Molecular maps of synovial cells in inflammatory arthritis using an optimized synovial tissue dissociation protocol.

In this study, we optimized the dissociation of synovial tissue biopsies for single-cell omics studies and created a single-cell atlas of human synovium in inflammatory arthritis. The optimized protocol allowed consistent isolation of highly viable cells from tiny fresh synovial biopsies, minimizing the synovial biopsy drop-out rate. The synovium scRNA-seq atlas contained over 100,000 unsorted synovial cells from 25 synovial tissues affected by inflammatory arthritis, including 16 structural, 11 lymphoid, and 15 myeloid cell clusters. This synovial cell map expanded the diversity of synovial cell types/states, detected synovial neutrophils, and broadened synovial endothelial cell classification. We revealed tissue-resident macrophage subsets with proposed matrix-sensing (FOLR2+COLEC12high) and iron-recycling (LYVE1+SLC40A1+) activities and identified fibroblast subsets with proposed functions in cartilage breakdown (SOD2highSAA1+SAA2+SDC4+) and extracellular matrix remodeling (SERPINE1+COL5A3+LOXL2+). Our study offers an efficient synovium dissociation method and a reference scRNA-seq resource, that advances the current understanding of synovial cell heterogeneity in inflammatory arthritis.

Gastric Adenocarcinoma Presenting as a Rheumatoid Factor and Anti-cyclic Citrullinated Protein Antibody-Positive Polyarthritis: A Case Report and Review of Literature.

A 64-year-old male presented with a 6-month history of symmetric polyarthritis involving proximal interphalangeal joints and metacarpophalangeal joints of the hands, wrists, and ankles. Associated symptoms included vomiting, progressive fatigue, and weight loss. Laboratory results showed microcytic anemia, leukocytosis, thrombocytosis, elevated C-reactive protein and erythrocyte sedimentation rate, and rheumatoid factor (RF) and anti-cyclic citrullinated protein (ACPA) antibody positivity. Joints radiographs were normal, without erosions. Upper endoscopy and gastric endoscopic ultrasonography showed a gastric adenocarcinoma with lymphatic involvement. Intraoperatively, peritoneal carcinomatosis was documented, and the patient started palliative chemotherapy. A paraneoplastic seropositive arthritis was assumed, and treatment with low-dose prednisolone and hydroxychloroquine was started. Arthritis remission was achieved and sustained up to 18 months of follow-up, although gastric cancer progression was documented. We describe a unique phenotype of paraneoplastic arthritis (PA) presenting as a seropositive (RF and ACPA positivity) rheumatoid arthritis (RA) with a good response to both low dose corticosteroids and hydroxychloroquine therapy. We also review the literature of PA, mostly the RA-like pattern, and the association between PA and ACPA positivity. This case highlights the importance of considering underlying cancer in elderly male patients, presenting with polyarthritis and systemic symptoms, even in those with ACPA-positive RA-like arthritis.

Efficacy, Safety, and Sample Quality of Ultrasound-Guided Synovial Needle Biopsy in Clinical Practice and Research: A Prospective Observational Study.

OBJECTIVE: To study the efficacy, tolerability, safety, and sampling variation of ultrasound (US)-guided synovial biopsies performed in clinical practice and research. METHODS: We included all patients who had a US-guided synovial needle biopsy from November 2013 to January 2018. Patients were evaluated for procedure safety and tolerability. Usefulness of synovial biopsy was considered based on contribution for achieving the proposed aims. We analyzed samples for presence and quality of synovial tissue, synovitis score/grade, and pathotype. Variation across patients, samples, section levels, and sampling order was assessed. RESULTS: A total of 64 US-guided synovial biopsies were performed (n = 52 in clinical practice, n = 12 in research). Patient tolerability (70% no/mild discomfort) was remarkably high. There was no significant aggravation of symptoms or US synovitis in the biopsied joint. Procedures were overall safe, with few minor, 2 moderate, and no major adverse events. Usefulness of US-guided synovial biopsies was high, both in clinical practice (37% direct diagnostic impact, 100% positive/95% negative predictive values for infection) and in research (92% success). Synovial tissue was retrieved in 88% of biopsies, with a median of 75% gradable samples. There was significant variation in sample quality and synovitis features across patients and samples, but not between different section levels. Samples collected later in the procedure had a lower frequency of synovial tissue and were poorly concordant in pathotype with those collected earlier. CONCLUSION: US-guided synovial needle biopsy is an effective, safe, and well-tolerated means to collect good quality synovial tissue for clinical and research purposes. Samples collected for different aims should be retrieved in parallel, rather than sequentially.

