Influence of Dextran Sulphate, Fibrin, and Ubiquitin on the Development of Casein-Induced Experimental AA Amyloidosis in C57BL/6 mice

The influence of subcutaneous injections of dextran sulphate (DS) and fibrin (F), as well as of an intraperitoneal  injection of ubiquitin (Ub), was investigated on 48 male C57BL/6 mice subjected to conventional  casein (C) induced amyloidosis. Histopathological examination of spleen and kidney tissue 3 and 5 weeks  after termination of the amyloidogenic stimulus showed that the amount of amyloid deposited (rated trace,  minimal, moderate or heavy) increased progressively with the duration of the amyloidogenic stimulus.  After 3 weeks of stimulation, 16.7% of mice injected with C had some perifollicular amyloid deposits in  the spleen while all had traces of amyloid in the kidney. Some amyloid was detected in the spleen of 33.3%  of the mice treated with C+DS and C+Ub and 83.3% treated with C+F. Half the latter group also showed  traces and half minimal amyloid deposits in their kidneys. In the other test groups, the incidence of kidney  amyloidosis was less. The most extensive tissue deposits were seen at 5 weeks postinjection (p.i.) with most in the C+F-treated  animals, all showing significantly more than the control C-treated group. Thus half the C+F-treated animals  had moderate and half heavy deposits throughout their spleens. Glomerulonephritis, kidney tubular  edema and some amyloid deposits were present in all of the animals. C+Ub resulted in a similar incidence  of amyloid accumulation in the spleen but in the kidneys 66.7% of animals had only traces of amyloid and  33.3%, minimal amyloid deposits. Amyloid was deposited in the mouse kidneys predominantly in the arterial  walls but also occurred in the basement membrane and interstitial tissues. A post-mortem examination  of the internal organs revealed splenomegaly in all the test groups and increased liver weight in the C-,  C+F-, and C+Ub-treated groups. The leukocyte count and ESR (erythrocyte sedimentation rate) were also  higher in all the experimental groups. Thus, the results indicated that F and Ub play a role in the amyloid deposition process in the experimentally  induced disorder in C57BL/6 mice and could enhance this pathological process.

S.6.1 β-catenin is a central mediator in SSc

Background. β-catenin is the central integrator of canonical Wnt signalling. Since recent evidence suggests a central role of Wnts in fibrosis, we examined the β-catenin/Wnt pathway in SSc and focused on the role of β-catenin in fibroblast activation. Methods. We performed qPCR for several Wnt ligands and axin-2 to examine Wnt expression in SSc skin. We further studied protein levels of Wnt-1, -4, -10b and β-catenin by IHC. To establish the effects of β-catenin/Wnt signalling on collagen release, we created mice with fibroblast-specific stabilization of β-catenin (dEx3 β-catenin (wt/fl) × Col1a2; Cre-ER) as well as mice carrying fibroblast-specific deletion of β-catenin [Ctnnb1(fl/fl) × Col1a2; Cre-ER]. Summary of the results. We could demonstrate mRNA overexpression of Wnt-1, -2, -9a, -9b, -10a, -10b and -16 in SSc skin. Wnt-1, -4 and -10b consistently showed strong expression in SSc skin when compared with healthy skin. On protein level, however, Wnt-4 was indistinguishable between SSc patients and healthy controls, whereas Wnt-1 and Wnt-10b protein levels were increased in SSc skin. The overexpression of Wnt-1 and Wnt-10b resulted in a prominent nuclear accumulation of β-catenin in fibroblasts. Finally, increased mRNA levels of the target gene axin-2 confirmed the activation of canonical Wnt signalling. In dEx3 β-catenin (wt/ex) mice, we addressed the consequences of enhanced Wnt signalling and increased accumulation of β-catenin in SSc. We selectively targeted β-catenin in fibroblasts. Cre-activated dEx3 β-catenin (wt/fl) × Col1a2; Cre-ER mice showed massive and spontaneous dermal thickening even 2 weeks after Cre activation. Eight weeks after Cre-activation, skin thickening cumulated at 102.6% (P < 0.001). In line with the dermal thickening, hydroxyproline content and myofibroblast counts showed strong increases. To test the therapeutic potential of targeting β-catenin/Wnt signaling, we created Ctnnb1(fl/fl) x Col1a2;Cre-ER mice to specifically delete β-catenin in fibroblasts. After Cre activation and β-catenin deletion in fibroblasts, mice were challenged with bleomycin subcutaneously for 4 weeks. We found that Cre-activated Ctnnb1(fl/fl) × Col1a2; Cre-ER mice were protected from bleomycin-induced dermal with a reduction of skin thickening by 71% (P < 0.05). Conclusions. We demonstrated a prominent activation of canonical Wnt signalling in SSc with nuclear accumulation of β-catenin in fibroblasts and activation of the target gene axin-2. Our results showed that fibroblast-specific stabilization of β-catenin resulted in enhanced collagen release, whereas deletion of β-catenin potently reduced collagen production. Together, our findings highlight a key role of β-catenin in fibroblast activation and fibrosis. Thus, β-catenin may be promising molecular target for anti-fibrotic therapie

