Analysis of proteins released from osteoarthritic cartilage by compressive loading.

In osteoarthritis (OA), synovial pathology may be induced by proteins released from degenerated cartilage. This study was conducted to identify the proteins released from OA cartilage. OA cartilage was obtained from OA knees at macroscopically preserved areas (PRES) and degenerated areas (DEG), while control cartilage (CONT) was collected from non-arthritic knees. Released proteins were obtained from these cartilage samples by repeatedly applying compressive loading, which simulated loading on cartilage in vivo. The released proteins were analyzed comprehensively by antibody array analyses and a quantitative proteomic analysis. For several proteins, the exact amounts released were determined by Luminex assays. The amount of active TGF-ß that was released was determined by an assay using genetically-engineered HEK cells. The results of the antibody array and proteomic analyses revealed that various biologically active proteins are released from OA cartilage, particularly from DEG, by loading. The Luminex assay confirmed that several alarmins, complement proteins C3a and C5a, and several angiogenic proteins including FGF-1, FGF-2 and VEGF-A were released in greater amounts from DEG than from CONT. The HEK cell assay indicated that active TGF-ß was released from DEG at biologically significant levels. These findings may be helpful in understanding the pathology of OA.

Accumulation of polyunsaturated fatty acid-derived metabolites in the sarcopenic muscle of aging mice.

AIM: Although it is known that advanced age alters skeletal muscle lipid metabolism, the role(s) of polyunsaturated fatty acid-derived metabolites (mostly eicosanoids and docosanoids) in sarcopenia are not clear. We therefore examined the changes in the metabolites of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid in the sarcopenic muscle of aged mice. METHODS: We used 6- and 24-month-old male C57BL/6J mice as healthy and sarcopenic muscle models, respectively. Skeletal muscles were removed from the lower limb and subjected to a liquid chromatography-tandem mass spectrometry analysis. RESULTS: The liquid chromatography-tandem mass spectrometry analysis detected distinct changes of metabolites in the muscles of the aged mice. Of the 63 metabolites identified, nine were significantly higher in the sarcopenic muscle of aged mice compared with the healthy muscle of young mice. In particular, prostaglandin E2 , prostaglandin F2a , thromboxane B2 , 5-hydroxyeicosatetraenoic acid, and 15-oxo-eicosatetraenoic acid (arachidonic acid-derived metabolites), 12-hydroxy-eicosapentaenoic acid and 14,15-epoxy-eicosatetraenoic acid (eicosapentaenoic acid-derived metabolites) and 10-hydroxydocosa-hexaenoic acid and 14-hydroxyoctadeca-pentaenoic acid (docosahexaenoic acid-derived metabolites) were significantly higher in aged tissue compared with young tissue (all P < 0.05). CONCLUSIONS: We observed the accumulation of metabolites in the sarcopenic muscle of aged mice. Our results may provide new insights into the pathogenesis and progression of aging- or disease-related sarcopenia. Geriatr Gerontol Int 2023; 23: 297-303.

Cutaneous methotrexate-related T-cell lymphoproliferative disorder with CD4, CD30, CD56, EBV-positive tumor cell infiltration: a case illustration and a brief review.

Methotrexate (MTX) is a commonly used anti-metabolite agent. Long-term MTX treatment can cause MTX-related lymphoproliferative disorder (MTX-LPD). T-cell LPDs comprise a small fraction of MTX-LPDs. Epstein-Barr virus (EBV)+ tumor cells are rarely detected in MTX-related T-cell LPDs (MTX T-LPDs). Therefore, there have been very few reports of EBV+ MTX T-LPD. We encountered a case of cutaneous MTX T-LPD with a unique cellular phenotype. The patient was a 71-year-old Japanese man with rheumatoid arthritis treated with MTX for 6 years. He was referred to our department with a 6-month history of red plaques and ulcerated lesions in both lower legs and a 2-week history of high fever and fatigue. Cutaneous specimens showed that medium-sized atypical lymphocytes were positive for CD3, CD4, CD30, CD56, and in situ hybridization for EBV-encoded RNA. The patient was diagnosed with cutaneous MTX T-LPD. Four months after discontinuation of MTX, the skin lesions had disappeared. This is the first report of cutaneous MTX T-LPD with CD4+CD30+CD56+EBV+ tumor cells.

The attenuation of insulin-like growth factor signaling may be responsible for relative reduction in matrix synthesis in degenerated areas of osteoarthritic cartilage.

