The role of proteasome in muscle wasting of experimental arthritis.

BACKGROUND: Rheumatoid arthritis is an autoimmune inflammatory disease that often leads patients to muscle impairment and physical disability. This study aimed to evaluate changes in the activity of proteasome system in skeletal muscles of mice with collagen-induced arthritis (CIA) and treated with etanercept or methotrexate. METHODS: Male DBA1/J mice were divided into four groups (n = 8 each): CIA-Vehicle (treated with saline), CIA-ETN (treated with etanercept, 5.5 mg/kg), CIA-MTX (treated with methotrexate, 35 mg/kg) and CO (healthy control group). Mice were treated two times a week for 6 weeks. Clinical score and hind paw edema were measured. Muscles were weighted after euthanasia and used to quantify proteasome activity, gene (MuRF-1, PMSα4, PSMß5, PMSß6, PSMß7, PSMß8, PSMß9, and PSMß10), and protein (PSMß1, PSMß5, PSMß1i, PSMß5i) expression of proteasome subunits. RESULTS: Both treatments slowed disease development, but only CIA-ETN maintained muscle weight compared to CIA-MTX and CIA-Vehicle groups. Etanercept treatment showed caspase-like activity of 26S proteasome similar to CO group, while CIA-Vehicle and CIA-MTX had higher activity compared to CO group (p: 0.0057). MuRF-1 mRNA expression was decreased after etanercept administration compared to CIA-Vehicle and CO groups (p: 0.002, p: 0.007, respectively). PSMß8 and PSMß9 mRNA levels were increased in CIA-Vehicle and CIA-MTX compared to CO group, while CIA-ETN presented no difference from CO. PMSß6 mRNA expression was higher in CIA-Vehicle and CIA-MTX groups than in CO group. Protein levels of the PSMß5 subunit were increased in CO group compared to CIA-Vehicle; after both etanercept and methotrexate treatments, PSMß5 expression was higher than in CIA-Vehicle group and did not differ from CO group expression (p: 0.0025, p: 0.001, respectively). The inflammation-induced subunit ß1 (LMP2) was enhanced after methotrexate treatment compared to CO group (p: 0.043). CONCLUSIONS: The results of CIA-Vehicle show that arthritis increases muscle proteasome activation by enhanced caspase-like activity of 26S proteasome and increased PSMß8 and PSMß9 mRNA levels. Etanercept treatment was able to maintain the muscle weight and to modulate proteasome so that its activity and gene expression were compared to CO after TNF inhibition. The protein expression of inflammation-induced proteasome subunit was increased in muscle of CIA-MTX group but not following etanercept treatment. Thus, anti-TNF treatment may be an interesting approach to attenuate the arthritis-related muscle wasting.

