a novel approach to resolve the elusive immune pathophysiology of MCD.
... Although these synovial changes lead to shared clinical and biological manifestations, hence the common diagnostic denomination, it is possible that the molecular profiles identified in patients with UA translate into distinct patterns of disease evolution and response to therapy, and, therefore, into different needs of medical intervention. Accordingly, we recently identified synovial markers of response to methotrexate, TNF blockade, tocilizumab or rituximab therapy in patients with established RA, a demonstration that a "molecular" diagnosis and characterization of arthritis can lead to specific and clinically relevant decisions [14,15,19,20]. Undoubtedly, such predictive information about disease progression and response to therapy matters for the clinician as much as, if not more than a diagnostic label. ...
Article
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Objectives: Early diagnosis of rheumatoid arthritis (RA) is an unmet medical need in the field of rheumatology. Previously, we performed high-density transcriptomic studies on synovial biopsies from patients with arthritis, and found that synovial gene expression profiles were significantly different according to the underlying disorder. Here, we wanted to further explore the consistency of the gene expression signals in synovial biopsies of patients with arthritis, using low-density platforms. Methods: Low-density assays (cDNA microarray and microfluidics qPCR) were designed, based on the results of the high-density microarray data. Knee synovial biopsies were obtained from patients with RA, spondyloarthropathies (SA) or osteoarthritis (OA) (n = 39), and also from patients with initial undifferentiated arthritis (UA) (n = 49). Results: According to high-density microarray data, several molecular pathways are differentially expressed in patients with RA, SA and OA: T and B cell activation, chromatin remodelling, RAS GTPase activation and extracellular matrix regulation. Strikingly, disease activity (DAS28-CRP) has a significant influence on gene expression patterns in RA samples. Using the low-density assays, samples from patients with OA are easily discriminated from RA and SA samples. However, overlapping molecular patterns are found, in particular between RA and SA biopsies. Therefore, prediction of the clinical diagnosis based on gene expression data results in a diagnostic accuracy of 56.8%, which is increased up to 98.6% by the addition of specific clinical symptoms in the prediction algorithm. Similar observations are made in initial UA samples, in which overlapping molecular patterns also impact the accuracy of the diagnostic algorithm. When clinical symptoms are added, the diagnostic accuracy is strongly improved. Conclusions: Gene expression signatures are overall different in patients with OA, RA and SA, but overlapping molecular signatures are found in patients with these conditions. Therefore, an accurate diagnosis in patients with UA requires a combination of gene expression and clinical data.
... In order to gauge the usefulness of cell-centered information, we attempted to extract the information for differential roles of cell types before and after treatments of three agents including infliximab (IFX; anti-TNF-α), tocilizumab (TCZ; anti-IL-6), and rituximab (RTX; anti-CD20) from three mRNA expression datasets from synovial tissues of RA patients. [44][45][46] The information was extracted based on the cell type enrichment analysis (CTen) software. 31 Figure. ...
Chapter
Human autoimmune diseases arise from complex interplays between innate and acquired immune systems. Molecular and cellular interactions underlying the interplays are highly complex. Different types of cells, such as macrophages, dendritic cells, and T and B cells, cross-talk via secretion of various cytokines, called cytokine networks. Furthermore, in the individual cells, several signaling pathways, such as toll-like receptor (TLR) and JAK-STAT signaling pathways, are collectively or serially activated during disease progression and interact with each other. The complex interplays at both molecular and cellular levels define autoimmune disease phenotypes. Thus, understanding the complex interplays at molecular and cellular levels is crucial because they will allow us to develop new therapeutic strategies for the autoimmune diseases or to accurately determine the state of the autoimmune diseases for optimizing therapeutic options
... A longitudinal study of synovial tissue showed downregulation of genes, encoding immunoglobulins, chemotaxis, leucocyte activation, and immune response after RTX therapy, whereas gene expression associated with cell developmental processes and tissue regeneration increased [25]. Another longitudinal study has shown that good responders demonstrated increased expression of type I IFN-response genes [18]. ...
Article
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Objective. To clarify molecular mechanisms for the response to rituximab in a longitudinal study. Methods. Peripheral blood from 16 RA patients treated with rituximab for a single treatment course and 26 healthy controls, blood and knee articular cartilages from 18 patients with long-standing RA, and cartilages from 14 healthy subjects were examined. Clinical response was assessed using ESR, ACPA, CRP, RF, DAS28 levels, CD19+ B-cell counts, bone erosion, and joint space narrowing scores. Protein expression in PBMCs was quantified using ELISA. Gene expression was performed with quantitative real-time PCR. Results. A decrease ( p < 0.05 ) in DAS28, ESR, and CRP values after rituximab treatment was associated with the downregulation of MTOR, p21, caspase 3, ULK1, TNFα, IL-1β, and cathepsin K gene expression in the peripheral blood to levels found in healthy subjects. MMP-9 expression remained significantly higher compared to controls although decreased ( p < 0.05 ) versus baseline. A negative correlation between baseline ULK1 gene expression and the number of tender joints at the end of follow-up was observed. Conclusions. The response to rituximab was associated with decreased MTOR, p21, caspase 3, ULK1, TNFα, IL-1β, and cathepsin K gene expression compared to healthy subjects. Residual increased expression in MMP-9, IFNα, and COX2 might account for remaining inflammation and pain. High baseline ULK1 gene expression indicates a good response in respect to pain.
... Исследования динамики экспрессии генов на фоне терапии РТМ малочисленны. Так, в синовиальной ткани отмечалось снижение экспрессии генов, кодирующих иммуноглобулины, хемотаксис, активацию лейкоцитов и иммунный ответ; напротив, экспрессия генов, связанных с онтогенезом клеток и регенерацией тканей, усиливалась в ходе лечения РТМ [26]. Кроме того, у хорошо отвечавших на терапию РТМ в крови наблюдалось увеличение экспрессии генов кластера ИФН I типа [27]. ...