Ultrasound-Guided Biopsies: Medium and Large Joints.

Ultrasound-guided needle synovial biopsies are useful for clinical practice and research in rheumatology. With the emergence of personalized medicine for the treatment of inflammatory rheumatic diseases, it is predicted that this technique will be increasingly used in the near future. Standardized characterization of the technical aspects of ultrasound-guided needle synovial biopsies is needed in order to produce solid evidence on the safety and effectiveness of the technique.

Treatment of Persistent Cutaneous Atrophy After Corticosteroid Injection With Fat Graft.

Subcutaneous fat atrophy and hypopigmentation are potential adverse side effects of local corticosteroid injection that may resolve spontaneously within 1-2 years. This report shows that fat grafting provides a simple, effective and safe correction of corticosteroid induced cutaneous atrophy with very satisfying esthetic and functional results.

B-cell phenotype and IgD-CD27- memory B cells are affected by TNF-inhibitors and tocilizumab treatment in rheumatoid arthritis.

BACKGROUND: The use of TNF-inhibitors and/or the IL-6 receptor antagonist, tocilizumab, in rheumatoid arthritis (RA) have pleiotropic effects that also involve circulating B-cells. The main goal of this study was to assess the effect of TNF-inhibitors and tocilizumab on B-cell phenotype and gene expression in RA. METHODS: Blood samples were collected from untreated early RA (ERA) patients, established RA patients under methotrexate treatment, established RA patients before and after treatment with TNF-inhibitors and tocilizumab, and healthy donors. B-cell subpopulations were characterized by flow cytometry and B-cell gene expression was analyzed by real-time PCR on isolated B-cells. Serum levels of BAFF, CXCL13 and sCD23 were determined by ELISA. RESULTS: The frequency of total CD19+ B cells in circulation was similar between controls and all RA groups, irrespective of treatment, but double negative (DN) IgD-CD27- memory B cells were significantly increased in ERA and established RA when compared to controls. Treatment with TNF-inhibitors and tocilizumab restored the frequency of IgD-CD27- B-cells to normal levels, but did not affect other B cell subpopulations. TACI, CD95, CD5, HLA-DR and TLR9 expression on B-cells significantly increased after treatment with either TNF-inhibitors and/ or tocilizumab, but no significant changes were observed in BAFF-R, BCMA, CD69, CD86, CXCR5, CD23, CD38 and IgM expression on B-cells when comparing baseline with post-treatment follow-ups. Alterations in B-cell gene expression of BAFF-R, TACI, TLR9, FcγRIIB, BCL-2, BLIMP-1 and ß2M were found in ERA and established RA patients, but no significant differences were observed after TNF-inhibitors and tocilizumab treatment when comparing baseline and follow-ups. Serum levels of CXCL13, sCD23 and BAFF were not significantly affected by treatment with TNF-inhibitors and tocilizumab. CONCLUSIONS: In RA patients, the use of TNF-inhibitors and/ or tocilizumab treatment affects B-cell phenotype and IgD-CD27- memory B cells in circulation, but not B-cell gene expression levels.

Portuguese recommendations for the use of methotrexate in rheumatic diseases - 2016 update.