BACKGROUND: In osteoarthritis (OA), cartilage matrix is lost gradually despite enhanced matrix synthesis by chondrocytes. This paradox may be explained, at least partly, by reduced chondrocyte anabolism in degenerated area of OA cartilage. However, to date, it is not known why chondrocyte anabolism is suppressed in those areas. METHODS: Cartilage was obtained from control knees and end-stage OA knees in macroscopically preserved areas and degenerated areas, and gene expression was analyzed in respective regions of cartilage using laser capture microdissection and qPCR. For the cartilage protein analysis, cartilage was obtained from preserved areas and degenerated areas of OA knees in pairs, and proteins were extracted using urea buffer. Protein concentrations were determined by Luminex and compared between the areas. Cartilage explants prepared from preserved areas and degenerated areas of OA knees were cultured in the presence or absence of an AKT inhibitor, and the gene expression was evaluated by qPCR. Finally, the expression of SP1 was evaluated in OA and control cartilage, and the significance of Sp1 on the expression of IGF1R and IRS1 was investigated in experiments using primary cultured chondrocytes. RESULTS: Within OA cartilage, the expression of IGF-1, IGF-2, IGF1R and IRS1 was reduced in degenerated areas compared to preserved areas, while the expression of all six IGF-binding protein genes examined was enhanced in the former areas. Consistent results were obtained by a protein analysis. In explant culture, the inhibition of AKT signaling abrogated the abundant matrix gene expression in the preserved areas over the degenerated areas, indicating that suppressed matrix synthesis in degenerated areas may be ascribed, at least partly, to attenuated IGF signaling. Within OA cartilage, the expression of Sp1 was considerably reduced in severely degenerated areas compared to preserved areas, which correlated well with the expression of IGF1R and IRS1. In experiments using primary cultured chondrocytes, the expression of IGF1R and IRS1 was enhanced by the induction of Sp1 expression and reduced by the suppression of Sp1 expression. CONCLUSIONS: The results of this study suggest that attenuated IGF signaling may be responsible, at least partly, for the reduced matrix synthesis in degenerated areas of OA cartilage.

Related factors, increased mortality and causes of death in patients with rheumatoid arthritis-associated interstitial lung disease.

Objectives: Interstitial lung disease (ILD) is a life-threatening extra-articular manifestation of rheumatoid arthritis (RA). We aimed to clarify the relationship between chronic ILD with a pattern of usual interstitial pneumonia (UIP) or non-UIP and mortality in RA patients.Methods: We retrospectively surveyed information of consecutive RA patients who visited our hospital from 2009 to 2014. The relationship between their mortality and chronic ILD (UIP or non-UIP) detected by high-resolution computed tomography was examined.Results: Of 2702 patients enrolled, 261 (9.7%) had chronic ILD and among these 120 had a UIP pattern. At the onset of RA, the prevalence of chronic ILD was 6%. Patients with chronic ILD had a higher mortality than those without. The most frequent cause of death was pneumonia including acute exacerbation (AE) of chronic ILD. Lung cancer death was frequently identified in deceased patients with chronic ILD with a UIP pattern compared with the other decedents (p=.062). The estimated mortality of lung cancer in patients with chronic ILD with a UIP pattern was five times higher than the general population.Conclusion: RA patients with ILD with a UIP pattern have a high mortality rate and are prone to die of AE or lung cancer.

A proteomic analysis of serum-derived exosomes in rheumatoid arthritis.

BACKGROUND: To understand the roles of serum exosomes in rheumatoid arthritis (RA), we comprehensively investigated the protein profiles of serum exosomes in patients with RA. METHODS: Exosomes were isolated from serum samples obtained from 33 patients (12 with active RA [aRA], 11 with inactive RA [iRA], 10 with osteoarthritis [OA]) and 10 healthy donors (HLs). Proteins extracted from the exosomes were separated by two-dimensional differential gel electrophoresis (2D-DIGE) and identified by mass spectrometry. RESULTS: In total, 204 protein spots were detected by 2D-DIGE. In the aRA, iRA, and OA groups, 24, 5, and 7 spots showed approximately ≥ ±1.3-fold intensity differences compared with the HL group, respectively. We were able to identify proteins in six protein spots. Among them, the protein spot identified as Toll-like receptor 3 (TLR3) showed approximately 6-fold higher intensity in the aRA group than in the other groups. CONCLUSIONS: Patients with active RA possessed considerably different protein profiles of serum exosomes from patients with iRA, patients with OA, and healthy donors. The unique protein profile of serum exosomes, such as the possession of abundant TLR3 fragments, may reflect the pathophysiology of active RA.

Biomarker for nontuberculous mycobacterial pulmonary disease in patients with rheumatoid arthritis: Anti-glycopeptidolipid core antigen immunoglobulin A antibodies.

OBJECTIVE: Nontuberculous mycobacterial (NTM) pulmonary disease is occasionally associated with rheumatoid arthritis (RA), influencing the therapeutic strategy of RA. Since chronic lung diseases are frequently associated with RA, the diagnosis of NTM pulmonary disease is quite difficult in RA patients. Recently, a serological diagnostic test detecting serum immunoglobulin A against the glycopeptidolipid (GPL) core antigen was developed. We investigated the serum levels of anti-GPL antibodies in RA patients to determine the usefulness for detecting NTM pulmonary disease. METHODS: Anti-GPL antibodies were detected in the sera from RA patients with or without NTM pulmonary disease. RESULTS: The positivity of anti-GPL antibodies in RA patients with NTM pulmonary disease was higher than in RA without (p = 1.76 × 10-14, odds ratio 70.29, 95% confidence interval [CI] 22.28-221.83). Anti-GPL Ab titers were increased in RA with NTM pulmonary disease (mean titer ± standard deviation [U/ml], RA with NTM pulmonary disease: 4.1 ± 7.0, RA without NTM pulmonary disease: 0.4 ± 1.6, p = 1.51 × 10-10). The area under the curve (AUC) value of the receiver operating characteristic (ROC) curve for anti-GPL antibodies was 0.917 (95%CI 0.860-0.974, p = 3.32 × 10-47). CONCLUSIONS: Serum anti-GPL antibodies are useful for detecting NTM pulmonary disease in RA patients.

Incidence and Risk Factors for Infections Requiring Hospitalization, Including Pneumocystis Pneumonia, in Japanese Patients with Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) may be complicated by different infections, but risk factors for these are not fully elucidated. Here, we assessed the incidence of and risk factors for infections requiring hospitalization (IRH) including pneumocystis pneumonia (PCP) in patients with RA. METHODS: We retrospectively surveyed all RA patients treated at our hospital from 2009 to 2013, for whom data were available on demographic features, medications, comorbidities, and severity of RA. Multivariate logistic regression analysis was applied to calculate adjusted odds ratios (ORs) for factors associated with the occurrence of IRH. RESULTS: In a total of 9210 patient-years (2688 patients), there were 373 IRH (3.7/100 patient-years). Respiratory tract infections were most frequent (n = 154, and additionally 16 PCP), followed by urinary tract infections (n = 50). Significant factors for PCP included higher age (≥70 years; OR 3.5), male sex (6.6), underlying lung disease (3.0), use of corticosteroids (4.8), and use of biologics (5.4). Use of methotrexate (5.7) was positively associated with PCP but negatively with total infections (0.7). Additionally, functional disorders and higher RA disease activity were also related to total infections. CONCLUSIONS: Risk factors for infection should be taken into account when deciding treatment for the individual RA patient.

Neutrophil CD64 is upregulated in RA patients with lymphoma but not in other solid cancers.

OBJECTIVE: To evaluate the utility of quantifying CD64 expression on neutrophils in rheumatoid arthritis patients with malignancy, especially its diagnostic role in lymphoma. METHODS: We used flow cytometry to quantify CD64 expression on neutrophils from patients diagnosed with malignancy during the follow-up period prior to initiating treatment. RESULTS: Neutrophils from 18 patients with lymphoma expressed significantly higher levels of CD64 (9635.6 ± 2123.7 molecules/cell) than those from 32 patients with other solid cancers (carcinoma) (1250.5 ± 91.1 molecules/cell) (p < 0.001). When the cutoff value was set at 2060 molecules/cell, the sensitivity and specificity of CD64 for diagnosing lymphoma was 88.9% and 94.4%, respectively. CONCLUSIONS: The quantitative measurement of neutrophil CD64 by flow cytometry may be useful as a subsidiary diagnostic marker in patients with suspected lymphoma. Although neutrophil CD64 is currently a well-known marker of infection, it is necessary to bear in mind that lymphoma is also a candidate in differential diagnosis when CD64 expression on neutrophils is upregulated.

Effects of methotrexate and salazosulfapyridine on protein profiles of exosomes derived from a human synovial sarcoma cell line of SW982.