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Objective: to determine the predictors of the efficiency of rituximab (RTM) therapy through analysis of blood gene expressions in patients with rheumatoid arthritis (RA). Subjects and methods. Sixteen patients (mean age 53.4±10.8 years) with RA (mean duration 8.2±7.1 years) who had previ- ously received disease-modifying antirheumatic drugs and tumor necrosis factor-α (TNF-α) inhibitors without effects were examined. Each patient underwent a treatment cycle with RTM in a dose of 0.5-1 g. A control group included 26 healthy individuals. Clinical response was assessed with DAS28. Erythrocyte sedimentation rate (ESR), serum levels of anti-cyclic citrullinated peptide antibodies, C-reactive protein (CRP), and rheumatoid factor (RF) were estimated. Bone erosions and joint space narrowing were evaluated radiologically. RNA was isolated from blood and used to estimate the expression of the mTOR, ULK1, caspase 3, p21, TNF-α, cathepsin K, matrix metalloproteinase-9 (MMP-9), interleukin-1β (IL-1β), inter- feron-γ (IFN-γ), and cyclooxygenase-2 (COX-2) genes by real-time reverse transcriptase polymerase chain reaction. Results. At the beginning of the investigation, the expression of all the genes under study was increased (p < 0.05) in the patients with RA versus the healthy individuals. RTM therapy resulted in reductions in DAS28, ESR, CRP levels, and CD10+ B lymphocyte depletion (p < 0.05). There were no changes in the number of erosions and the width of the joint space during RTM therapy. The blood expression of the mTOR, p21, caspase 3, ULK1, TNF-α, IL-1β, and cathepsin K genes was suppressed to that of healthy individuals. As compared to the beginning of the investigation, the expression of MMP-9 was also reduced (p < 0.05); however, it remained far higher than that in the controls and no drastic changes occurred in the expression of the IFN-р and COX-2 genes. Conclusion. Blood gene expression analysis may serve as a source of information on the status of patients with RA dur- ing RTM therapy. The higher residual expression of MMP-9, IFN-γ, and COX-2 may be a reason for the preserved activity of RA and its exacerbation.
... Rituximab (RTX) is an anti-CD20 targeting B cells. No clear reduction in the infiltration of MP has been reported [96]. However, despite not directly targeting MP, in treated patients, RTX alters monocyte-derived MP functions with an increase in B cell activating factor (BAFF), IL10, and CD86 expression, as well as a decrease in TNF secretion [97]. ...
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Monocytes and their tissue counterpart macrophages (MP) constitute the front line of the immune system. Indeed, they are able to rapidly and efficiently detect both external and internal danger signals, thereby activating the immune system to eradicate the disturbing biological, chemical, or physical agents. They are also in charge of the control of the immune response and account for the repair of the damaged tissues, eventually restoring tissue homeostasis. The balance between these dual activities must be thoroughly controlled in space and time. Any sustained unbalanced response of MP leads to pathological disorders, such as chronic inflammation, or favors cancer development and progression. In this review, we take advantage of our expertise in chronic inflammation, especially in rheumatoid arthritis, and in cancer, to highlight the pivotal role of MP in the physiopathology of these disorders and to emphasize the repolarization of unbalanced MP as a promising therapeutic strategy to control these diseases.
... In order to gauge the usefulness of cell-centered information, we attempted to extract the information for differential roles of cell types before and after treatments of three agents including infliximab (IFX; anti-TNF-α), tocilizumab (TCZ; anti-IL-6), and rituximab (RTX; anti-CD20) from three mRNA expression datasets from synovial tissues of RA patients. [44][45][46] The information was extracted based on the cell type enrichment analysis (CTen) software. 31 Figure. ...
... GSE24742 [13] is from a study investigating the global molecular effects of rituximab in synovial biopsies obtained from 12 anti-TNF resistant rheumatoid arthritis (RA) patients before and after administration of the drug (rituximab). For each of the 24 samples, the expression levels of 54,675 gene probes were measured by Affymetrix Human Genome U133 Plus 2.0 array. ...
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Detecting disease-associated genomic outcomes is one of the key steps in precision medicine research. Cutting-edge high-throughput technologies enable researchers to unbiasedly test if genomic outcomes are associated with disease of interest. However, these technologies also include the challenges associated with the analysis of genome-wide data. Two big challenges are (1) how to reduce the effects of technical noise; and (2) how to handle the curse of dimensionality (i.e., number of variables are way larger than the number of samples). To tackle these challenges, we propose a constrained mixture of Bayesian hierarchical models (MBHM) for detecting disease-associated genomic outcomes for data obtained from paired/matched designs. Paired/matched designs can effectively reduce effects of confounding factors. MBHM does not involve multiple testing, hence does not have the problem of the curse of dimensionality. It also could borrow information across genes so that it can be used for whole genome data with small sample sizes.
... This dataset analyses the rheumatoid arthritis synovium response to rituximab (RTX) therapy. The dataset contains paired samples from 12 Homo Sapiens, one sample prior to the start of the therapy and one sample after 12 weeks of the RTX therapy [43]. The total numbers of probe-sets in the data set are 54,675, whose behaviour was studied and analysed across all the 24 samples. ...
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Gene Regulatory Networks (GRNs) are reconstructed from the microarray gene expression data through diversified computational approaches. This process ensues in symmetric and diagonal interaction of gene pairs that cannot be modelled as direct activation, inhibition, and self-regulatory interactions. The values of gene co-expressions could help in identifying co-regulations among them. The proposed approach aims at computing the differences in variances of co-expressed genes rather than computing differences in values of mean expressions across experimental conditions. It adopts multivariate co-variances using principal component analysis (PCA) to predict an asymmetric and non-diagonal gene interaction matrix, to select only those gene pair interactions that exhibit the maximum variances in gene regulatory expressions. The asymmetric gene regulatory interactions help in identifying the controlling regulatory agents, thus lowering the false positive rate by minimizing the connections between previously unlinked network components. The experimental results on real as well as in silico datasets including time-series RTX therapy, Arabidopsis thaliana, DREAM-3, and DREAM-8 datasets, in comparison with existing state-of-the-art approaches demonstrated the enhanced performance of the proposed approach for predicting positive and negative feedback loops and self-regulatory interactions. The generated GRNs hold the potential in determining the real nature of gene pair regulatory interactions.
