Peptidyl arginine deiminase inhibition suppresses arthritis via decreased protein citrullination in joints and serum with the downregulation of interleukin-6.

Objective: To explore the relevance of citrullinated proteins and anti-citrullinated protein antibodies (ACPA) via protein arginine deiminase (PAD) inhibition in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA).Methods: Cl-amidine, a PAD inhibitor, was injected into pGIA. Clinical scores and histopathological findings of ankle joints were assessed. Serum ACPA titers were analyzed using ELISA. Citrullinated protein expression in joints and sera were examined with immunohistochemistry and Western blot analysis, respectively. Serum levels of IL-6, TNFα, and IL-1ß were measured with cytometric bead array (CBA). Gene expression levels of IL-6 and TNFα in joints, lymph nodes, and spleens were analyzed with quantitative PCR. GPI-specific productions of IFNγ and IL-17 from T cells in lymph nodes were evaluated.Results: Cl-amidine treatment significantly reduced arthritis severity while ACPA titers tended to be lower, but not significantly different compared to the control. Citrullinated proteins in joints and sera from treated mice were clearly decreased. With Cl-amidine treatment, serum IL-6 levels were significantly decreased, and IL-6 and TNFα gene expression were significantly reduced in joints. IL-17 production from GPI-specific T cells tended to be lower in Cl-amidine-treated mice, but not significantly different.Conclusion: Our results suggested that PAD-mediated citrullinated protein was involved in the pathogenesis of arthritis via IL-6.

Intractable Hemophagocytic Syndrome Associated with Systemic Lupus Erythematosus Resistant to Corticosteroids and Intravenous Cyclophosphamide That Was Successfully Treated with Cyclosporine A.

Hemophagocytic syndrome (HPS) associated with systemic lupus erythematosus (SLE), dubbed acute lupus hemophagocytic syndrome (ALHS), is an intractable complication of SLE. A 24-year-old man who had been diagnosed with SLE three months previously, presented with fever, rash, hallucination, and pancytopenia accompanied with hyperferritinemia and bone marrow hemophagocytosis. He was diagnosed with ALHS and neuropsychiatric (NP)-SLE. Although 4 courses of methylprednisolone pulse therapy and 1 course of intravenous cyclophosphamide (IVCY) improved his NP-SLE, his ALHS did not respond. However, the addition of cyclosporine A (CsA) led to a rapid remission from ALHS. This suggests the usefulness of CsA in the treatment of intractable, corticosteroid- and IVCY-resistant ALHS.

Identification of novel biomarker as citrullinated inter-alpha-trypsin inhibitor heavy chain 4, specifically increased in sera with experimental and rheumatoid arthritis.

BACKGROUND: Anticitrullinated protein antibodies (ACPA) and citrullinated proteins play key roles in the pathogenesis of rheumatoid arthritis (RA). Many candidate citrullinated antigens have been identified in joints, but citrullinated proteins in sera are mostly uncertain in patients with RA. We explored the expression of citrullinated proteins in joints and sera of experimental arthritis, and we further investigated their specific expression correlated with the disease activity in patients with RA. METHODS: Citrullinated protein expression in tissues was examined by IHC in peptide glucose-6-phosphate isomerase-induced arthritis (pGIA). Serum citrullinated proteins from pGIA were examined by Western blotting, and the sequence was identified by MS. With the same methods, serum citrullinated proteins were analyzed in patients with RA, primary Sjögren's syndrome, systemic lupus erythematosus, and osteoarthritis as well as in healthy subjects, by Western blotting and MS. In patients with RA, the relationship between the expression of the identified protein (inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and clinical features was evaluated, and the levels of citrullinated ITIH4 were compared before and after biological treatment. The antibody response against citrullinated ITIH4 peptide was measured by enzyme-linked immunosorbent assay. RESULTS: Citrullinated proteins were detected specifically in arthritic joints and sera from pGIA relative to controls. In sera, a common band of citrullinated protein at 120 kDa was revealed, and it fluctuated in parallel with arthritis score of pGIA by Western blotting. Interestingly, in 82% of RA patient sera, similar bands of citrullinated protein were specifically detected. These proteins were identified as citrullinated ITIH4, and especially the R438 site was commonly citrullinated between mice and humans. Citrullinated ITIH4 levels were associated with clinical parameters such as C-reactive protein (CRP), rheumatoid factor, and Disease Activity Score in 28 joints as measured by CRP in patients with RA. Its levels were decreased in correlation with the reduction of disease activity score after effective treatment in patients with RA. Moreover, antibody response to citrullinated epitope in ITIH4 was specifically observed in patients with RA. CONCLUSIONS: Our results suggest that serum citrullinated ITIH4 was specifically increased in patients with RA and could be a novel biomarker for assessing disease activity in patients with RA.