BACKGROUND: Methotrexate (MTX) is the first-line drug in the treatment of rheumatoid arthritis (RA) and the most commonly prescribed disease modifying anti-rheumatic drug. Moreover, it is also used as an adjuvant drug in patients under biologic therapies, enhancing the efficacy of biologic agents. OBJECTIVES: To review the literature and update the Portuguese recommendations for the use of MTX in rheumatic diseases first published in 2009. METHODS: The first Portuguese guidelines for the use of MTX in rheumatic diseases were published in 2009 and were integrated in the multinational 3E Initiative (Evidence Expertise Exchange) project. The Portuguese rheumatologists based on literature evidence and consensus opinion formulated 13 recommendations. At a national meeting, the recommendations included in this document were further discussed and updated. The document resulting from this meeting circulated to all Portuguese rheumatologists, who anonymously voted online on the level of agreement with the updated recommendations. RESULTS: Results presented in this article are mainly in accordance with previous guidelines, with some new information regarding hepatitis B infection during MTX treatment, pulmonary toxicity monitoring, hepatotoxicity management, association with hematologic neoplasms, combination therapy and tuberculosis screening during treatment. CONCLUSION: The present recommendations combine scientific evidence with expert opinion and attained desirable agreement among Portuguese rheumatologists. The regular update of these recommendations is essential in order to keep them a valid and useful tool in daily practice.

Comparative Effectiveness of Tocilizumab and TNF Inhibitors in Rheumatoid Arthritis Patients: Data from the Rheumatic Diseases Portuguese Register, Reuma.pt.

OBJECTIVES: To compare the effectiveness of TNF inhibitors (TNFi) and tocilizumab in rheumatoid arthritis (RA) treatment, according to different response criteria. METHODS: We included RA patients registered in the Rheumatic Diseases Portuguese Register treated with TNFi or tocilizumab for at least 6 months, between January 2008 and July 2013. We assessed remission/low disease activity (LDA) at 6 months according to DAS28, CDAI, and SDAI, as well as Boolean ACR/EULAR remission and EULAR response rate, adjusting for measured confounders. RESULTS: Tocilizumab-treated patients (n = 95) presented higher baseline disease activity and were less frequently naïve to biologics compared to TNFi users (n = 429). Multivariate logistic regression analysis including the propensity score for receiving tocilizumab showed that patients treated with tocilizumab were more likely to achieve remission or LDA according to DAS28 (OR = 11.0/6.2, 95% CI 5.6-21.6/3.2-12.0), CDAI (OR = 2.8/2.6, 95% CI 1.2-6.5/1.3-5.5), or SDAI (OR = 3.6/2.5, 95% CI 1.5-8.7/1.1-5.5), as well as a good EULAR response (OR = 6.4, 95% CI 3.4-12.0). However, both groups did not differ in Boolean remission (OR = 1.9, 95% CI 0.8-4.8) or good/moderate EULAR response (OR = 1.8, 95% CI 0.8-4.5). CONCLUSIONS: Compared with TNFi, tocilizumab was associated with greater likelihood of achieving DAS28, CDAI, and SDAI remission/LDA and EULAR good response. Boolean remission and EULAR good/moderate response did not differ significantly between groups.

TRAF1/C5 but not PTPRC variants are potential predictors of rheumatoid arthritis response to anti-tumor necrosis factor therapy.

BACKGROUND: The aim of our work was to replicate, in a Southern European population, the association reported in Northern populations between PTPRC locus and response to anti-tumor necrosis factor (anti-TNF) treatment in rheumatoid arthritis (RA). We also looked at associations between five RA risk alleles and treatment response. METHODS: We evaluated associations between anti-TNF treatment responses assessed by DAS28 change and by EULAR response at six months in 383 Portuguese patients. Univariate and multivariate linear and logistic regression analyses were performed. In a second step to confirm our findings, we pooled our population with 265 Spanish patients. RESULTS: No association was found between PTPRC rs10919563 allele and anti-TNF treatment response, neither in Portuguese modeling for several clinical variables nor in the overall population combining Portuguese and Spanish patients. The minor allele for RA susceptibility, rs3761847 SNP in TRAF1/C5 region, was associated with a poor response in linear and logistic univariate and multivariate regression analyses. No association was observed with the other allellic variants. Results were confirmed in the pooled analysis. CONCLUSION: This study did not replicate the association between PTPRC and the response to anti-TNF treatment in our Southern European population. We found that TRAF1/C5 risk RA variants potentially influence anti-TNF treatment response.

BAFF and TACI gene expression are increased in patients with untreated very early rheumatoid arthritis.