... After 3 months of treatment with adalimumab, patients with RA and an inadequate response to methotrexate had a marked decrease in CD 68 + cells (58). Finally, 3 months of treatment with rituximab resulted in a significant reduction in both B cells and IL-17-producing T cells in patients with RA (59). ...
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Ultrasound-guided synovial biopsy is a safe, well-tolerated, and effective method to collect good-quality synovial tissue from all types of joints for clinical and research purposes. Although synovial biopsy cannot be used to distinguish between types of inflammatory rheumatic disease, analysis of synovial tissue has led to remarkable advances in the understanding of the pathobiology of rheumatoid arthritis and other inflammatory rheumatic diseases. Synovitis is the hallmark of these diseases; hence, accessing the core of the pathological process, synovial tissue, provides an opportunity to gather information with potential diagnostic and prognostic utility.
... Таким образом, применение РТМ сопровождается снижением уровня всего спектра медиаторов, принимающих участие в различных звеньях патогенеза РА. Учитывая выраженное уменьшение показателей цитокинового профиля, можно говорить о подавлении активации CD4+ Тлимфоцитов на фоне терапии РТМ, что подтверждается результатами работ других авторов [46,47]. ...
Article
The aim of the investigation was to study the changes of cytokine profile parameters in patients with rheumatoid arthritis (RA) 12 and 24 weeks after initiation of therapy with rituximab (RTM) biosimilar at a total dose of 1200 mg, in comparison with the original drug Material and methods. The study included 54 patients with a reliable diagnosis of RA. Depending on the therapy, all patients were divided into two groups: 34 patients received the original RTM (group 1) and 20 patients – biosimilar (group 2) in a total dose of 1200 mg according to the standard scheme. The concentration of 27 cytokines in blood serum was determined by multiplex xMAP technology on the analyzer Bio-Plex Array System (BIO-RAD, USA). Results and discussion. The use of the original drug has been accompanied by reliable and significant reduction (over 30%) by 24 weeks of treatment levels of proinflammatory [interleukin (IL) 1â, IL2, IL6, IL12, IL15, interferon ã (IFN-ã), tumor necrosis factor á (TNF-á)], IL1 receptor antagonist (IL1ra), IL5, IL9, IL10, IL13 cytokines, growth factors (IL7, granulocyte-macrophage colony stimulating factor, fibroblast growth factor) and chemokines (monocyte chemoattractant protein 1 – MCP1). During the treatment with Acellbia a rapid and marked reduction in the concentration of practically the whole range of investigated parameters already 12–24 weeks after the first infusion was achieved. After 24 weeks a decrease in the concentration IL1â, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, eotaxin, granulocyte colony-stimulating factor, IFN-ã, IFN-ã-induced protein 10, MCP1, macrophage inflammation protein 1â, TNF-á, vascular endothelial growth factor was recorded (p<0.05). Conclusion. Analysis of the effectiveness of two infusions of RTM biosimilar Acellbia («BIOCAD», Russia) 24 weeks after the start of therapy shows its ability to cause a decrease of levels of proinflammatory cytokines, chemokines and growth factors in the blood serum. Changes of the cytokine profile during the therapy with Acellbia are not significantly different from that during the treatment with the original drug.
... Data supporting this idea derive from independent observational studies based on patients' stratification through either histological parameters or molecular signatures. Associations between synovial pathologic traits and clinical response to specific treatments has been obtained in studies focusing on agent targeting different molecular pathways, including anti-TNF (95,(98)(99)(100)(101)(102)(103)(104), IL-6 inhibitors (105), or B cell depleting agents (106)(107)(108), pointing at a wide spectrum of applicability. The assessment of synovial patho-biology in single joints has been shown also to hold an intrinsic potential for the development of prognostic biomarkers, as it can be inferred, for example, by the association between B cell-rich/lymphoid synovitis (109) and radiographic progression, recently confirmed in independent RA cohorts (85,97,104). ...
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The synovial tissue is a primary target of multiple diseases characterized by different pathogenic mechanisms, including infective, deposition, neoplastic, and chronic immune-inflammatory pathologies. Synovial biopsy can have a relevant role in differential diagnosis of specific conditions in clinical practice, although its exploitation remains relatively limited. In particular, no validated synovial-tissue-derived biomarkers are currently available in the clinic to aid in the diagnosis and management in most frequent forms of chronic inflammatory arthropathies, namely rheumatoid arthritis (RA) and the spondyloarthritides (SpA). In this brief review, we will discuss the current spectrum of clinical applications of synovial biopsy in routine rheumatologic care and will provide an analysis of the perspectives for its potential exploitation in patients with chronic inflammatory arthritides.
... This was also confirmed at the tissue level, where patients with a high inflammatory gene score, overexpressing macrophage and T-cell-related genes and under-expressing IFN and remodelling genes, responded better to rituximab [37]. Also in line with this, another study found responders to have upregulation of synovium immunoglobulin genes and of genes involved in antigen processing and MHC class II presentation [120]. ...
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Individualising biologic disease-modifying anti-rheumatic drugs (bDMARDs) to maximise outcomes and deliver safe and cost-effective care is a key goal in the management of rheumatoid arthritis (RA). Investigation to identify predictive tools of bDMARD response is a highly active and prolific area of research. In addition to clinical phenotyping, cellular and molecular characterisation of synovial tissue and blood in patients with RA, using different technologies, can facilitate predictive testing. This narrative review will summarise the literature for the available bDMARD classes and focus on where progress has been made. We will also look ahead and consider the increasing use of ‘omics’ technologies, the potential they hold as well as the challenges, and what is needed in the future to fully realise our ambition of personalised bDMARD treatment.