[The pathogenic role of ACPA in rheumatoid arthritis].

  In rheumatoid arthritis (RA), ACPA (anti-citrullinated protein/peptide antibody) is elevated with high specificity, and clinically, anti-CCP (cyclic citrullinated peptide) antibody is widely used for diagnosis of RA. It is thought ACPAs are produced with genetic background such as HLA-DR, environmental factors such as periodontal disease and smoking, however, the pathogenic role of ACPA in RA has not been elucidated. These were showed immune complexes including ACPA or ACPA itself promoted inflammatory cytokine production such as TNF. PADs (peptidylarginine deiminases) were expressed and citrullinated proteins existed in RA synovium. ACPAs were deposited on the site of citrulline in CD68 positive cells of RA synovium. The damage of bone and cartilage is observed in RA. It was also suggested that deposition of ACPAs caused osteoclastogenesis and bone loss. We introduce several findings about the pathogenic role of ACPA in RA.

TIARP attenuates autoantibody-mediated arthritis via the suppression of neutrophil migration by reducing CXCL2/CXCR2 and IL-6 expression.

TNFα-induced adipose-related protein (TIARP) is a six-transmembrane protein expressed on macrophages, neutrophils and synoviocytes. We reported recently that mice deficient in TIARP (TIARP-/-) spontaneously develop arthritis and are highly susceptible to collagen-induced arthritis (CIA) with enhanced interleukin (IL)-6 production. However, the effects of TIARP on neutrophils and fibroblast-like synoviocytes (FLS) have not been elucidated. We analyzed the roles of TIARP in K/BxN serum transfer model using TIARP-/- mice. Arthritis in TIARP-/- mice transferred with K/BxN serum was significantly exacerbated compared with WT mice. We characterized the differences in neutrophils between wild-type (WT) and TIARP-/- mice by DNA microarray. Overexpression of CXCR1 and CXCR2 was noted in TIARP-/- neutrophils. Neutrophils of TIARP-/- mice showed strong migration activity, which was markedly facilitated by CXCL2 in vitro and in vivo. Moreover, enhanced production of CXCL2 and IL-6 and cell proliferation was noted in TIARP-/- TNFα-stimulated FLS. Blockade of IL-6R significantly attenuated serum-transferred TIARP-/- arthritis with diminished neutrophil recruitment in joints. Our findings suggested that TIARP independently down-regulated CXCL2 and IL-6 production by FLS, and the expression of chemokine receptors (CXCR1 and CXCR2) in neutrophils, with resultant reduction of neutrophil migration into arthritic joints.

Effectiveness of abatacept for patients with Sjögren's syndrome associated with rheumatoid arthritis. An open label, multicenter, one-year, prospective study: ROSE (Rheumatoid Arthritis with Orencia Trial toward Sjögren's syndrome Endocrinopathy) trial.

OBJECTIVE: To clarify the efficacy and safety of abatacept for secondary Sjögren's syndrome (SS) associated with rheumatoid arthritis (RA). METHODS: The primary endpoint of this open-labeled, prospective, observational multicenter study for secondary SS with RA was the remission rate of Simplified Disease Activity Index (SDAI) at 52 weeks after initiation of abatacept. The secondary endpoints included Saxon's test and Schirmer's test. Adverse events and adherence rate during the study period were also analyzed. RESULTS: Thirty-six patients (all females) were enrolled in this study. The mean SDAI decreased significantly from 20.6 ± 11.2 (±SD) at baseline to 10.0 ± 10.5 at 52 weeks (p < 0.05). Patients with SDAI remission increased from 0 (0 week) to 12 patients (33.3%) at 52 weeks. Saliva volume assessed by Saxon's test increased significantly from 2136 ± 1809 (0 week) to 2397 ± 1878 (24 weeks) mg/2 min (n = 34, p < 0.05). Saliva volume increased significantly from 2945 ± 2090 (0 week) to 3419 ± 2121 (24 weeks) mg/2 min in 11 patients with Greenspan grade 1 or 2 of labial salivary gland biopsy (p < 0.05), but no change was noted in 18 patients with Greenspan grade 3 or 4. Tear volume by Schirmer's test increased significantly from 4.2 ± 4.8 (0 week) to 6.4 ± 7.8 (24 weeks) mm/5 min (n = 30, p < 0.05). The adherence rate to abatacept was 80.6% (29/36) over the 52-week period. Twelve adverse events occurred in 10 of the 36 patients, and 7 of these events were infections. CONCLUSION: Abatacept seems to be effective for both RA and SS related manifestations.