Infliximab plus methotrexate is superior to methotrexate alone in the treatment of psoriatic arthritis in methotrexate-naive patients: the RESPOND study

Objective: To compare the efficacy and safety of treatment with infliximab plus methotrexate with methotrexate alone in methotrexate-naive patients with active psoriatic arthritis (PsA). Methods: In this open-label study, patients 18 years and older with active PsA who were naive to methotrexate and not receiving disease-modifying therapy (N=115) were randomly assigned (1:1) to receive either infliximab (5 mg/kg) at weeks 0, 2, 6 and 14 plus methotrexate (15 mg/week); or methotrexate (15 mg/week) alone. The primary assessment was American College of Rheumatology (ACR) 20 response at week 16. Secondary outcome measures included psoriasis area and severity index (PASI), disease activity score in 28 joints (DAS28) and dactylitis and enthesitis assessments. Results: At week 16, 86.3% of patients receiving infliximab plus methotrexate and 66.7% of those receiving methotrexate alone achieved an ACR20 response (p<0.02). Of patients whose baseline PASI was 2.5 or greater, 97.1% receiving infliximab plus methotrexate compared with 54.3% receiving methotrexate alone experienced a 75% or greater improvement in PASI (p<0.0001). Improvements in C-reactive protein levels, DAS28 response and remission rates, dactylitis, fatigue and morning stiffness duration were also significantly greater in the group receiving infliximab. In the infliximab plus methotrexate group, 46% (26/57) had treatment-related adverse events (AE) and two patients had serious AE, compared with 24% with AE (13/54) and no serious AE in the methotrexate-alone group. Conclusions: Treatment with infliximab plus methotrexate in methotrexate-naive patients with active PsA demonstrated significantly greater ACR20 response rates and PASI75 improvement compared with methotrexate alone and was generally well tolerated. This trial is registered in the US National Institutes of Health clinicaltrials.gov database, identifier NCT00367237

Predictors of disease worsening defined by progression of organ damage in diffuse systemic sclerosis: a European Scleroderma Trials and Research (EUSTAR) analysis.

Objectives Mortality and worsening of organ function are desirable endpoints for clinical trials in systemic sclerosis (SSc). The aim of this study was to identify factors that allow enrichment of patients with these endpoints, in a population of patients from the European Scleroderma Trials and Research group database. Methods Inclusion criteria were diagnosis of diffuse SSc and follow-up over 12\ub13 months. Disease worsening/organ progression was fulfilled if any of the following events occurred: new renal crisis; decrease of lung or heart function; new echocardiography-suspected pulmonary hypertension or death. In total, 42 clinical parameters were chosen as predictors for the analysis by using (1) imputation of missing data on the basis of multivariate imputation and (2) least absolute shrinkage and selection operator regression. Results Of 1451 patients meeting the inclusion criteria, 706 had complete data on outcome parameters and were included in the analysis. Of the 42 outcome predictors, eight remained in the final regression model. There was substantial evidence for a strong association between disease progression and age, active digital ulcer (DU), lung fibrosis, muscle weakness and elevated C-reactive protein (CRP) level. Active DU, CRP elevation, lung fibrosis and muscle weakness were also associated with a significantly shorter time to disease progression. A bootstrap validation step with 10 000 repetitions successfully validated the model. Conclusions The use of the predictive factors presented here could enable cohort enrichment with patients at risk for overall disease worsening in SSc clinical trial

Progressive skin fibrosis is associated with a decline in lung function and worse survival in patients with diffuse cutaneous systemic sclerosis in the European Scleroderma Trials and Research (EUSTAR) cohort.