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Important advances have occurred during the last decade in the understanding of the pathogenesis of rheumatoid arthritis (RA). However, we are still far from having a clear picture of the molecular network that predisposes an individual to develop the disease, to worsen the symptoms after that, or to successfully respond to a specific treatment. In this sense, different -omics fields (including transcriptomics, proteomics, metabolomics, genomics and epigenomics) have recently produced promising insights that could definitively help us to sharpen such picture if integrated trough a systems biology approach. In this review we will summarise and discuss the recent progress achieved in those fields and its possible impact on the discovery of suitable biomarkers for RA diagnosis, prognosis and treatment response.
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Synovial tissue is a key structure in diarthrodial joints and is the primary target of inflammation in autoimmune arthritis. The study of synovial tissue has developed significantly in the last two decades as arthroscopic and ultrasonographic techniques have allowed visualization and access to synovial biopsy. Further progress in synovial tissue processing and analysis has improved studies of disease pathogenesis, biomarker discovery, and molecular therapeutic targeting with increasingly specialized analytical and technological approaches. In September 2018 the first course on Synovial Tissue Biopsies was convened in Brussels, in this Mini Review these approaches will be described and I will summarize how synovial tissue research advanced.
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Predictors of response to biologics in rheumatoid arthritis (RA) is an important issue in the current era. Rituximab (RTX) has been demonstrated effective and safe in active RA, resistant to traditional or biologic DMARDs. Fifty-seven patients with active longstanding RA were treated with RTX after traditional DMARD or anti-TNF alpha therapy failure. Number of anti-TNF treatment previously failed (p=0.005), HAQ (p=0.013), rheumatoid factor (RF) (p=0.0002) and anti-CCP (p=0.006) were associated with an ACR response > or =50 at the end of 6th month by univariate analysis. Multivariate analysis confirmed that the number of anti-TNF previously failed, baseline HAQ and RF, but not anti-CCP were associated with an ACR response > or =50. EULAR moderate/good response was associated with ESR value (p=0.036), HAQ (p=0.032), and RF (p=0.01) by univariate analysis, while only RF positivity was associated with EULAR moderate/good response by multivariate analysis. RF positivity rather than anti-CCP positivity is a predictor of response to RTX, suggesting that RF-positive patients with low disability may obtain a clinical response when treated to RTX after the first anti-TNF agent failure or after traditional DMARD therapies. Larger studies are required to confirm these results.
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To identify markers and mechanisms of resistance to adalimumab therapy, we studied global gene expression profiles in synovial tissue specimens obtained from severe rheumatoid arthritis (RA) patients before and after initiation of treatment. Paired synovial biopsies were obtained from the affected knee of 25 DMARD (disease-modifying antirheumatic drug)-resistant RA patients at baseline (T0) and 12 weeks (T12) after initiation of adalimumab therapy. DAS28-CRP (disease activity score using 28 joint counts-C-reactive protein) scores were computed at the same time points, and patients were categorized as good, moderate, or poor responders according to European League Against Rheumatism criteria. Global gene expression profiles were performed in a subset of patients by means of GeneChip Human Genome U133 Plus 2.0 Arrays, and confirmatory immunohistochemistry experiments were performed on the entire cohort. Gene expression studies performed at baseline identified 439 genes associated with poor response to therapy. The majority (n = 411) of these genes were upregulated in poor responders and clustered into two specific pathways: cell division and regulation of immune responses (in particular, cytokines, chemokines, and their receptors). Immunohistochemistry experiments confirmed that high baseline synovial expression of interleukin-7 receptor alpha chain (IL-7R), chemokine (C-X-C motif) ligand 11 (CXCL11), IL-18, IL-18 receptor accessory (IL-18rap), and MKI67 is associated with poor response to adalimumab therapy. In vitro experiments indicated that genes overexpressed in poor responders could be induced in fibroblast-like synoviocytes (FLS) cultures by the addition of tumor necrosis factor-alpha (TNF-alpha) alone, IL-1beta alone, the combination of TNF-alpha and IL-17, and the combination of TNF-alpha and IL-1beta. Gene expression studies of the RA synovium may be useful in the identification of early markers of response to TNF blockade. Genes significantly overexpressed at baseline in poor responders are induced by several cytokines in FLSs, thereby suggesting a role for these cytokines in the resistance to TNF blockade in RA.
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B-cell depletion with rituximab, a chimeric anti-CD20 antibody, is a novel treatment for refractory and relapsing ANCA-associated small-vessel vasculitis. Data are limited and most reports describe single patients or small numbers of patients followed prospectively. We report a single-centre experience with 15 patients who received rituximab for refractory or relapsing ANCA-associated vasculitis. All patients had been treated with corticosteroids and cyclophosphamide and a variety of other second-line immunosuppressive agents. None of the patients had evidence of infection and received four infusions of 375 mg/m(2) of rituximab. Disease activity was assessed in accordance with the Birmingham Vasculitis Activity Score (BVAS). BVAS, C-reactive protein and ANCA titres were recorded at baseline and during follow-up. B-cell depletion was achieved in all patients. Partial or complete remission was seen in 14 of 15 patients with a significant decline in BVAS compared to baseline (P < 0.007). One patient with granulomatous ANCA-associated vasculitis did not respond to rituximab. There were no side effects during rituximab infusion. Transient leucopenia was observed in two patients. One patient with bronchial stenosis died of pneumonia 5.5 months after the initiation of rituximab treatment. One initially anti-HBc-positive/HBsAg-negative patient experienced a reactivation of hepatitis B, developed end-stage renal failure and died after refusal of dialysis. We report the largest case series of rituximab use for ANCA-associated vasculitis so far. Our data support that the drug is capable of inducing partial or complete remission in refractory or relapsing patients. Leucopenia and infectious complications remain a matter of concern.
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Murine monoclonal antibody 2B8 specifically recognizes the CD20 phosphoprotein expressed on the surface of normal B lymphocytes and B-cell lymphomas. The light- and heavy-chain variable regions of 2B8 were cloned, after amplification by the polymerase chain reaction, into a cDNA expression vector that contained human IgG1 heavy chain and human kappa-light chain constant regions. High-level expression of chimeric-2B8 antibody (C2B8) was obtained in Chinese hamster ovary cells. Purified C2B8 exhibited antigen binding affinity and human-tissue reactivity similar to the native murine antibody. In vitro studies showed the ability of C2B8 to bind human C1q, mediate complement-dependent cell lysis of human B-lymphoid cell lines, and lyse human target cells through antibody-dependent cellular cytotoxicity. Infusion of macaque cynomolgus monkeys with doses ranging from 1.6 mg/kg to 6.4 mg/kg resulted in greater than 98% depletion of peripheral blood (PB) B cells and 40% to 70% depletion of lymph node B cells. Recovery of PB B cells usually started at 2 weeks after treatment and required 60 to greater than 90 days to reach normal levels. As much as 95% depletion of B cells in peripheral lymph nodes and bone marrow was observed following weekly injections of 16.8 mg/kg antibody. No toxicity was observed in any of the animals. These results offer the possibility of using an "immunologically active" chimeric anti-CD20 antibody as an alternative approach in the treatment of B-cell lymphoma.
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Functional annotation of differentially expressed genes is a necessary and critical step in the analysis of microarray data. The distributed nature of biological knowledge frequently requires researchers to navigate through numerous web-accessible databases gathering information one gene at a time. A more judicious approach is to provide query-based access to an integrated database that disseminates biologically rich information across large datasets and displays graphic summaries of functional information. Database for Annotation, Visualization, and Integrated Discovery (DAVID; http://www.david.niaid.nih.gov) addresses this need via four web-based analysis modules: 1) Annotation Tool - rapidly appends descriptive data from several public databases to lists of genes; 2) GoCharts - assigns genes to Gene Ontology functional categories based on user selected classifications and term specificity level; 3) KeggCharts - assigns genes to KEGG metabolic processes and enables users to view genes in the context of biochemical pathway maps; and 4) DomainCharts - groups genes according to PFAM conserved protein domains. Analysis results and graphical displays remain dynamically linked to primary data and external data repositories, thereby furnishing in-depth as well as broad-based data coverage. The functionality provided by DAVID accelerates the analysis of genome-scale datasets by facilitating the transition from data collection to biological meaning.
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Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3+ T cells, CD38+ plasma cells and CD68+ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland-Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.
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Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
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To investigate the synovial tissue in patients with rheumatoid arthritis (RA) treated with rituximab and to identify possible predictors of clinical response. A total of 24 patients with RA underwent synovial biopsy before, 4 and 16 weeks after initiation of rituximab treatment (without peri-infusional corticosteroids to prevent bias). Immunohistochemical analysis was performed and stained sections were analysed by digital image analysis. Linear regression analysis was used to identify predictors of clinical response. The 28-joint Disease Activity Score (DAS28) was unaltered at 4 weeks, but significantly reduced at 16 and 24 weeks. Serum levels of IgM-rheumatoid factor (RF) decreased significantly at 24 weeks and anti-citrullinated peptide antibody (ACPA) levels at 36 weeks. Peripheral blood B cells were depleted at 4 weeks and started to return at 24 weeks. Synovial B cells were significantly decreased at 4 weeks, but were not completely depleted in all patients; there was a further reduction at 16 weeks in some patients. We found a significant decrease in macrophages at 4 weeks, which was more pronounced at 16 weeks. At that timepoint, T cells were also significantly decreased. The reduction of plasma cells predicted clinical improvement at 24 weeks. The results support the view that B cells orchestrate local cellular infiltration. The kinetics of the serological as well as the tissue response in clinical responders are consistent with the notion that rituximab exerts its effects in part by an indirect effect on plasma cells associated with autoantibody production, which could help explain the delayed response after rituximab treatment.
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There is increasing evidence that B lymphocytes are involved in the pathogenesis of multiple sclerosis, and they may be a therapeutic target. Rituximab, a monoclonal antibody, selectively targets and depletes CD20+ B lymphocytes. In a phase 2, double-blind, 48-week trial involving 104 patients with relapsing-remitting multiple sclerosis, we assigned 69 patients to receive 1000 mg of intravenous rituximab and 35 patients to receive placebo on days 1 and 15. The primary end point was the total count of gadolinium-enhancing lesions detected on magnetic resonance imaging scans of the brain at weeks 12, 16, 20, and 24. Clinical outcomes included safety, the proportion of patients who had relapses, and the annualized rate of relapse. As compared with patients who received placebo, patients who received rituximab had reduced counts of total gadolinium-enhancing lesions at weeks 12, 16, 20, and 24 (P<0.001) and of total new gadolinium-enhancing lesions over the same period (P<0.001); these results were sustained for 48 weeks (P<0.001). As compared with patients in the placebo group, the proportion of patients in the rituximab group with relapses was significantly reduced at week 24 (14.5% vs. 34.3%, P=0.02) and week 48 (20.3% vs. 40.0%, P=0.04). More patients in the rituximab group than in the placebo group had adverse events within 24 hours after the first infusion, most of which were mild-to-moderate events; after the second infusion, the numbers of events were similar in the two groups. A single course of rituximab reduced inflammatory brain lesions and clinical relapses for 48 weeks. This trial was not designed to assess long-term safety or to detect uncommon adverse events. The data provide evidence of B-cell involvement in the pathophysiology of relapsing-remitting multiple sclerosis. (ClinicalTrials.gov number, NCT00097188 [ClinicalTrials.gov].).
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Whether rituximab could effectively and safely avoid splenectomy for adults with chronic immune thrombocytopenic purpura (ITP) remains unresolved. A multicenter, prospective, open-label, single-arm, phase 2 trial was conducted to assess rituximab safety and efficacy in adult splenectomy candidates with chronic ITP. Sixty patients with chronic (>or= 6 months) ITP and platelet counts less than 30 x 10(9)/L received a weekly intravenous infusion of rituximab (375 mg/m(2)) for 4 weeks. All other ITP treatments were stopped. A good response was defined as a platelet count 50 x 10(9)/L or more, with at least a doubling of the initial value at 1 and 2 years after the first rituximab infusion. Patients who required another treatment during follow up were considered nonresponders. Sixteen patients experienced transient side effects that necessitated treatment discontinuation for only 1. Good 1-year responses were obtained in 40% of the patients (24/60 [95% confidence interval: 28%-52%]). At 2 years, 33.3% (20/60 patients) had good responses and 6.7% (4/60) had sustained platelet counts of 30 x 10(9)/L or more without treatment. Thirty-six (60%) patients failed to respond; 25 underwent splenectomy. Based on these results, rituximab was an apparently safe and effective splenectomy-avoiding option in some adults with chronic ITP. This trial is registered at http://clinicaltrials.gov as NCT00225875.
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The revised criteria for the classification of rheumatoid arthritis (RA) were formulated from a computerized analysis of 262 contemporary, consecutively studied patients with RA and 262 control subjects with rheumatic diseases other than RA (non-RA). The new criteria are as follows: 1) morning stiffness in and around joints lasting at least 1 hour before maximal improvement; 2) soft tissue swelling (arthritis) of 3 or more joint areas observed by a physician; 3) swelling (arthritis) of the proximal interphalangeal, metacarpophalangeal, or wrist joints; 4) symmetric swelling (arthritis); 5) rheumatoid nodules; 6) the presence of rheumatoid factor; and 7) radiographic erosions and/or periarticular osteopenia in hand and/or wrist joints. Criteria 1 through 4 must have been present for at least 6 weeks. Rheumatoid arthritis is defined by the presence of 4 or more criteria, and no further qualifications (classic, definite, or probable) or list of exclusions are required. In addition, a “classification tree” schema is presented which performs equally as well as the traditional (4 of 7) format. The new criteria demonstrated 91–94% sensitivity and 89% specificity for RA when compared with non-RA rheumatic disease control subjects.
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Objective. The development and validation of Modified Disease Activity Scores (DAS) that include different 28-joint counts. Methods. These scores were developed by canonical discriminant analyses and validated for criterion, correlational, and construct validity. The influence of disease duration on the composition of the DAS was also investigated. Results. No influence of disease duration was found. The Modified DAS that included 28-joint counts were able to discriminate between high and low disease activity (as indicated by clinical decisions of rheumatologists). Conclusion. The Modified DAS are as valid as disease activity scores that include more comprehensive joint counts.
Article
Objective. To validate the European League Against Rheumatism (EULAR), the American College of Rheumatology (ACR), and the World Health Organization (WHO)/International League Against Rheumatism (ILAR) response criteria for rheumatoid arthritis (RA). Methods. EULAR response criteria were developed combining change from baseline and level of disease activity attained during followup. In a trial comparing hydroxychloroquine and sulfasalazine, we studied construct (radiographic progression), criterion (functional capacity), and discriminant validity. Results. EULAR response criteria had good construct, criterion, and discriminant validity. ACR and WHO/ILAR criteria showed only good criterion validity. Conclusion. EULAR response criteria showed better construct and discriminant validity than did the ACR and the WHO/ILAR response criteria for RA.
Article
Rituximab is an effective treatment in patients with established rheumatoid arthritis (RA). The objective of the IMAGE study was to determine the efficacy of rituximab in the prevention of joint damage and its safety in combination with methotrexate (MTX) in patients initiating treatment with MTX. In this double-blind randomised controlled phase III study, 755 MTX-naïve patients with active RA were randomly assigned to MTX alone, rituximab 2×500 mg + MTX or rituximab 2×1000 mg + MTX. The primary end point at week 52 was the change in joint damage measured using a Genant-modified Sharp score. 249, 249 and 250 patients were randomly assigned to MTX alone, rituximab 2×500 mg + MTX or rituximab 2×1000 mg + MTX, respectively. At week 52, treatment with rituximab 2×1000 mg + MTX compared with MTX alone was associated with a reduction in progression of joint damage (mean change in total modified Sharp score 0.359 vs 1.079; p=0.0004) and an improvement in clinical outcomes (ACR50 65% vs 42%; p<0.0001); rituximab 2×500 mg + MTX improved clinical outcomes (ACR50 59% vs 42%; p<0.0001) compared with MTX alone but did not significantly reduce the progression of joint damage. Safety outcomes were similar between treatment groups. Treatment with rituximab 2×1000 mg in combination with MTX is an effective therapy for the treatment of patients with MTX-naïve RA. ClinicalTrials.gov identifier NCT00299104.
Article
The development and validation of Modified Disease Activity Scores (DAS) that include different 28-joint counts. These scores were developed by canonical discriminant analyses and validated for criterion, correlational, and construct validity. The influence of disease duration on the composition of the DAS was also investigated. No influence of disease duration was found. The Modified DAS that included 28-joint counts were able to discriminate between high and low disease activity (as indicated by clinical decisions of rheumatologists). The Modified DAS are as valid as disease activity scores that include more comprehensive joint counts.