Association of anti-Ro/SSA antibody with response to biologics in patients with rheumatoid arthritis.

OBJECTIVE: To compare the effectiveness of three different biologics in anti-Ro/SSA antibody-positive and antibody-negative patients with rheumatoid arthritis (RA). METHODS: The study subjects were 110 biologics naïve patients with RA who started treatment with biologics and examined for anti-Ro/SSA antibody between December 2003 and March 2014. For patients treated with intravenous infliximab (IFX), tocilizumab (TCZ), or abatacept (ABT), we compared the clinical characteristics and changes in composite disease activity index, such as DAS28, SDAI, and CDAI, for 12 months in anti-Ro/SSA antibody-positive and antibody-negative patients. RESULTS: We examined 59 patients (nine were positive and 50 were negative for anti-Ro/SSA antibody) treated with IFX, 27 patients (5 positive and 22 negative) treated with TCZ, and 24 patients (13 positive and 11 negative) treated with ABT. For patients treated with IFX, parameters of disease activity did not change significantly from baseline in anti-Ro/SSA antibody-positive patients, whereas they improved in antibody-negative patients. On the other hand, treatment with TCZ and ABT significantly decreased disease activity, relative to baseline, in both anti-Ro/SSA antibody-positive and antibody-negative patients. Anti-Ro/SSA antibody-positive patients treated with IFX showed higher frequency of HACA and seroconversion of ANA, and lower serum TGF-ß levels. CONCLUSIONS: Positivity to anti-Ro/SSA in RA seems to confer resistance to IFX via production of HACA and ANA, and low serum TGF-ß levels, but not to TCZ and ABT.

Prevalence of soluble peptidylarginine deiminase 4 (PAD4) and anti-PAD4 antibodies in autoimmune diseases.

The objectives of this study are to investigate the prevalence of PAD4 and anti-PAD4 antibodies (Abs) in autoimmune diseases and to clarify their association with anticitrullinated protein antibodies (ACPAs) and shared epitope (SE) in patients with rheumatoid arthritis (RA). Levels of human PAD4 and anti-PAD4 Abs in serum or plasma were measured using sandwich ELISA. Samples were obtained from patients with RA (n = 148), SLE (n = 36), or SS (n = 37) and from healthy controls (HCs; n = 40). Antibodies against cyclic citrullinated glucose-6-phosphate isomerase (GPI) (CCG)-2, CCG-7, anti-CEP-1, and anti-CCP Abs were also measured using ELISA. Patients with RA were genotyped for HLA-DRB1. The human PAD4 and anti-PAD4 Ab levels were compared with the ACPA and SE in patients with RA. The PAD4 levels were 111.9 U/ml in the RA, 30.4 U/ml in the SLE, 81.9 U/ml in the SS patients, and 46.6 U/ml in the HCs. The PAD4 levels were significantly higher in the RA than in the SLE patients or the HCs. Anti-PAD4 Abs were detected in 29.7 % of the patients with RA, but not in the patients with SLE or SS, nor in the HCs. In the RA patients, the PAD4 levels in the anti-PAD4 Ab-negative group were significantly higher than those in the anti-PAD4 Ab-positive group. Moreover, anti-CCG-2, CCG-7, CEP-1, and anti-CCP Ab levels were significantly higher in the anti-PAD4 Ab-positive group than in the anti-PAD4 Ab-negative group. In the RA patients, the PAD4 levels were not correlated with ACPAs. Neither PAD4 nor anti-PAD4 Abs were significantly correlated with the presence of SE alleles. The PAD4 levels were higher in RA than in SLE or HC. Anti-PAD4 Abs appeared specifically in patients with RA. Moreover, anti-PAD4 Abs were associated with ACPAs.