OBJECTIVE: B cells play important roles in rheumatoid arthritis (RA). Given the beneficial effect of B cell depletion therapy in RA as well as the observed alterations in B cell subpopulations in this disease, we evaluated whether changes in the expression of genes related to B cell survival and activation were already present in patients with untreated very early RA (VERA; < 6 weeks of disease duration). METHODS: The expression of a group of B cell-related activation and survival genes was quantified in peripheral blood mononuclear cells from patients with VERA by real-time PCR and compared with untreated early RA (< 1 year), established treated RA, and other untreated early arthritis conditions. Serum B cell-activating factor belonging to the tumor necrosis factor family (BAFF) was quantified by ELISA. RESULTS: BAFF gene expression and serum levels were highest in patients with VERA. The expression of BAFF receptor (BAFF-R) increased with disease progression, while transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) was elevated since the first weeks of RA onset. Paired box 5 gene expression was also increased at all RA stages. Chemokine (C-X-C motif) receptor 5 was elevated only in established RA. No differences were observed in B cell maturation antigen, activation-induced cytidine deaminase, B lymphocyte-induced maturation protein, and B cell lymphoma 2 expression. CONCLUSION: Disturbances in the expression of B cell-related activation and survival genes, particularly BAFF and TACI, occur from the onset of RA and precede changes in BAFF-R. These alterations can lead to the development of autoreactive B cells from the first weeks of RA onset.

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures.

INTRODUCTION: Osteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization. We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event. METHODS: In this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined. Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis. RESULTS: 64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p<0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~0; p=0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p=0.029). ucOC levels were higher in the fracture group although not significantly (p=0.058). No differences were found regarding total OC (p=0.602), apoE (p=0.467) and Vitamin K (p=0.371). In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (ß=0.607; p=0.013) and stiffness (ß=0.693; p=0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (ß=-0.411; p=0.030) and stiffness (ß=-0.487; p=0.009). CONCLUSION: We demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.

Smoking is a predictor of worse trabecular mechanical performance in hip fragility fracture patients.

Clinical risk factors (CRFs) are established predictors of fracture events. However, the influence of individual CRFs on trabecular mechanical fragility is still a subject of debate. In this study, we aimed to assess differences, adjusted for CRFs, between bone macrostructural parameters measured in ex-vivo specimens from hip fragility fracture patients and osteoarthritis patients, and to determine whether individual CRFs could predict trabecular bone mechanical behavior in hip fragility fractures. Additionally, we also looked for associations between the 10-year risk of major and hip fracture calculated by FRAX and trabecular bone mechanical performance. In this case-control study, a group of fragility fracture patients were compared with a group of osteoarthritis patients, both having undergone hip replacement surgery. A clinical protocol was applied in order to collect CRFs [body mass index (BMI), prior fragility fracture, parental history of hip fracture, long-term use of oral glucocorticoids, rheumatoid arthritis, current smoking, alcohol consumption, age and gender]. The 10-year probability of fracture was calculated. Serum bone turnover markers were determined and dual X-ray absorptiometry performed. Femoral head diameter was evaluated and trabecular bone cylinders were drilled for mechanical testing to determine bone strength, stiffness and toughness. We evaluated 40 hip fragility fracture and 52 osteoarthritis patients. Trabecular bone stiffness was significantly lower (p = 0.042) in hip fragility fracture patients when compared to osteoarthritic individuals, adjusted for age, gender and BMI. No other macrostructural parameter was statistically different between the groups. In hip fragility fracture patients, smoking habits (ß = -0.403; p = 0.018) and female gender (ß = -0.416; p = 0.008) were independently associated with lower stiffness. In addition, smoking was also independently associated with worse trabecular strength (ß = -0.323; p = 0.045), and toughness (ß = -0.403; p = 0.018). In these patients, the 10-year risk of major (r = -0.550; p = 0.012) and hip fracture (r = -0.513; p = 0.021) calculated using only CRFs was strongly correlated with femoral neck bone mineral density but not with mechanical performance. Our data showed that among fragility fracture patients active smoking is a predictor of worse intrinsic trabecular mechanical performance, and female gender is also independently associated with lower stiffness. In this population, the 10-year risk of fracture using CRFs with different weights only reflects bone mass loss but not trabecular mechanical properties.