Article
Rituximab, a chimeric monoclonal antibody that binds specifically to the CD20 antigen, induced objective responses in 50% of patients with low-grade or follicular B-cell lymphoma. Because most nonfollicular B-cell lymphomas also express the CD20 antigen, we conducted a phase II study to evaluate the efficacy and tolerability of this new agent in patients with more aggressive types of lymphoma. Patients with diffuse large B-cell lymphoma (DLCL), mantle cell lymphoma (MCL), or other intermediate- or high-grade B-cell lymphomas according to the Working Formulation were included in this prospective randomized phase II study if they were in first or second relapse, if they were refractory to initial therapy, if they progressed after a partial response to initial therapy, or if they were elderly (age >60 years) and not previously treated. The patients received 8 weekly infusions of rituximab at the dose of 375 mg/m2 in arm A or one infusion of 375 mg/m2 followed by 7 weekly infusions of 500 mg/m2 in arm B. Patients were evaluated 2 months after the last rituximab infusion. Fifty-four patients were randomized from 9 centers in Europe and Australia (28 in arm A and 26 in arm B). A total of 5 complete responses (CR) and 12 partial responses (PR) were observed among the 54 enrolled patients, with no difference between the two doses. In an intent-to-treat analysis, the CR rate was 9% (CI95%, 3% to 20%) and the PR rate was 22% (CI95%, 12% to 36%), for an overall response rate of 31% (CI95%, 20% to 46%). An analysis of prognostic factors showed that response rates were lower in patients with refractory disease, patients with lymphoma not classified as DLCL, and patients with a tumor larger than 5 cm in diameter. DLCL and MCL patients had response rates of 37% and 33%, respectively. The median time to progression exceeded 246 days for the 17 responding patients. The most frequently reported adverse events were related to an infusion syndrome and were mild: 19% of the patients had a grade 3 related adverse event, slightly more in arm B, and only 1 patient had a grade 4 related adverse event in arm A. Two patients (3.7%) withdrew from treatment because of severe adverse events, one patient in each arm. In this first trial of rituximab in DLCL and MCL, patients experienced a significant clinical activity with a low toxicity. Rituximab has significant activity in DLCL and MCL patients and should be tested in combination with chemotherapy in such patients.
Article
B lymphocyte depletion therapy in rheumatoid arthritis can provide major clinical benefits. Widespread use in the future will depend on continued evidence of safety, particularly in the context of long term use. Rituximab is a highly effective agent, but it may be best used in combination with other agents. Substantial improvement following a single course of therapy has been found to last up to 42 months, and it is reasonable to hope that further development of strategies targeting B cells will extend this toward the original aim of truly long-term remission.
Article
To examine the efficacy and safety of different rituximab doses plus methotrexate (MTX), with or without glucocorticoids, in patients with active rheumatoid arthritis (RA) resistant to disease-modifying antirheumatic drugs (DMARDs), including biologic agents. A total of 465 patients were randomized into 9 treatment groups: 3 rituximab groups (placebo [n = 149], 500 mg [n = 124], or 1,000 mg [n = 192] on days 1 and 15) each also taking either placebo glucocorticoids, intravenous methylprednisolone premedication, or intravenous methylprednisolone premedication plus oral prednisone for 2 weeks. All patients received MTX (10-25 mg/week); no other DMARDs were permitted. Significantly more patients who received 2 500-mg or 2 1,000-mg infusions of rituximab met the American College of Rheumatology 20% improvement criteria (achieved an ACR20 response) at week 24 (55% and 54%, respectively) compared with placebo (28%; P < 0.0001). ACR50 responses were achieved by 33%, 34%, and 13% of patients, respectively (P < 0.001), and ACR70 responses were achieved by 13%, 20%, and 5% of patients (P < 0.05). Changes in the Disease Activity Score in 28 joints (-1.79, -2.05, -0.67; P < 0.0001) and moderate to good responses on the European League Against Rheumatism criteria (P < 0.0001) reflected the ACR criteria responses. Glucocorticoids did not contribute significantly to the primary efficacy end point, ACR20 response at 24 weeks. Intravenous glucocorticoid premedication reduced the frequency and intensity of first infusion-associated events; oral glucocorticoids conferred no additional safety benefit. Rituximab was well tolerated; the type and severity of infections was similar to those for placebo. Both rituximab doses were effective and well tolerated when added to MTX therapy in patients with active RA. The primary end point (ACR20 response) was independent of glucocorticoids, although intravenous glucocorticoid premedication improved tolerability during the first rituximab infusion.
Article
To determine the efficacy and safety of treatment with rituximab plus methotrexate (MTX) in patients with active rheumatoid arthritis (RA) who had an inadequate response to anti–tumor necrosis factor (anti-TNF) therapies and to explore the pharmacokinetics and pharmacodynamics of rituximab in this population. We evaluated primary efficacy and safety at 24 weeks in patients enrolled in the Randomized Evaluation of Long-Term Efficacy of Rituximab in RA (REFLEX) Trial, a 2-year, multicenter, randomized, double-blind, placebo-controlled, phase III study of rituximab therapy. Patients with active RA and an inadequate response to 1 or more anti-TNF agents were randomized to receive intravenous rituximab (1 course, consisting of 2 infusions of 1,000 mg each) or placebo, both with background MTX. The primary efficacy end point was a response on the American College of Rheumatology 20% improvement criteria (ACR20) at 24 weeks. Secondary end points were responses on the ACR50 and ACR70 improvement criteria, the Disease Activity Score in 28 joints, and the European League against Rheumatism (EULAR) response criteria at 24 weeks. Additional end points included scores on the Functional Assessment of Chronic Illness Therapy–Fatigue (FACIT-F), Health Assessment Questionnaire (HAQ) Disability Index (DI), and Short Form 36 (SF-36) instruments, as well as Genant-modified Sharp radiographic scores at 24 weeks. Patients assigned to placebo (n = 209) and rituximab (n = 311) had active, longstanding RA. At week 24, significantly more (P < 0.0001) rituximab-treated patients than placebo-treated patients demonstrated ACR20 (51% versus 18%), ACR50 (27% versus 5%), and ACR70 (12% versus 1%) responses and moderate-to-good EULAR responses (65% versus 22%). All ACR response parameters were significantly improved in rituximab-treated patients, who also had clinically meaningful improvements in fatigue, disability, and health-related quality of life (demonstrated by FACIT-F, HAQ DI, and SF-36 scores, respectively) and showed a trend toward less progression in radiographic end points. Rituximab depleted peripheral CD20+ B cells, but the mean immunoglobulin levels (IgG, IgM, and IgA) remained within normal ranges. Most adverse events occurred with the first rituximab infusion and were of mild-to-moderate severity. The rate of serious infections was 5.2 per 100 patient-years in the rituximab group and 3.7 per 100 patient-years in the placebo group. At 24 weeks, a single course of rituximab with concomitant MTX therapy provided significant and clinically meaningful improvements in disease activity in patients with active, longstanding RA who had an inadequate response to 1 or more anti-TNF therapies.