Evaluation of changes in magnetic resonance images following 24 and 52 weeks of treatment of rheumatoid arthritis with infliximab, tocilizumab, or abatacept.

OBJECTIVES: To compare MRI findings in rheumatoid arthritis (RA) patients treated with biologic disease-modifying anti-rheumatic drugs (DMARDs). METHODS: The study subjects were 43 RA patients treated with biologic DMARDs (13 with infliximab, 15 with tocilizumab, and 15 with abatacept). They were evaluated using Simplified Disease Activity Index (SDAI) and low-field extremity MRI at baseline, and at 24 weeks and 52 weeks of treatment. RESULTS: Synovitis scores were significantly lower by 24 weeks in all groups, compared with baseline (P < 0.05). Significant improvement in bone marrow edema (BME) scores were noted from baseline to 24 weeks in infliximab and abatacept groups (P < 0.05), but from 24 weeks to 52 weeks in tocilizumab group (P < 0.01). No significant change was found in erosion score. The synovitis score at baseline correlated significantly with SDAI at 24 weeks (P < 0.05), and the score at 24 weeks correlated significantly with SDAI at 52 weeks (P < 0.05). CONCLUSIONS: The results suggest that the inflammatory improvement by infliximab and abatacept may express earlier than those by tocilizumab, despite similar improvement in SDAI. MRI-detected synovitis could be a useful predictor of SDAI at 24 weeks of treatment. The MRI remains the best tool to detect and assess the effects of biologic DMARDs in RA.

CTRP6 is an endogenous complement regulator that can effectively treat induced arthritis.

The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6(-/-) mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6(-/-) mice and C1qtnf6(-/-) embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H2O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases.

Successful Treatment with Infliximab for Refractory Uveitis in a Hemodialysis Patient with Behçet's Disease and a Review of the Literature for Infliximab Use in Patients on Hemodialysis.

A 36-year-old man with a 16-year history of refractory Behçet's disease (BD)-associated uveitis and chronic renal failure requiring hemodialysis suffered from frequent ocular attacks despite treatment with systemic corticosteroids and cyclosporine A. Following infliximab administration, the patient's BD ocular attack score 24 and visual acuity improved. Although he developed mild acute gastroenteritis, he did not experience any other adverse events. In our review of the literature, we identified seven patients on hemodialysis with inflammatory disease successfully treated with infliximab. Infliximab may be effective and safe in cases of BD and other diseases, including in patients under hemodialysis.

Efficacy and safety of abatacept for patients with Sjögren's syndrome associated with rheumatoid arthritis: rheumatoid arthritis with orencia trial toward Sjögren's syndrome Endocrinopathy (ROSE) trial-an open-label, one-year, prospective study-Interim analysis of 32 patients for 24 weeks.

Abstract Objective. To assess the efficacy and safety of abatacept for secondary Sjögren's syndrome (SS) associated with rheumatoid arthritis (RA). Methods. The primary endpoint of this 1-year, open-labeled, prospective, observational multicenter study of RA-associated secondary SS was the rate of SDAI remission at 52 weeks after initiation of abatacept therapy. The secondary endpoints included that of Saxson's test and Schirmer's test. Adverse events during the study period were also analyzed. Results. Thirty-two patients (all females) were enrolled in this study. Interim analysis at 24 weeks included assessment of efficacy (n = 31) and safety (n = 32). The mean SDAI decreased from 19.8 ± 11.0 (± SD) at baseline to 9.9 ± 9.9 at 24 weeks (P < 0.05). Patients with clinical remission, as assessed by SDAI, increased from 0 patient (0 week) to 8 patients (25.8%) at 24 weeks. Saliva volume (assessed by Saxson's test) increased slightly from 2232 ± 1908 (0 week) to 2424 ± 2004 (24 weeks) mg/2 min (n = 29). In 11 patients with Greenspan grading 1/2 of labial salivary glands biopsy, saliva volume increased from 2945 ± 2090 (0 week) to 3419 ± 2121 (24 weeks) mg/2 min (P < 0.05). Schirmer's test for tear volume showed increase from 3.6 ± 4.6 (0 week) to 5.5 ± 7.1 (24 weeks) mm/5 min (n = 25; P < 0.05). Five adverse events occurred in five of 32 patients (15.6%), and three of these events were infections. Conclusion. Abatacept seems to be effective for both RA and RA-related secondary SS.

Clinical features of patients with IgG4-related disease complicated with perivascular lesions.