The role of proteasome in muscle wasting of experimental arthritis

Abstract Background Rheumatoid arthritis is an autoimmune inflammatory disease that often leads patients to muscle impairment and physical disability. This study aimed to evaluate changes in the activity of proteasome system in skeletal muscles of mice with collagen-induced arthritis (CIA) and treated with etanercept or methotrexate. Methods Male DBA1/J mice were divided into four groups (n = 8 each): CIA-Vehicle (treated with saline), CIA-ETN (treated with etanercept, 5.5 mg/kg), CIA-MTX (treated with methotrexate, 35 mg/kg) and CO (healthy control group). Mice were treated two times a week for 6 weeks. Clinical score and hind paw edema were measured. Muscles were weighted after euthanasia and used to quantify proteasome activity, gene (MuRF-1, PMSα4, PSMβ5, PMSβ6, PSMβ7, PSMβ8, PSMβ9, and PSMβ10), and protein (PSMβ1, PSMβ5, PSMβ1i, PSMβ5i) expression of proteasome subunits. Results Both treatments slowed disease development, but only CIA-ETN maintained muscle weight compared to CIA-MTX and CIA-Vehicle groups. Etanercept treatment showed caspase-like activity of 26S proteasome similar to CO group, while CIA-Vehicle and CIA-MTX had higher activity compared to CO group (p: 0.0057). MuRF-1 mRNA expression was decreased after etanercept administration compared to CIA-Vehicle and CO groups (p: 0.002, p: 0.007, respectively). PSMβ8 and PSMβ9 mRNA levels were increased in CIA-Vehicle and CIA-MTX compared to CO group, while CIA-ETN presented no difference from CO. PMSβ6 mRNA expression was higher in CIA-Vehicle and CIA-MTX groups than in CO group. Protein levels of the PSMβ5 subunit were increased in CO group compared to CIA-Vehicle; after both etanercept and methotrexate treatments, PSMβ5 expression was higher than in CIA-Vehicle group and did not differ from CO group expression (p: 0.0025, p: 0.001, respectively). The inflammation-induced subunit β1 (LMP2) was enhanced after methotrexate treatment compared to CO group (p: 0.043). Conclusions The results of CIA-Vehicle show that arthritis increases muscle proteasome activation by enhanced caspase-like activity of 26S proteasome and increased PSMβ8 and PSMβ9 mRNA levels. Etanercept treatment was able to maintain the muscle weight and to modulate proteasome so that its activity and gene expression were compared to CO after TNF inhibition. The protein expression of inflammation-induced proteasome subunit was increased in muscle of CIA-MTX group but not following etanercept treatment. Thus, anti-TNF treatment may be an interesting approach to attenuate the arthritis-related muscle wasting.

Finding the best fit for improving reproducibility: reflections from the QUEST Center for Responsible Research.

Increasing the reproducibility and trustworthiness of biomedical research requires engaging stakeholders from all levels in an institutional setting. The QUEST Center for Responsible Research aims to develop and implement new approaches to improve the culture and practice of research, tailored to the needs of these stakeholders. Members of the QUEST Center organised a brainstorm to reflect on the challenges and new opportunities encountered in implementing different projects through QUEST and share the lessons that working groups have learned over the first five years. The authors informally surveyed and interviewed working groups where relevant and highlight common themes that have influenced the success of many projects, including top-down and bottom-up engagement, managing expectations, the availability of expertise, ensuring sustainability, and considering incentives. The commentary authors conclude by encouraging the research community to view initiatives that promote reproducibility not as a one-size-fits-all undertaking, but rather as an opportunity to unite stakeholders and customise drivers of cultural change.

Improving preclinical studies through replications.

The purpose of preclinical research is to inform the development of novel diagnostics or therapeutics, and the results of experiments on animal models of disease often inform the decision to conduct studies in humans. However, a substantial number of clinical trials fail, even when preclinical studies have apparently demonstrated the efficacy of a given intervention. A number of large-scale replication studies are currently trying to identify the factors that influence the robustness of preclinical research. Here, we discuss replications in the context of preclinical research trajectories, and argue that increasing validity should be a priority when selecting experiments to replicate and when performing the replication. We conclude that systematically improving three domains of validity - internal, external and translational - will result in a more efficient allocation of resources, will be more ethical, and will ultimately increase the chances of successful translation.

Response to abatacept is associated with the inhibition of proteasome ß1i expression in T cells of patients with rheumatoid arthritis.

OBJECTIVE: Abatacept is a biological disease-modifying antirheumatic drug (DMARD) used for the treatment of rheumatoid arthritis (RA) and modulates the costimulatory signal by cluster of differentiation (CD)28:CD80/CD86 interaction required for T cell activation. Since CD28-mediated signalling regulates many T cell functions including cytokine production of, for example, interferons (IFNs), it is of interest to clarify, whether response to abatacept has an effect on the IFN inducible immunoproteasome, as a central regulator of the immune response. METHODS: Effects of abatacept on the proteasome were investigated in 39 patients with RA over a period of 24 weeks. Using real-time PCR, transcript levels of constitutive and corresponding immunoproteasome catalytic subunits were investigated at baseline (T0), week 16 (T16) and week 24 (T24) in sorted blood cells. Proteasomal activity and induction of apoptosis after proteasome inhibition were also evaluated. RESULTS: Abatacept achieved remission or low disease activity in 55% of patients at T16 and in 70% of patients at T24. By two-way analysis of variance (ANOVA), a significant reduction of proteasome immunosubunit ß1i was shown only in CD4+ and CD8+ T cells of sustained responders at both T16 and T24. One-way ANOVA analysis for each response group confirmed the results and showed a significant reduction at T24 in CD4+ and CD8+ T cells of the same group. Abatacept did not influence chymotrypsin-like activity of proteasome and had no effect on induction of apoptosis under exposure to a proteasome inhibitor in vitro. CONCLUSION: The reduction of proteasome immunosubunit ß1i in T cells of patients with RA with sustained response to abatacept suggests association of the immunoproteasome of T cells with RA disease activity.

Assessment of the association of baseline anti-CarbV and anti-MCV antibodies with response to treatment and radiographic progression in an RA population treated with either methotrexate or baricitinib: post-hoc analyses from RA-BEGIN.

BACKGROUND: The development of autoantibodies in patients with rheumatoid arthritis (RA) has potential as a marker of treatment response. This analysis assessed the association of an autoantibody response to carbamylated vimentin (anti-CarbV) and to vimentin modified by citrullination (anti-MCV) with response to treatment and structural damage progression in the phase III study RA-BEGIN. METHODS: Data from patients in the modified intent-to-treat population of RA-BEGIN were included for analysis; these patients received methotrexate (MTX), baricitinib 4 mg once daily, or baricitinib plus MTX during the 52-week study period. Endpoints analyzed were clinical response to treatment, assessed using change from baseline (CFB) in Simplified Disease Activity Index (SDAI) and Disease Activity Score for 28-joint count with serum high-sensitivity C-reactive protein (DAS28-hsCRP), and structural damage progression, assessed using CFB greater than the smallest detectable change in the van der Heijde-modified Total Sharp Score. The anti-CarbV and anti-MCV isotypes assessed were immunoglobulin (Ig) A, IgG, and IgM. Multivariable mixed-effect models for repeated measures (MMRMs) were used for the longitudinal analysis of treatment response, and multivariable logistic regression models were used for the analysis of structural damage progression at week 52. RESULTS: Analysis of the association between autoantibodies and treatment response showed that high titers of anti-CarbV (IgA and IgG) were associated with a greater clinical response as measured by SDAI and DAS28-hsCRP. Anti-CarbV IgA and IgG, but not IgM, demonstrated an association after adjustment for other factors included in the MMRMs. High titers of anti-CarbV IgM were associated with a poor response to MTX monotherapy, whereas a nonsignificant trend toward a better response to baricitinib and baricitinib plus MTX was observed. There was no association between anti-MCV antibodies and treatment response. High titers of anti-CarbV IgA were associated with a greater probability of radiographic progression, but no association between anti-MCV antibodies and radiographic progression was observed. CONCLUSIONS: High titers of anti-CarbV IgA and IgG isotypes, but not anti-MCV isotypes, may be useful prognostic biomarkers for identifying the likelihood of the response to treatment and structural damage progression in patients with RA.

Carbamylated vimentin represents a relevant autoantigen in Latin American (Cuban) rheumatoid arthritis patients.

