Ultrasound efficacy of targeted-synthetic disease-modifying anti-rheumatic drug treatment in rheumatoid arthritis: a multicenter prospective cohort study in Japan.

OBJECTIVE: This study investigated the effectiveness of treatment with Janus kinase (JAK) inhibitors in rheumatoid arthritis (RA) assessed by ultrasonography (US) activity, and the influence of patient characteristics and previous treatments. METHOD: This prospective study assessed 60 treatment initiations among 53 Japanese patients diagnosed with RA who underwent treatment with JAK inhibitors during June 2013 to February 2020. Of the 53 patients, seven patients were enrolled in duplicate because they were treated with two different JAK inhibitors at different periods. For each case, the improvement rate on the power Doppler (PD) score was assessed at 6 month follow-up. Median improvement rate of PD score was used to classify cases as either US responders or non-responders, and patient characteristics were compared between the two groups. RESULTS: All indicators of clinical disease activity and US activity showed a significant improvement at 3 months compared with baseline. Although the JAK inhibitor-cycler group and the interleukin-6 (IL-6) inhibitor inadequate response (IR) group tended to show a later improvement for US activity, all indicators of clinical disease activity and US activity showed a significant improvement at 6 months compared with baseline for both groups. Multivariate analysis showed that concomitant methotrexate use and an IR to the previous biologic or targeted-synthetic disease-modifying anti-rheumatic drug (b/tsDMARD) treatment were independently and significantly associated with US responders. CONCLUSION: Use of a JAK inhibitor in combination with methotrexate and an absence of IR to any previous b/tsDMARDs demonstrated superior effectiveness for patients with RA.

Evolution of anti-modified protein antibody responses can be driven by consecutive exposure to different post-translational modifications.

BACKGROUND: Besides anti-citrullinated protein antibodies (ACPA), rheumatoid arthritis patients (RA) often display autoantibody reactivities against other post-translationally modified (PTM) proteins, more specifically carbamylated and acetylated proteins. Immunizing mice with one particular PTM results in an anti-modified protein antibody (AMPA) response recognizing different PTM-antigens. Furthermore, human AMPA, isolated based on their reactivity to one PTM, cross-react with other PTMs. However, it is unclear whether the AMPA-reactivity profile is "fixed" in time or whether consecutive exposure to different PTMs can shape the evolving AMPA response towards a particular PTM. METHODS: Longitudinally collected serum samples of 8 human individuals at risk of RA and 5 with early RA were tested with ELISA, and titers were analyzed to investigate the evolution of the AMPA responses over time. Mice (13 per immunization group in total) were immunized with acetylated (or carbamylated) protein (ovalbumin) twice or cross-immunized with an acetylated and then a carbamylated protein (or vice versa) and their serum was analyzed for AMPA responses. RESULTS: Human data illustrated dynamic changes in AMPA-reactivity profiles in both individuals at risk of RA and in early RA patients. Mice immunized with either solely acetylated or carbamylated ovalbumin (AcOVA or CaOVA) developed reactivity against both acetylated and carbamylated antigens. Irrespective of the PTM-antigen used for the first immunization, a booster immunization with an antigen bearing the other PTM resulted in increased titers to the second/booster PTM. Furthermore, cross-immunization skewed the overall AMPA-response profile towards a relatively higher reactivity against the "booster" PTM. CONCLUSIONS: The relationship between different reactivities within the AMPA response is dynamic. The initial exposure to a PTM-antigen induces cross-reactive responses that can be boosted by an antigen bearing this or other PTMs, indicating the formation of cross-reactive immunological memory. Upon subsequent exposure to an antigen bearing another type of PTM, the overall reactivity pattern can be skewed towards better recognition of the later encountered PTM. These data might explain temporal differences in the AMPA-response profile and point to the possibility that the PTM responsible for the initiation of the AMPA response may differ from the PTM predominantly recognized later in time.

Discrepancy between clinical and ultrasound remissions in rheumatoid arthritis: a multicentre ultrasound cohort study in Japan.

Objectives: Using multicentre ultrasound (US) cohort data among patients with rheumatoid arthritis (RA), we aimed to identify baseline factors that permit differentiation between two patient cohorts achieving US remission and clinical remission, and to determine the factors contributing to the discrepancy.Method: We reviewed 248 Japanese patients diagnosed with RA who underwent treatment with biological disease-modifying anti-rheumatic drugs at 13 centres. We performed US assessments of the synovia of 22 joints. We assessed the percentages of patients with clinical remission and US remission, defined as total power Doppler scores of 0 at 12 months.Results: The 87 patients who achieved US remission were divided into a group that achieved both clinical and US remission (n = 53) and a group that achieved US remission only (n = 34). Baseline factors that were significantly and independently associated with clinical remission at 12 months among patients who also achieved US remission included short disease duration, the presence of concomitant methotrexate use, and low patient global assessment score (p < 0.05, p < 0.05, and p < 0.005, respectively).Conclusions: RA patients with baseline high patient global assessment scores and long disease duration at baseline were unlikely to achieve clinical remission even after achieving US remission. Objective joint assessments using US provide additional information of potential importance for the management of RA.

Investigation of immune complexes formed by mitochondrial antigens containing a new lipoylated site in sera of primary biliary cholangitis patients.

Primary biliary cholangitis (PBC) is characterized by the presence of serum anti-mitochondrial autoantibodies (AMAs). To date, four antigens among the 2-oxo-acid dehydrogenase complex family, which commonly have lipoyl domains as an epitope, have been identified as AMA-corresponding antigens (AMA-antigens). It has recently been reported that AMAs react more strongly with certain chemically modified mimics than with the native lipoyl domains in AMA-antigens. Moreover, high concentrations of circulating immune complexes (ICs) in PBC patients have been reported. However, the existence of ICs formed by AMAs and their antigens has not been reported to date. We hypothesized that AMAs and their antigens formed ICs in PBC sera, and analyzed sera of PBC and four autoimmune diseases (Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, and rheumatoid arthritis) using immune complexome analysis, in which ICs are separated from serum and are identified by nano-liquid chromatography-tandem mass spectrometry. To correctly assign MS/MS spectra to peptide sequences, we used a protein-search algorithm that including lipoylation and certain xenobiotic modifications. We found three AMA-antigens, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) and dihydrolipoamide dehydrogenase binding protein (E3BP), by detecting peptides containing lipoylation and xenobiotic modifications from PBC sera. Although the lipoylated sites of these peptides were different from the well-known sites, abnormal lipoylation and xenobiotic modification may lead to production of AMAs and the formation ICs. Further investigation of the lipoylated sites, xenobiotic modifications, and IC formation will lead to deepen our understanding of PBC pathogenesis.

Effects of cigarette smoking and human T-cell leukaemia virus type 1 infection on anti-citrullinated peptide antibody production in Japanese community-dwelling adults: the Nagasaki Islands Study.

Objectives: We investigated whether the positivity of anti-citrullinated peptide antibody (ACPA) is associated with cigarette-smoking status and human T-cell leukaemia virus type 1 (HTLV-1) infection in a general population in Nagasaki, Japan, which is an ageing and HTLV-1-endemic area.Method: Baseline data from community-dwelling people in the Nagasaki Islands Study (NaIS) were included in this cross-sectional analysis. ACPA and HTLV-1 were measured in 3887 subjects without a history of treatment for rheumatoid arthritis. A logistic regression analysis was performed to assess the relationship between ACPA positivity and candidates of correlation with ACPA, i.e. the cigarette-smoking status quantified by Brinkman's index (BI) and HTLV-1 positivity.Results: Fifty-one subjects (1.3%) showed ACPA positivity, and 650 subjects (16.6%) were HTLV-1 carriers. In an age- and gender-adjusted logistic regression analysis, the BI [odds ratio (OR) 1.09, 95% confidence interval (CI)1.02-1.14, p = 0.0031] and a BI value > 500 (OR 3.92, 95% CI 1.72-9.22, p = 0.0014) were each significantly associated with ACPA positivity. HTLV-1 positivity did not show any association with ACPA positivity.Conclusion: A significant effect of cigarette-smoking status on ACPA production was revealed, whereas HTLV-1 positivity was not associated with ACPA production in this general population.

Significance of anti-Ro/SSA antibodies in the response and retention of abatacept in patients with rheumatoid arthritis: a multicentre cohort study.

Objective: To determine whether the positivity of baseline anti-Ro/Sjögren's syndrome antigen A (SSA) antibodies influences the response to abatacept, we compared therapeutic responses between anti-Ro/SSA antibody-negative and -positive patients with rheumatoid arthritis (RA) using a multicentre RA ultrasonography prospective cohort. Method: We reviewed Japanese patients with RA who started abatacept as the first biological disease-modifying anti-rheumatic drug between June 2013 and April 2018. We assessed 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) change between baseline and 6 or 12 months after treatment in RA patients treated with abatacept, and European League Against Rheumatism (EULAR) response at 6 and 12 months. The Global OMERACT-EULAR Synovitis Score (GLOESS) was calculated at baseline and at 6 and 12 months. Results: Overall, 51 patients were enrolled and divided into anti-Ro/SSA antibody-negative and -positive groups of 35 and 16, respectively. Median age at baseline was significantly higher in the anti-Ro/SSA antibody-negative group (p = 0.04). The retention rate and percentage of EULAR good responders at 12 months were significantly higher in the anti-Ro/SSA antibody-negative group (both p = 0.02). Anti-Ro/SSA antibody-negative patients exhibited larger decreases in both DAS28-ESR and DAS28-C-reactive protein at 12 months than anti-Ro/SSA antibody-positive patients (p = 0.02 and 0.04, respectively). GLOESS decreased significantly at 6 months in anti-Ro/SSA antibody-negative patients (p = 0.03). Multivariate analyses showed that anti-Ro/SSA antibody positivity was an independent factor associated with change in the DAS28-ESR at 6 months (p < 0.05). Conclusion: Anti-Ro/SSA antibody positivity predicts a poor response to abatacept and low retention rate.

IL-15 is a biomarker involved in the development of rapidly progressive interstitial lung disease complicated with polymyositis/dermatomyositis.

BACKGROUND: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease that is sometimes complicated with rapidly progressive interstitial lung disease (RPILD). However, serum and lung biomarkers that can predict RPILD development remain unclear. OBJECTIVES: To determine potential serum and lung biomarkers that can predict RPILD development in patients with PM/DM-ILD. METHODS: In total, 49 patients with PM/DM-ILD were enrolled. We measured the serum levels of 41 cytokines/chemokines, ferritin and anti-MDA5 antibody, compared them between the RPILD (n = 23) and non-RPILD (n = 26) groups, and ranked them by their importance through random forest analysis. To distinguish the two groups, we determined biomarker combinations by logistic regression analysis. We also measured the bronchoalveolar lavage fluid (BALF) levels of 41 cytokines/chemokines. Using immunohistochemistry, we examined IL-15 expression in lung tissues. The IL-15 production was also investigated using A549 and BEAS-2B cells. RESULTS: The RPILD group had significantly higher IL-15, IL-1RA, IL-6, CXCL10, VCAM-1, anti-MDA5 antibody and ferritin serum levels than the non-RPILD group, but it had a significantly low CCL22 level. Meanwhile, anti-MDA5 antibody, IL-15, CXCL8, CCL22, IL-1RA and ferritin were the best combination to distinguish the two groups. IL-15 and CCL22 were also predictive marker for RPILD development in anti-MDA5 antibody-positive patients. Additionally, the RPILD group had significantly high IL-15 levels in BALF. The lung tissues expressed IL-15, which increased after cytokine stimulation in the A549 cells. CONCLUSION: This study identified a combination of biomarkers predicting PM/DM-RPILD progression, and IL-15 is an important cytokine for predicting RPILD development and reflecting ILD severity.

Fluorescence optical imaging in patients with active rheumatoid arthritis: a comparison with ultrasound and an association with biomarkers.

Objectives: This study compared indocyanine green (ICG)-enhanced fluorescence optical imaging (FOI) and musculoskeletal ultrasound (MSUS), and explored the significance of the FOI findings based on the association between the FOI and MSUS findings and serum biomarkers in patients with rheumatoid arthritis (RA). The study also explored the association between the FOI findings and patients' joint destruction at the joint-area level.Method: We enrolled 50 consecutive patients with active RA from among the patients hospitalized from May 2014 to March 2016 at Nagasaki University Hospital, Japan. FOI images were acquired with the Xiralite® fluorescence imaging system and compared with the patients' clinical examination results and MSUS findings. On the same day, the patients' clinical disease activity and levels of serum biomarkers (including vascular endothelial growth factor) were obtained.Results: Although the FOI detected synovitis with high sensitivity, the frequency of positive findings and the diagnostic performance with MSUS as the reference standard for FOI differed considerably among the phases of FOI as well as among the affected joint regions. The FOI scores were positively correlated with clinical disease activity, MSUS scores, and serum biomarkers. The severity of FOI-proven synovitis was associated with the presence of MSUS-proven bone erosion.Conclusion: FOI is effective for detecting joint inflammation in RA patients, with high accuracy. The severity of the FOI score was closely associated with the joint destruction at the joint-area level. However, the significance of positive FOI findings differed depending on not only the phase of FOI but also the affected joint regions.

Anti-citrullinated protein antibody titre as a predictor of abatacept treatment persistence in patients with rheumatoid arthritis: a prospective cohort study in Japan.

Objective: Successful rheumatoid arthritis (RA) outcome depends on treatment efficacy in the early stages of the disease and its sustainability. It is thus critical to identify factors predicting treatment persistence with biological agents, such as abatacept. We compared clinical profiles, including early changes in autoantibody titres at 3 months, between patients with RA demonstrating sustained persistence and those discontinuing abatacept treatment.Method: We prospectively enrolled 71 and 78 active RA patients treated with abatacept and tumour necrosis factor inhibitors (TNF-Is), respectively, who had previous disease-modifying anti-rheumatic drug) failure. Clinical characteristics were compared between non-continuation and continuation groups stratified according to abatacept or TNF-I persistence for at least 12 months from treatment initiation.Results: Significantly larger decreases in rheumatoid factor titre and anti-citrullinated protein autoantibody (ACPA) titre were observed in the continuation group of abatacept therapy at 3 months, and early reduction in ACPA titre remained a significant and independent predictor of sustained persistence with abatacept in multivariate analysis. In addition, we obtained the area under the receiver operator characteristics curve of 0.904 from a model including baseline ACPA titre and reduction of ACPA titre at 3 months. Sustained reduction of RA disease activity score at 12 months was significantly and independently associated with reduced ACPA titre at 3 months.Conclusions: Persistence with abatacept and sustained therapeutic response are associated with an early reduction in ACPA titre. Prediction of abatacept continuation and efficacy will facilitate the optimal design of therapy in the early stages of RA.

Factors predictive of long-term mortality in lupus nephritis: a multicenter retrospective study of a Japanese cohort.

BACKGROUND: Lupus nephritis (LN) is a major determinant of mortality in systemic lupus erythematosus (SLE). Here we evaluated the association between complete renal response (CR) and mortality in LN. METHODS: We retrospectively analyzed the cases of 172 of 201 patients with LN for whom data on the therapeutic response at 6 and 12 months after induction therapy were available. The patients underwent a renal biopsy at Nagasaki University Hospital and community hospitals in Nagasaki between the years 1990 and 2016. We determined the CR rates at 6 and 12 months after induction therapy initiation and evaluated the predictive factors for CR and their relationship with mortality. We performed univariate and multivariable competing risks regression analyses to determine the factors predictive of CR. The patients' survival data were analyzed by the Kaplan-Meier method with a log-rank test. RESULTS: The median follow-up duration after renal biopsy was 120 months (interquartile range: 60.3-191.8 months). The 5-, 10-, 15- and 20-year survival rates of our cohort were 99.3, 94.6, 92.0 and 85.4%, respectively. During follow-up, nine patients (5.2%) died from cardiovascular events, infection, malignancy and other causes. The multivariate analysis revealed that the following factors were predictive of CR. At 6 months: male gender (odds ratio (OR) 0.23, 95% confidence interval (CI) 0.08-0.65, p = 0.0028), proteinuria (g/gCr) (OR 0.83, 95% CI 0.71-0.97, p = 0.0098) and index of activity (0-24) (OR 0.84, 95% CI 0.71-0.99, p = 0.0382). At 12 months: male gender (OR 0.25, 95% CI 0.09-0.67, p = 0.0043) and index of activity (0-24) (OR 0.82, 95% CI 0.69-0.98, p = 0.0236). The Kaplan-Meier analysis showed that compared to not achieving CR at 12 months, achieving CR at 12 months was significantly correlated with the survival rate (OR 0.18, 95% CI 0.04-0.92, p = 0.0339). CONCLUSIONS: Our results suggest that the survival rate of patients with LN is associated with the achievement of CR at 12 months after induction therapy, and that male gender and a higher index of activity (0-24) are the common predictive factors for failure to achieve CR at 6 and 12 months.

Complete renal response at 12 months after induction therapy is associated with renal relapse-free rate in lupus nephritis: a single-center, retrospective cohort study.

BACKGROUND: Lupus nephritis (LN) is a major risk factor for overall morbidity and mortality in systemic lupus erythematosus (SLE). METHODS: We retrospectively analyzed cases of proliferative and membranous LN patients who underwent a renal biopsy at our hospital in 1993-2016. We analyzed the association between complete renal response (CR) rates at 12 months after induction therapy and predictive factors for CR and their association with renal flares. RESULTS: Of the 95 cases analyzed, we were able to track the therapeutic responses of 81 patients at 12 months after their induction therapy. The median follow-up duration after renal biopsy was 51 months (interquartile range: 16.5-154.5 months). The Cox proportional hazards model showed that, compared to not attaining CR at 12 months, the attainment of CR at 12 months was correlated with being free from renal flares. The multivariate logistic analysis revealed that the predictive factors for CR at 12 months were the anti-La/SSB antibodies (U/ml) (odds ratio (OR) 1.22, 95% confidence interval (CI) 1.01-1.63, p = 0.0220), blood urea nitrogen (BUN) (OR 0.68, 95% CI 0.44-0.90, p = 0.00048) and serum ß2 microglobulin (MG) (OR 0.26, 95% CI 0.06-0.74, p = 0.00098) levels. CONCLUSIONS: Among LN patients, being free from renal flares was associated with attaining CR at 12 months after induction therapy. Anti-La/SSB antibodies were a positive predictive factor, and BUN and serum ß2MG levels were negative predictive factors of CR at 12 months.

The efficacy of adjunct tacrolimus treatment in pregnancy outcomes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) involves multiple organ systems and primarily affects women during their reproductive years. Pregnancy in a woman with SLE may lead to higher rates of disease flares. Little is known regarding which medications are safe to maintain remission and/or treat flares throughout such pregnancies. Here we retrospectively analyzed the efficacy of tacrolimus (TAC) in the pregnancy outcomes of SLE patients. We studied the 54 deliveries of 40 SLE patients over an eight-year period from 2008 to 2016. We used analyses of covariance with adjustments for the propensity score and inverse probability of treatment weights to compare the patient backgrounds between the TAC users and non-TAC users. TAC was administered to the patient in 15 of the 54 (27.8%) pregnancies, and these patients had a significantly higher dose of prednisolone, hypocomplementemia, lower estimated glomerular filtration rate, past history of lupus nephritis, and complication with antiphospholipid syndrome. In the adjusted background of the TAC deliveries, the risks of decreased fetal body weight, low birth weight infant, non-reassuring fetal status (NRFS), and preterm birth were not increased compared to the non-TAC deliveries. Thrombocytopenia and hypertension during the pregnancy were extracted as independent predictive risk factors for decreased fetal body weight and NRFS, respectively. We had anticipated that the maternal and fetal outcomes in the TAC-use deliveries would be poor before the analysis; however, the TAC-use group showed no significant difference in risks contributing to outcomes compared to the non-TAC group, suggesting that adjunct TAC treatment corrected various risk factors during the lupus pregnancies.

TGFß1 synergizes with FLT3 ligand to induce chemoresistant quiescence in acute lymphoblastic leukemia with MLL gene rearrangements.

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in mixed-lineage leukemia (MLL) gene-rearranged acute lymphoblastic leukemia (MLL+ALL) with a dismal prognosis. We previously reported that FLT3 ligand (FL) stimulation induced cell cycle arrest in MLL+ALL cells leading to resistance against anti-leukemic agents. Given that FL stimulation enhanced transforming growth factor (TGF)ß1 mRNA levels in MLL+ALL cells, we extensively examined the effect of TGFß1 on the cell cycle progression and chemosensitivity in MLL+ALL cells, and found that TGFß1 stimulation induced MLL+ALL cells into cell cycle arrest resistant to arabinosyl cytosine; its effect was markedly enhanced in synergy with FL. Thus, it is likely that TGFß1 and FL, both abundantly produced by bone marrow stromal cells, function in a coordinated manner to render MLL+ALL cells chemoresistant, which should lead to the development of minimal residual disease (MRD) resulting in relapse. The use of inhibitors against FLT3 and TGFß1 may become a useful strategy for eradicating MRD in MLL+ALL.

Circulating CD34-Positive Cells Are Associated with Handgrip Strength in Japanese Older Men: The Nagasaki Islands Study.

BACKGROUND: Handgrip strength is a simple measurement of overall muscular strength and is used to detect sarcopenia. It also predicts adverse events in later life. Many mechanisms of sarcopenia development have been reported. A hypertensive status impairs endothelial dysfunction, which might deteriorate skeletal muscle if vascular angiogenesis is not maintained. OBJECTIVES: This study investigated muscle strength and circulating CD34-positive cells as a marker of vascular angiogenesis. DESIGN: Cross-sectional study. PARTICIPANTS: 262 male Japanese community dwellers aged 60 to 69 years. MEASUREMENTS: The participants' handgrip strength, medical history, and blood samples were taken. We stratified the participants by hypertensive status to investigate the association between handgrip strength and circulating CD34-positive cells according to hypertensive status. Pearson correlation and linear regression analyses were used. RESULTS: In the Pearson correlation analysis, handgrip strength and the logarithm of circulating CD34-positive cells were significantly associated in hypertensive participants (r=0.22, p=0.021), but not in non-hypertensive participants (r=-0.01, p=0.943). This relationship was only significant in hypertensive participants (ß=1.94, p=0.021) in the simple linear regression analysis, and it remained significant after adjusting for classic cardiovascular risk factors (ß=1.92, p=0.020). The relationship was not significant in non-hypertensive participants (ß=-0.09, p=0.903). CONCLUSIONS: We found a positive association between handgrip strength and circulating CD34-positive cells in hypertensive men. Vascular maintenance attributed by circulating CD34-positive cells is thought to be a background mechanism of this association after hypertension-induced vascular injury in skeletal muscle.

Local and systemic responses following intravitreous injection of AAV2-encoded modified Volvox channelrhodopsin-1 in a genetically blind rat model.

We previously designed a modified channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa.

MEFV gene polymorphisms and TNFRSF1A mutation in patients with inflammatory myopathy with abundant macrophages.

Inflammatory myopathy with abundant macrophages (IMAM) has recently been proposed as a new clinical condition. Although IMAM shares certain similarities with other inflammatory myopathies, the mechanisms responsible for this condition remain unknown. Patients with familial Mediterranean fever (FMF) and tumour necrosis factor receptor-associated periodic syndrome (TRAPS) also often develop myalgia. We therefore investigated the polymorphisms or mutations of MEFV and TNFRSF1A genes in patients with IMAM to identify their potential role in this condition. We analysed the clinical features of nine patients with IMAM and sequenced exons of the MEFV and TNFRSF1A genes. The patients with IMAM had clinical symptoms such as myalgia, muscle weakness, erythema, fever and arthralgia. Although none of the patients were diagnosed with FMF or TRAPS, seven demonstrated MEFV polymorphisms (G304R, R202R, E148Q, E148Q-L110P and P369S-R408Q), and one demonstrated a TNFRSF1A mutation (C43R). These results suggest that MEFV gene polymorphisms and TNFRSF1A mutation are susceptibility and modifier genes in IMAM.

Notch signaling pathway regulates proliferation and differentiation of immortalized Müller cells under hypoxic conditions in vitro.

Previous studies have indicated that Müller glia in chick and fish retinas can re-enter the cell cycle, express progenitor genes, and regenerate neurons via the Notch signaling pathway in response to retinal damage or growth factors. Here, we investigated the role of Notch signaling and the effect of hypoxia, as a means to induce retinal damage, on the proliferation of an immortalized Müller cell line (rMC-1 cells). Our data showed that rMC-1 cells expressed Müller glia and neural and retinal progenitor markers but did not express neuronal or retinal markers. Hypoxia increased rMC-1 cell proliferation by activating the positive cell-cycle regulators, cyclins A and D1, as well as the neural and retinal progenitor markers, Notch1, Hes1, nestin, Sox2, Msi1, Pax6, and NeuroD1. However, hypoxia did not significantly influence the expression of Müller glial markers GS, CRALBP, and cyclin D3 or the death of the rMC-1 cells. The increase in cell proliferation induced by hypoxia was greatly attenuated by blocking Notch signaling with the inhibitor DAPT, resulting in the reduced expression of positive cell-cycle regulators (cyclins A and D1) and neural and retinal progenitor markers (Notch1, Hes1, Sox2, Pax6, and NeuroD1). Blockade of the Notch signaling pathway by DAPT after hypoxia promoted the differentiation of rMC-1 cells to neurons, as demonstrated by the induction of neural marker (Tuj1), retinal amacrine (Syntaxin1), and retinal ganglion cell (Brn3b) markers, although the expression of the latter marker was low. Taken together, our data indicate that Notch signaling is required for proliferation under hypoxic conditions either by activating the positive cell-cycle regulators or by skewing their de-differentiation towards a neural progenitor lineage. These findings indicate that the Notch signaling pathway regulates hypoxia-induced proliferation and differentiation of Müller glia.

Adipogenesis of the mesenchymal stromal cells and bone oedema in rheumatoid arthritis.

OBJECTIVES: Bone oedema is a pathological change in rheumatoid arthritis (RA) that is detectable by magnetic resonance imaging (MRI). Recent histological analyses revealed that a prominent feature of bone oedema is the replacement of adipose tissue with inflammatory cells. Here, we demonstrate the possible roles of mesenchymal stromal cells (MSCs) in bone oedema formation and the pathogenic potential of the cells in RA. METHODS: Adipogenesis of bone marrow-derived human MSCs was induced by a standard adipogenic induction medium in the presence or absence of cytokines. The cytokine productions from MSCs were screened by an antibody array system and confirmed by ELISA. The migration assay was performed to determine the locomotive abilities of undifferentiated MSCs or MSCs after adipogenesis. The expression of α smooth muscle actin (SMA) and F-actin was examined by immunostaining and phalloidin staining, respectively. RESULTS: TNF-α, interleukin (IL)-1ß, IL-6, and TGF-ß clearly inhibited the adipogenesis of MSCs. Production of IL-6 was markedly reduced, and IL-8 secretion was augmented in MSCs after adipogenesis. The mobility of MSCs after adipogenesis was clearly reduced in migration assays compared to that of undifferentiated MSCs. Consistent with these findings, SMA and F-actin expressions were clearly suppressed in MSCs committed to adipogenesis. CONCLUSIONS: Our data suggest that the inflammatory milieu promotes bone oedema by blocking adipogenesis of MSCs. In bone oedema, the enhanced IL-6 production and the increased mobility of MSCs may contribute to the progression of RA. Therefore, bone oedema may be an important target lesion in the treatment of RA.

Immune responses to adeno-associated virus type 2 encoding channelrhodopsin-2 in a genetically blind rat model for gene therapy.

We had previously reported that transduction of the channelrhodopsin-2 (ChR2) gene into retinal ganglion cells restores visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats. In this study, we attempted to reveal the safety and influence of exogenous ChR2 gene expression. Adeno-associated virus (AAV) type 2 encoding ChR2 fused to Venus (rAAV-ChR2V) was administered by intra-vitreous injection to dystrophic RCS rats. However, rAAV-ChR2 gene expression was detected in non-target organs (intestine, lung and heart) in some cases. ChR2 function, monitored by recording visually evoked potentials, was stable across the observation period (64 weeks). No change in retinal histology and no inflammatory marker of leucocyte adhesion in the retinal vasculature were observed. Although antibodies to rAAV (0.01-12.21 µg ml(-1)) and ChR2 (0-4.77 µg ml(-1)) were detected, their levels were too low for rejection. T-lymphocyte analysis revealed recognition by T cells and a transient inflammation-like immune reaction only until 1 month after the rAAV-ChR2V injection. In conclusion, ChR2, which originates from Chlamydomonas reinhardtii, can be expressed without immunologically harmful reactions in vivo. These findings will help studies of ChR2 gene transfer to restore vision in progressed retinitis pigmentosa.