OBJECTIVE: To define the clinical features of IgG4-related disease (IgG4-RD) complicated with perivascular lesions. METHODS: The clinical features of seven patients with IgG4-RD and perivascular lesions diagnosed at the University of Tsukuba Hospital between October 2008 and October 2013, were analyzed, including clinical background, results of imaging studies, satisfaction of the 2011 comprehensive diagnostic criteria (CDC) for IgG4-RD, laboratory data, distribution of perivascular lesions, involvement of other organs, and response to steroid therapy. RESULTS: We studied six men and one woman with a mean age of 66.9 ± 6.7 years (± SD). Six of seven patients were diagnosed as definite IgG4-RD, while the seventh was considered possible IgG4-RD, based on the CDC for IgG4-RD. Serum IgG4 levels at diagnosis were higher than 135 mg/dl in all seven patients (mean, 933 ± 527). Serum C-reactive protein (CRP) levels were elevated in two only (mean, 1.42 ± 3.56 mg/dl). The perivascular lesions were located in the pulmonary artery (n = 1), thoracic aorta (n = 2), abdominal aorta (n = 6), coronary (n = 1), celiac (n = 1), superior mesenteric (n = 1), renal (n = 2), inferior mesenteric (n = 5), and iliac (n = 3) arteries. In addition to perivascular lesions, six patients showed involvement of other organs. All seven patients were treated with prednisolone (0.6 mg/kg/day), which rapidly improved the perivascular and other organ lesions in six patients (the other one patient have not yet been evaluated due to the short follow-up). CONCLUSION: Perivascular lesions show wide distribution in patients with IgG4-RD. Serum CRP levels are not necessarily elevated in these patients. Steroid therapy is effective in IgG4-RD and results in resolution of lesions.

Pulmonary nocardiosis in patients with connective tissue disease: A report of two cases.

Reported here are 2 patients with connective tissue disease who developed pulmonary nocardiosis. Case 1 involved a 73-year-old man with malignant rheumatoid arthritis treated with prednisolone 25 mg/day. Chest X-rays revealed a pulmonary cavity and bronchoscopy detected Nocardia species. The patient was successfully treated with trimethoprim/sulfamethoxazole. Case 2 involved a 41-year-old woman with systemic lupus erythematosus. The patient received remission induction therapy with 50 mg/day of prednisolone and tacrolimus. Six weeks later, a chest CT scan revealed a pulmonary cavity; bronchoscopy resulted in a diagnosis of pulmonary nocardiosis. The patient had difficulty tolerating trimethoprim/sulfamethoxazole, so she was switched to and successfully treated with imipenem/cilastatin and amikacin.

Involvement of CD161+ Vδ1+ γδ T cells in systemic sclerosis: association with interstitial pneumonia.

OBJECTIVE: Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with high mortality and poor prognosis. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of CD161(+) Vδ1(+) γδ T cells in SSc patients with IP. METHODS: The proportion of CD161(+) Vδ1(+) γδ T cells in peripheral blood mononuclear cells (PBMCs) and serum sialylated carbohydrate antigen (KL-6) levels were determined. GeneChip analysis was performed with CD161(-) and CD161(+) Vδ1(+) γδ T cells. Cytokine and chemokine expression from CD161(+) Vδ1(+) γδ T cells was measured and used to evaluate the effect of culture supernatant on fibroblast proliferation. RESULTS: The proportion of CD161(+) Vδ1(+) γδ T cells was significantly higher in SSc than healthy controls (HCs) and correlated negatively with serum KL-6 levels in IP-positive SSc patients. The gene and mRNA expression level of chemokine ligand 3 (CCL3) was markedly higher in CD161(+) Vδ1(+) γδ T cells than in CD161(-) Vδ1(+) γδ T cells. CD161(+) Vδ1(+) γδ T cells in IP-positive SSc patients showed higher production of CCL3 and lower production of IFN-γ than in HCs. Culture supernatant derived from IP-negative and IP-positive SSc patients promoted fibroblast proliferation, whereas that from HCs did not. CONCLUSION: The small proportion and the altered cell functions of CD161(+) Vδ1(+) γδ T cells among PBMCs in SSc patients play a role in the pathogenesis of IP. These findings suggest that CD161(+) Vδ1(+) γδ T cells may play a regulatory role in the pathogenesis of IP in SSc patients via IFN-γ production.