Smoking produces substances that activate proinflammatory, prothrombotic and vasoconstrictive mediators via posttranslational carbamylation of proteins. As a new consequence of carbamylation, induction of anti-carbamylated autoantibodies were observed in rheumatoid arthritis (RA) patients, sometimes prior to onset of the disease. The overall aim of this study was to characterize the reactivity of different isotypes of autoantibodies against carbamylated antigens of vimentin in relation to established rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA) and markers of disease activity in a so far largely uncharacterized population of Latin American (Cuban) patients with RA. Antigenic properties of carbamylated vimentin as well as vimentin peptides were analyzed in 101 patients with RA, 50 disease controls and 51 healthy controls. The diagnostic performance was compared with established commercial ELISA rheumatoid factor, anti-cyclic citrullinated peptide antibodies of second generation (anti-CCP2) and anti-mutated citrullinated vimentin (anti-MCV) antibodies. Prevalence of anti-MCV IgG (86 %), anti-carbamylated vimentin (carbVIM) IgG (77 %) and anti-carbamylated MCV (carbMCV) IgG antibodies (65 %) was higher than the classical RF IgM (60 %) and anti-CCP2 IgG (52 %) in this RA cohort. Of note, smoking status was associated with positive IgG antibody reactivity against CCP2 in 75.0 % and against MCV in 90 % of patients. Furthermore, IgM antibody response against carbMCV and carbVIM was observed in 80 and 90.0 % of smokers, respectively. Due to a high sensitivity of the IgM antibody isotype of anti-carbVIM of 85.2 %, the combination of ACPA with anti-carbVIM IgM provided the best diagnostic performance so far achieved in a RA cohort of this ethnic origin. We demonstrate a high prevalence of anti-carbVIM antibodies and correlation with smoking in Latin American (Cuban) RA patients. Anti-carbVIM IgM represents an useful marker in ACPA-negative patients and, in combination with ACPA IgG assays, optimizes the strategy for autoantibody testing.

Restrictive IgG antibody response against mutated citrullinated vimentin predicts response to rituximab in patients with rheumatoid arthritis.

INTRODUCTION: Antibodies against mutated citrullinated vimentin (AMCV) represent a useful diagnostic marker with correlation to disease activity in patients with rheumatoid arthritis (RA). Since seropositivity for citrullinated autoantibodies was predictive for response to B-cell depleting therapy (BCDT) with rituximab (RTX), we investigated whether differences in antibody fine reactivity and immunoglobulin (Ig) isotype kinetics among AMCV-positive patients could provide additional information about outcome. METHODS: A total of 50 AMCV IgG-positive RA patients (RTX responders (RRs) n = 37 and non-responders (NRRs) n = 13) were analyzed for reactivity against MCV epitopes and co-existent AMCV isotypes IgM and IgA. Antibody titers were determined by enzyme-linked immunosorbent assay at baseline and 24 weeks after the first cycle of RTX, and compared to kinetics of rheumatoid factor (RF) and antibodies against cyclic citrullinated peptide (ACCP). RESULTS: Recognized MCV epitopes by AMCV IgG of RRs and NRRs showed similar baseline patterns, with reducing reactivity in RRs and unchanged or even expanding reactivity in NRRs upon RTX treatment. At baseline, RRs were more frequently negative for AMCV subtypes, especially for IgA (68%), compared to NRRs (31%). Being AMCV IgA-negative at baseline indicated a good treatment response to RTX (negative predictive value = 0.86). Co-existence of AMCV IgA and IgG with stable titers upon treatment were associated with poorer responses to RTX. Furthermore, reductions of AMCV IgA levels upon RTX correlated with the improvement of 28-joint Disease Activity Score (DAS28). In comparison, subtypes of RF and ACCP were not of additional value for prediction of RTX response. CONCLUSIONS: Restrictive IgG seropositivity against MCV with treatment-associated decline in fine reactivity and titers was predictive for response to RTX. Double-positivity for AMCV IgG and IgA was associated with failure to respond to BCDT, suggesting a pathogenetic and less sensitive IgA-producing B-cell subset in NRRs.

Selected cyclic citrullinated peptides derived from the sequence of mutated and citrullinated vimentin (MCV) are targeted by different antibodies subclasses in patients with rheumatoid arthritis in Russian patients.