PURPOSE: To elucidate effects of salazosulfapyridine (SASP) and methotrexate (MTX), major anti-rheumatic drugs, on exosomes derived from SW982 of a human synovial sarcoma cell line. EXPERIMENTAL DESIGN: SW982 was treated with SASP and/or MTX under interleukin-1ß (IL-1ß)-treated or nontreated conditions. Exosomes were isolated from the culture media, and exosomal proteome was analyzed by 2D-DIGE. Protein spots whose intensity was significantly altered by the above treatments were identified by MS. RESULTS: Two hundred ninety-four protein spots were detected in the exosome preparations by 2D-DIGE. Compared to the nontreated cells, SASP-, MTX-, and (SASP + MTX)-treated cells displayed 8, 10, and 21 exosomal protein spots with more than ±2.0-fold intensity differences (p < 0.05), respectively. Similarly, the IL-1ß-treated cells displayed 58 exosomal protein spots with more than ±1.5-fold intensity differences (p < 0.05). In about half of the 58 spots, the IL-1ß-induced intensity changes were suppressed by simultaneous addition of SASP and/or MTX. Most of the identified proteins were immunity- or anti-oxidation-related proteins. CONCLUSIONS AND CLINICAL RELEVANCE: The SASP and/or MTX treatments altered the protein profiles of exosomes and suppressed the effects of IL-1ß on the exosomal proteome. Exosomes may play roles in the actions of these anti-rheumatic drugs.

Incidence of malignancy and the risk of lymphoma in Japanese patients with rheumatoid arthritis compared to the general population.

OBJECTIVE: Recent advances in the management of patients with rheumatoid arthritis (RA) increased the rates of disease remission and patient life expectancy, while malignancy has become a more common cause of death. Here, we report the incidence of malignancy in a nationwide survey of Japanese patients with RA compared to the general population, focusing on the risk of lymphoma, which often arises in patients with RA. METHODS: Data on the occurrence of malignancy were collected from patients registered in a nationwide Japanese cohort database, the National Database of Rheumatic Diseases by iR-net in Japan, from 2003 to 2012. To adjust for different population composition and to compare the incidence of malignancy with the general population, standardized incidence rates (SIR) were calculated. To identify risk factors for lymphoma, individual patient data were obtained for multivariate analysis for the year before lymphoma diagnosis. RESULTS: In 10 years, the cohort composed of 66,953 patient-years yielded 559 malignancies, most frequently lung cancer, followed by gastric cancer, breast cancer, and lymphoma. The overall incidence of malignancies in patients with RA was slightly lower than in the general population (SIR 0.89, 95% CI 0.82-0.97). However, lymphoma risk was significantly higher (SIR 3.43, 95% CI 2.59-4.28), whereas risk of colon, rectal, or liver cancer was lower. Significant risk factors for lymphoma were the use of methotrexate or tacrolimus, and higher age. CONCLUSION: Patients with RA had no higher overall incidence of malignancies, but lymphoma was significantly more frequent than in the general population.

Neutrophil CD64 for monitoring the activity of nontuberculous mycobacteria infection in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the utility of neutrophil CD64 as a marker for monitoring the activity of nontuberculous mycobacteria (NTM) infection in patients with rheumatoid arthritis (RA). METHODS: We compared neutrophil CD64 expression in nine RA patients with NTM infection in the active and inactive phase of NTM disease chronologically. "Active phase" was here defined as present in patients admitted to hospital to receive intensive treatment for NTM, as well as outpatients with an infectious episode showing positive acid- and alcohol-fast bacillus (AFB) staining of sputa (Grade 2-3) who needed to start treatment for NTM with a multiple antibiotics regimen. The cut-off value for CD64 positivity was 2000 molecules/cell. RESULTS: Neutrophils from patients with active-phase NTM infection expressed high levels of CD64 with a mean ± SEM of 7335 ± 784 molecules/cell. However, during the inactive phase of disease, this was significantly lower (1481 ± 103 molecules/cell, p < 0.001). The sensitivity and specificity of neutrophil CD64 to detect active-phase NTM infection was 96.3% and 84.6%, respectively. Expression of neutrophil CD64 was not affected by disease activity of the RA itself. CONCLUSIONS: Neutrophil CD64 is useful for monitoring disease activity in NTM infection of patients with RA.

[Successful early treatment in a case of Cogan's syndrome].

We report a 53-year-old male with Cogan's syndrome. He was admitted to our hospital because of a fever of 2-weeks duration, blurred vision for 10 days, hypoacusis, and numbness of the left hand for 3 days. In addition to uveitis, hypoacusis, and aseptic meningitis, multiple mononeuropathy was diagnosed based on a nerve conduction study. Furthermore, positron emission tomography/computed tomography (PET/CT) revealed diffuse aortitis. Accordingly, the patient was diagnosed with Cogan's syndrome. After starting steroid-pulse therapy followed by 1 mg oral prednisolone/kg/day, the uveitis and hypoacusis improved immediately, while the peripheral neuropathy persisted until effectively treated with intravenous gamma globulin therapy. Prompt steroid therapy for Cogan's syndrome based on a diagnosis made using PET/CT prevented progression of the hypoacusis.