Comparative effectiveness and predictors of response to tumour necrosis factor inhibitor therapies in rheumatoid arthritis.

OBJECTIVES: Adalimumab, etanercept and infliximab are effective TNF inhibitors (TNFis) in the treatment of RA, but no randomized clinical trials have compared the three agents. Prior observational data are not consistent. We compared their effectiveness over 1 year in a prospective cohort. METHODS: Analyses were performed on subjects' first episode of TNFi use in the Rheumatic Diseases Portuguese Register, Reuma.pt. The primary outcome was the proportion of patients with European League Against Rheumatism good response sustained at two consecutive observations separated by 3 months during the first year of TNFi use. Comparisons were performed using conventional adjusted logistic regression, as well as matching subjects across the three agents using a propensity score. In addition, baseline predictors of treatment response to TNFi were identified. RESULTS: The study cohort included 617 RA patients, 250 starting etanercept, 206 infliximab and 161 adalimumab. Good response was achieved by 59.6% for adalimumab, 59.2% for etanercept and 51.9% for infliximab (P = 0.21). The modelled probability of good response did not significantly differ across agents (etanercept vs adalimumab OR = 0.97, 95% CI 0.55, 1.71; etanercept vs infliximab OR = 1.25, 95% CI 0.74, 2.12; infliximab vs adalimumab OR = 0.80, 95% CI 0.47, 1.36). Matched propensity score analyses also showed no significant treatment response differences. Greater educational attainment was a predictor of better response, while smoking, presence of ACPA, glucocorticoid use and worse physician assessment of disease activity at baseline each predicted a reduced likelihood of treatment response. CONCLUSION: Over 1 year, we found no difference in effectiveness between adalimumab, etanercept and infliximab.

Primary biliary cirrhosis in a rheumatoid arthritis patient treated with rituximab, a case-based review.

Primary biliary cirrhosis (PBC) is an autoimmune disease in which intrahepatic bile ducts are targeted by an immune-mediated injury. This disease tends to progress to fibrosis and cirrhosis with hepatic failure. The authors report a case of a 50-year-old rheumatoid arthritis (RA) patient, with erosions and seropositive for rheumatoid factor and anti-citrullinated peptide antibodies, with 18 years disease duration refractory to prednisolone and several disease-modifying antirheumatic drugs, either conventional or biological (adalimumab and etanercept). In April 2007, she started therapy with rituximab (RTX) with good European League Against Rheumatism response achieved 9 months later. In June 2008, she was admitted with intrahepatic cholestasis, steatorrhea, and spontaneous fractures of various ribs. After excluding cholelitiasis, as well as infectious and neoplastic diseases a liver biopsy was performed that was compatible with the diagnosis of PBC. The antinuclear antibodies (1/160) were positive as well as the antimitochondrial antibodies (1/640). Other antibodies were negative such as anti-SSA and anti-SSB. Afterwards, the patient started ursodesoxycholic acid 15 mg kg(-1) day(-1) with progressive improvement of cholestatic markers. A labial salivary gland biopsy was performed and showed findings compatible with the concomitant diagnosis of Sjögren's syndrome. Based on this clinical report, a detailed review of the clinical aspects of PBC is presented as well as its association with other immune-mediated inflammatory diseases, particularly, with RA.

Cytokine pattern in very early rheumatoid arthritis favours B-cell activation and survival.

OBJECTIVES: B cells play an important role in the perpetuation of RA, particularly as autoantibody-producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process. However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum from very early polyarthritis patients, with <6 weeks of disease duration, who latter on evolved into very early RA (VERA). METHODS: A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) and IL-21 levels were measured by ELISA in the serum of VERA, other very early arthritis (VEA), established RA patients and controls. SF samples of established RA were also analysed. RESULTS: VERA patients have higher levels of APRIL and BAFF as compared with VEA, established RA and controls. Furthermore, APRIL and BAFF levels are also significantly elevated in RA-SF when compared with serum. CONCLUSIONS: The increased levels of APRIL and BAFF in VERA patients suggests that B-cell activation and the development of autoreactive B-cell responses might be crucial in early phases of RA. Therefore, APRIL and BAFF could be promising targets for therapy in the early phase of RA.