Article
Synovitis is a common feature of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but the pattern of joint involvement differs in each disease. This study was undertaken to investigate the global gene expression profiles in synovial biopsy tissue from the swollen knees of untreated SLE patients (n = 6), RA patients (n = 7), and osteoarthritis (OA) patients (n = 6). Synovial biopsy samples were obtained from the affected knees of patients in the 3 groups by needle arthroscopy. Half of the material was used for extraction of total RNA, amplification of complementary RNA, and high-density oligonucleotide spotted hybridization arrays. On the remaining tissue samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical experiments were performed to confirm the microarray data. SLE synovial biopsy tissue displayed a significant down-regulation of genes involved in extracellular matrix (ECM) homeostasis and a significant up-regulation of interferon-inducible (IFI) genes. Real-time RT-PCR experiments confirmed the up-regulation of selected IFI genes (IFI27, IFI44, and IFI44L) in the SLE synovial tissue. Immunohistochemical analyses showed that 3 molecules involved in ECM regulation, chondroitin sulfate proteoglycan 2, latent transforming growth factor beta binding protein 2, and fibroblast activation protein alpha, were significantly down-regulated in SLE synovium. In contrast, immunostaining for IFI27, Toll-like receptor 4, and STAT-1 resulted in higher quantitative scores in SLE synovial tissue, which could be attributed to the fact that the RA samples had a large population of inflammatory cell infiltrates that were negative for these markers. Arthritis in SLE has a very distinct molecular signature as compared with that in OA and RA, characterized by up-regulation of IFI genes and down-regulation of genes involved in ECM homeostasis.
Article
To characterise the bone morphogenetic protein (BMP) target cells positive for phosphorylated (P)-SMAD1/5, in rheumatoid arthritis (RA) synovium. Synovial biopsies were obtained by needle arthroscopy. Anti-P-SMAD1/5 antibodies were used for Western blot (WB) on protein extracts from RA and normal synovium and for immunostaining of synovial biopsy sections. Positive cells were further identified by double staining for CD3, CD20, CD68, CD138, CD90, alpha smooth muscle actin (SMA), endoglin (CD105) and von Willebrand factor (VWF). In sections from early patients with RA taken before and under antirheumatic treatment, the degree of inflammation and activation of the BMP pathway were quantified. P-SMAD1/5 protein was detected by WB in RA and to a lesser extent in normal synovium. Different P-SMAD1/5 positive cell populations were identified in RA synovium, mainly in perivascular and sublining cells. P-SMAD1/5 positive perivascular cells were alphaSMA positive and located around VWF positive endothelial cells. Some CD90 positive synovial fibroblasts were P-SMAD1/5 positive, as was part of the CD68 positive synovial cells but other cells of the haematopoietic lineage showed no SMAD1/5 phosphorylation. Treatment resulted in an absolute but not relative decrease in BMP activation in the synovium. BMP-activated cells belong to distinct stromal compartments in RA synovium and some of them express markers associated with the mesenchymal progenitor cell lineage. Antirheumatic treatment effectively downregulates synovial inflammation, but BMP activation in the synovium does persist albeit reduced.
Article
We evaluated the safety, tolerability, pharmacodynamics, and activity of B-cell depletion with rituximab in patients with relapsing-remitting multiple sclerosis, receiving two courses of rituximab 6 months apart, and followed for a total of 72 weeks. No serious adverse events were noted; events were limited to mild-to-moderate infusion-associated events, which tended to decrease with subsequent infusions. Infections were also mild or moderate, and none led to withdrawal. Fewer new gadolinium-enhancing or T2 lesions were seen starting from week 4 and through week 72. An apparent reduction in relapses was also observed over the 72 weeks compared with the year before therapy.
Database for Annotation, Visualization, and Inte-grated Discovery
  • Dennis Jr
  • Bt Sherman
  • Hosack
  • Yang J Da
  • W Gao
  • Lane
  • Hc
Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, et al. DAVID: Database for Annotation, Visualization, and Inte-grated Discovery. Genome Biol 2003;4:P3.
  • G Dennis
  • Jr
  • Bt Sherman
  • Da Hosack
  • Yang J Gao
  • W Lane
Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 2003;4:P3.
Clinical, radiographic and biomolecular features of B cell synovitis in rheumatoid arthritis
  • S Bugatti
  • A Manzo
  • B Vitolo
  • C Fusetti
  • R Caporali
  • C Pitzalis
Bugatti S, Manzo A, Vitolo B, Fusetti C, Caporali R, Pitzalis C, et al. Clinical, radiographic and biomolecular features of B cell synovitis in rheumatoid arthritis [abstract]. Arthritis Rheum 2010;62 Suppl:S260.
  • G Dennis
  • B T Sherman
  • D A Hosack
  • J Yang
  • W Gao
  • H C Lane
Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 2003;4:P3.
Clinical, radiographic and biomolecular features of B cell synovitis in rheumatoid arthritis
  • Bugatti