OBJECTIVES: Antibodies against citrullinated antigens (ACPA) represent one rheumatoid arthritis (RA) classification criteria. Recently, mutated and citrullinated vimentin (MCV), containing approx. 45 potentially citrullinated sites, was characterised as another modified autoantigenic RA target. Therefore, we wanted to screen, select and validate predominant MCV autoantigenic epitopes (called here MCE) as possible new diagnostic targets. METHODS: MCV-derived peptides with citrullinated sites were screened in healthy controls and patients. Based on this, twelve selected MCE were used for validation of ACPA isotypes (IgA/IgG/IgM) with ELISA in early RA (ERA, <12 months) and established RA (>12 months) Russian patients. Sensitivity of MCE reactivity was compared to commercially available ELISAs for anti-CCP IgG, anti-MCV IgG, and anti-RF IgA/IgM/IgG. RESULTS: Anti-MCE IgG/IgA//IgM antibodies were observed in 64.1%, 23.1%, and 15.4% ERA, and 63.9%, 26.7%, and 13.1% established RA patients, respectively. Anti-MCV IgG was present in 64.1% ERA and 55.0% RA patients. Furthermore, anti-CCP IgG and RF IgG/IgA/IgM were detectable in up to 76.9%, 71.8%, 71.8%, and 38.5% ERA, and 80.1%, 72.3%, 67.5%, and 43.0% RA patients. Anti-CCP IgG single positivity was observed in 7.7% ERA and 6.3% RA patients. Only one RA patient was anti-MCE single positive. CONCLUSIONS: MCV autoantigenic epitopes were emulated by cyclic citrullinated MCV-derived peptides and recognised by all autoantibody-Ig subclasses in RA. Tested MCE were recognized more frequently by IgG as the original MCV antigen. High antibody prevalence against CCP epitopes suggests a strong CCP-linkage to RA pathogenesis in the investigated Russian cohort.

Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

OBJECTIVE: In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. METHODS: Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. RESULTS: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. CONCLUSIONS: Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition.

Gene expression of catalytic proteasome subunits and resistance toward proteasome inhibition of B lymphocytes from patients with primary sjogren syndrome.

OBJECTIVE: Dysregulation of proteasome subunit ß1i expression has been shown in total blood mononuclear cells (PBMC) from patients with primary Sjögren syndrome (pSS), a B cell-driven systemic autoimmune disorder. METHODS: Proteasome activation was investigated in sorted blood cells from patients with pSS and controls by measuring transcript levels of constitutive (ß1/ß2/ß5) and corresponding immunoproteasome catalytic subunits (ß1i/ß2i/ß5i) using real-time PCR. At protein level, ß1i protein expression was analyzed by immunoblotting. Functional effects of proteasome inhibition on proteolytic activity and induction of apoptosis were also evaluated in cellular subsets. RESULTS: The proteasome was found to be activated in pSS, with upregulation of gene expression of catalytic proteasome subunits. Western blot analysis revealed decreased ß1i protein expression in pSS B lymphocytes, with decreased protein despite increased messenger RNA (mRNA) levels. After proteasome inhibition in vitro, proteolytic activity was less reduced and resistance to apoptosis was increased in B lymphocytes compared to other cells. CONCLUSION: In pSS, catalytic subunits of the proteasome are upregulated at the mRNA level, while dysregulation of subunit ß1i is attributed to B lymphocytes. B cell resistance after proteasome inhibition differs from the classical concept of increased susceptibility toward inhibition in activated cells, supporting the novel notion that susceptibility depends on cellular intrinsic factors and on proteasome activation.

Expression of proteasomal immunosubunit beta1i is dysregulated in inflammatory infiltrates of minor salivary glands in Sjogren's syndrome.

OBJECTIVE: Minor salivary gland specimens were analyzed to investigate dysregulation of the proteasome system in patients with Sjögren's syndrome (SS) and patients with sicca syndrome. METHODS: Labial biopsy specimens from 17 patients with SS and 11 patients with non-autoimmunesicca syndrome were analyzed by immunohistochemistry for expression of the inducible proteasomal subunits ss1i, ss2i, and ss5i. The infiltrating subsets of lymphocytes were characterized using immunofluorescence stainings against the cell-surface markers CD20 and CD27. Two-dimensional electrophoresis and immunoblotting were used for detection of the proteasomal subunits ss1 and ss1i in peripheral blood monocyte cells. Gene expression of the constitutive subunits ss1, ss2, and ss5 and the corresponding inducible subunits ss1i, ss2i, and ss5i was further investigated at the mRNA level in small lip biopsies using real-time polymerase chain reaction. RESULTS: The expression of ss1i in infiltrating and peripheral immune cells was altered in patients with SS compared to patients with non-autoimmune sicca syndrome and healthy controls. No significant differences were found in ss2i and ss5i expression between the same groups in small lip biopsies. Chisholm-Mason grade and ss1i expression were found to be inversely correlated (Spearman r = -0.461, p = 0.014). The phenotype and distribution of the lymphocytic infiltrate showed no differences between patients with primary and secondary SS regardless of ss1i expression. CONCLUSION: The proteasomal ss1i subunit is dysregulated in peripheral white blood cells and in inflammatory infiltrates of minor salivary glands in patients with SS.

Bronchoalveoloar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients.

INTRODUCTION: Interstitial lung disease (ILD) is a frequent manifestation of systemic sclerosis (SSc), and cytokines can contribute to the disease pathology. The aim of the current study was to identify specific changes in cytokine levels that may serve as disease markers and possible targets for therapy. METHODS: Cytokines were measured with bioplex analysis in 38 bronchoalveolar fluids (BALFs) from 32 SSc patients (27 with alveolitis and 11 without alveolitis) and 26 control patients. In the case of SSc patients, cytokines were correlated with the respective bronchoalveolar lavage (BAL) cell differentiation, lung function, and thoracic HR-CT score. For 35 BALF samples derived from 29 SSc patients, follow-up investigations of clinical data, lung-function parameter, or thoracic HR-CT scans were available to evaluate the predictive capacity of BALF cytokines and chemokines. RESULTS: High IL-7 levels were characteristic of SSc-associated interstitial lung disease (ILD) and, in addition, when compared with ILD-negative SSc patients, ILD-positive SSc patients revealed higher IL-4, IL-6, IL-8, and CCL2 (MCP-1) BALF levels. High CCL2 and IL-8 BALF concentrations were associated with neutrophilic and mixed alveolitis. Cytokine levels of IL-4, IL-8, and CCL2 correlated negatively with lung-function parameters; CCL2 concentrations also correlated with HR-CT scores. High concentrations of several cytokines were associated with the progress of ILD and end-stage ILD. Univariate analyses revealed high IL-2 and tumor necrosis factor-alpha (TNF-alpha) levels as the best predictors for progressive disease, together with lung-function parameters, young age, and neutrophilic alveolitis. Multivariate analyses partially confirmed these results but did not sufficiently converge because of the limited number of patients. CONCLUSIONS: The association of BALF cytokines with lung fibrosis and its progress suggests that cytokines contribute to the pathogenesis of ILD and hence could be regarded as potential therapeutic targets.

Role of the spleen in peripheral memory B-cell homeostasis in patients with autoimmune thrombocytopenia purpura.

The effect of splenectomy on circulating memory B cells in autoimmune thrombocytopenia purpura (AITP) patients has not yet been addressed. We therefore analyzed the distribution and phenotypic characteristics of B-cell subsets in non-splenectomized and splenectomized AITP patients and controls, as well as CD95 expression after B cell activation. Decreased frequencies of memory B cells in splenectomized individuals were observed, with a rapid decline of CD27+IgD+ and a slower decrease of CD27+IgD- and CD27-/IgD- cells. Similar results were noted following splenectomy in healthy donors (HD). CD95+ B cells were substantially increased in all subsets in patients with active AITP, indicating their enhanced activation status. After splenectomy, the percentage of CD95+ B cells were further increased in the CD27+IgD- post-switch memory population in AITP, but not in HD. CD95+CD27+ memory B cells largely reside in the region in the human spleen analogous to the murine marginal zone. Thus, the spleen plays a fundamental role in controlling peripheral memory B cell homeostasis in both AITP and HD and regulates activated CD95+ B cells in patients with AITP.

High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets are opsonised by autoantibodies and destroyed by macrophages. Therefore, ITP represents a prototype of a B-cell-mediated autoimmune disorder. B-cell activating factor (BAFF) is a member of the tumour necrosis factor family and an important regulator of B-cell development. BAFF levels were determined in serum samples from 53 patients with ITP. Serum BAFF levels in patients with an active ITP were increased when compared with the healthy control group (median 1620 pg/ml vs. 977 pg/ml; P < 0.001). Moreover, immunosuppressive treatment was associated with strongly suppressed BAFF levels (median 629 pg/ml; P < 0.01). In addition, a polymorphic site was detected in the BAFF promoter region (-871) that appeared to occur more frequently in ITP patients than in healthy persons. This promoter variant was associated with very high BAFF levels in ITP patients. Our data suggest that BAFF is an important pathogenetic factor in the development of ITP.

Immunopathologic role of B lymphocytes in rheumatoid arthritis: rationale of B cell-directed therapy.

Although the immunopathogenesis of rheumatoid arthritis (RA) remains unclear, recent advances have paved the way for new therapies, such as anti-cytokine and cell-directed therapies. Here, B cells have re-gained interest concerning the pathogenesis of a number of autoimmune diseases after observing that patients with RA and non-Hodgkin lymphoma, who received anti-CD20 therapy leading to B cell depletion, demonstrated remarkable improvements. The underlying modes of action appear to be related to B cell functions, such as deletion of memory B cells, interruption of immune activation, antigen-presentation and production of inflammatory cytokines. In many RA patients, synovial extrafollicular germinal centers develop, where B cells play an intimate role in local inflammation and the generation of memory B cells and plasma cells. These local processes lead to activation of the immune system and ultimately to joint destruction in RA. Recent data demonstrating the clinical value of B cell depletion in refractory RA patients substantiate the notion that B cells are important players in the pathogenesis of the disease. Future studies should clarify which functions are affected by B cell depletion, providing the promise of new avenues to patient-tailored therapies.

Tissue-specific up-regulation of the proteasome subunit beta5i (LMP7) in Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is characterized by autoimmune infiltration and focal accumulation of lymphocytes in the exocrine glands, with a predominance of CD4-positive T cells. Since these histologic findings are nonspecific, determination of clinical and serologic abnormalities contribute to the diagnosis. The aim of this study was to identify a novel, disease-specific, immunologically relevant marker for SS. METHODS: To analyze disease-related and tissue-specific expression of candidate markers, we examined biopsied minor salivary glands and peripheral blood mononuclear cells from patients with primary and secondary SS (n = 26) as well as from patients with sicca symptoms without autoimmune sialadenitis (n = 15). Expression of the Th1/Th2-related chemokines CCL3 (macrophage inflammatory protein 1alpha) and CCL2 (monocyte chemoattractant protein 1), CXCL7 (neutrophil-activating peptide 2 [NAP-2]), interleukin-1beta, inducible costimulator, and the proteasome subunits alpha3 (C9) and beta5i (LMP7) was analyzed at the messenger RNA (mRNA) level using real-time polymerase chain reaction techniques. Immunohistochemical analysis was used to identify the beta5i (LMP7)-expressing cell populations in minor salivary glands. RESULTS: The expression profiles revealed a significant up-regulation of beta5i (LMP7) exclusively in the salivary glands of SS patients. Immunohistochemistry confirmed expression of the immunoproteasome subunit beta5i (LMP7) within the acinar and ductal epithelial cells. No significant difference in the distinct histologic focus scores was evident for the expression of the markers investigated. In the peripheral blood compartment, the expression of CXCL7 was up-regulated both in primary and in secondary SS. CONCLUSION: Tissue-specific up-regulation of beta5i (LMP7) mRNA was shown to be characteristic of SS, indicating a disease-specific modulation of the proteasome system. Expression of beta5i (LMP7) represents an independent parameter that can be used in addition to the focus score to distinguish SS in biopsied labial salivary glands.

Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue.

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.