Objectives To determine whether progressive skin fibrosis is associated with visceral organ progression and mortality during follow-up in patients with diffuse cutaneous systemic sclerosis (dcSSc). Methods We evaluated patients from the European Scleroderma Trials and Research database with dcSSc, baseline modified Rodnan skin score (mRSS) ≥7, valid mRSS at 12±3 months after baseline and ≥1 annual follow-up visit. Progressive skin fibrosis was defined as an increase in mRSS &gt;5 and ≥25% from baseline to 12±3 months. Outcomes were pulmonary, cardiovascular and renal progression, and all-cause death. Associations between skin progression and outcomes were evaluated by Kaplan-Meier survival analysis and multivariable Cox regression. Results Of 1021 included patients, 78 (7.6%) had progressive skin fibrosis (skin progressors). Median follow-up was 3.4 years. Survival analyses indicated that skin progressors had a significantly higher probability of FVC decline ≥10% (53.6% vs 34.4%; p&lt;0.001) and all-cause death (15.4% vs 7.3%; p=0.003) than non-progressors. These significant associations were also found in subgroup analyses of patients with either low baseline mRSS (≤22/51) or short disease duration (≤15 months). In multivariable analyses, skin progression within 1 year was independently associated with FVC decline ≥10% (HR 1.79, 95% CI 1.20 to 2.65) and all-cause death (HR 2.58, 95% CI 1.31 to 5.09). Conclusions Progressive skin fibrosis within 1 year is associated with decline in lung function and worse survival in dcSSc during follow-up. These results confirm mRSS as a surrogate marker in dcSSc, which will be helpful for cohort enrichment in future trials and risk stratification in clinical practice

Phenotypes Determined by Cluster Analysis and Their Survival in the Prospective European Scleroderma Trials and Research Cohort of Patients With Systemic Sclerosis

Objective: Systemic sclerosis (SSc) is a heterogeneous connective tissue disease that is typically subdivided into limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) depending on the extent of skin involvement. This subclassification may not capture the entire variability of clinical phenotypes. The European Scleroderma Trials and Research (EUSTAR) database includes data on a prospective cohort of SSc patients from 122 European referral centers. This study was undertaken to perform a cluster analysis of EUSTAR data to distinguish and characterize homogeneous phenotypes without any a priori assumptions, and to examine survival among the clusters obtained. / Methods: A total of 11,318 patients were registered in the EUSTAR database, and 6,927 were included in the study. Twenty‐four clinical and serologic variables were used for clustering. / Results: Clustering analyses provided a first delineation of 2 clusters showing moderate stability. In an exploratory attempt, we further characterized 6 homogeneous groups that differed with regard to their clinical features, autoantibody profile, and mortality. Some groups resembled usual dcSSc or lcSSc prototypes, but others exhibited unique features, such as a majority of lcSSc patients with a high rate of visceral damage and antitopoisomerase antibodies. Prognosis varied among groups and the presence of organ damage markedly impacted survival regardless of cutaneous involvement. / Conclusion: Our findings suggest that restricting subsets of SSc patients to only those based on cutaneous involvement may not capture the complete heterogeneity of the disease. Organ damage and antibody profile should be taken into consideration when individuating homogeneous groups of patients with a distinct prognosis

Disease activity and health status in rheumatoid arthritis: a case-control comparison between Norway and Lithuania

Objective: To compare disease characteristics and health status in patients with rheumatoid arthritis (RA) from two countries, Norway and Lithuania. Methods: Patients were recruited from the RA registers in Vilnius (Lithuania) and Oslo (Norway). For each patient from Vilnius, a patient matched for age and sex from the Oslo register was selected. Sociodemographic characteristics, disease process, and health status were compared between the patient groups. Results: 201 Lithuanian patients and 201 Norwegian patients were included. Mean (SD) age in both groups was 55.9 (10.0) years, and 83% were women. Patients from Lithuania were less often employed (27% v 42%; p=0.001), had higher disease activity expressed by the disease activity score (DAS28; mean (SD) 5.3 (1.0) v 4.4 (1.4); p<0.001), had worse physical function by the modified Health Assessment Questionnaire (MHAQ; mean (SD) 2.3 (0.8) v 1.6 (0.5); p<0.001), had more often comorbidity (73% v 53%; p<0.001) and they reported worse general health measured by Short Form-36 Health Survey (SF-36; mean (SD) 23.2 (13.5) v 44.5 (21.3); p<0.001). The proportions of patients who had used disease modifying drugs were similar, but the pattern of use differed. Conclusion: Important differences in employment, disease activity, physical function, and self reported health status were observed in patients with RA from two northern European countries. Socioeconomic inequalities, differences in disease management, and access to specialised health care, as well as methodological issues regarding instruments and data collection are likely explanations. These data support the view that management of RA should be adapted to country-specific needs

Lack of inhibititory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analyzed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc