A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Recurrence in long-term survivor of anti-MDA5 antibody-positive clinically amyopathic dermatomyositis: case series and literature review.

Anti-MDA5 antibody-positive clinically amyopathic dermatomyositis (CADM) is often complicated by rapidly progressive interstitial lung disease and is associated with poor prognosis. However, even though recurrence is reported to be infrequent if successful medical treatment is administered, the long-term prognosis remains unclear. In this case report, we examined the clinical features and treatment details of three patients with anti-MDA5 antibody-positive CADM with multiple recurrences during long-term survival at Juntendo University Urayasu Hospital. Of the three patients, two failed to convert to an anti-MDA5 antibody-negative status, and one patient died. One of the remaining patients experienced two relapses but eventually tested negative for anti-MDA5 antibodies and showed a relatively stable clinical course. Although cases of recurring anti-MDA5 antibody-positive CADM rarely occur, they may occasionally be fatal. The prognosis for anti-MDA5 antibody-positive CADM has improved over time owing to its establishment as a disease. However, further information and research is necessary to ascertain its long-term prognosis.

Metformin repositioning in rheumatoid arthritis.

OBJECTIVES: Metformin is a known therapeutic agent for diabetes. Recently, several reports suggested the possibility of improvement in autoimmune disease and malignancy conditions through the effect of metformin on the immune system. Although there have been reports on the therapeutic effects of metformin on mouse models of collagen-induced arthritis, simulating human rheumatoid arthritis (RA), the effect of metformin on human RA remains unknown. Therefore, we investigated the inhibitory effect of metformin on the pathogenesis of human RA in vitro. METHODS: Osteoclastogenesis was evaluated with or without metformin. through tartrate-resistant acid phosphatase staining, osteoclast-specific enzyme expression analysis, and a bone resorption assay. Human fibroblast-like synoviocyte MH7A cells were stimulated with TNF-α, and the expression of proinflammatory cytokines and protease and growth factor genes was evaluated with or without metformin. Metformin has been used to evaluate their potential modulatory effects on cells treated with TNF-α. Moreover, we examined angiogenesis by performing a tube formation assay using human umbilical vein endothelial cells (HUVECs) with or without metformin. RESULTS: Osteoclastogenesis was suppressed in the presence of metformin, and the expression of osteoclast-specific genes was reduced. The TNF-α-induced expression of inflammatory cytokines and protease and growth factor genes in MH7A cells was downregulated by metformin. Additionally, the induced formation of tubular networks in HUVECs was also disrupted following treatment with metformin. CONCLUSIONS: These results suggest that metformin might improve the pathogenesis of RA, including joint inflammation and destruction. Thus, metformin might be utilised as a potential therapeutic agent in the treatment of RA.

MicroRNA-766-3p Contributes to Anti-Inflammatory Responses through the Indirect Inhibition of NF-κB Signaling.

MicroRNA (miRNA) is small RNA of 20 to 22 nucleotides in length and is stably present in plasma. Regulating the expression of miRNA taken into cells has been suggested as a general therapeutic approach. We identified the novel anti-inflammatory miRNA hsa-miR-766-3p and investigated its biological function in human rheumatoid arthritis (RA) fibroblast-like synoviocyte MH7A cells. To verify the function of the miRNA present in the plasma of RA patients, we performed a comprehensive analysis of the miRNA expression during abatacept treatment and identified eight miRNAs with significantly altered expression levels. Among these eight miRNAs, miR-766-3p was found to have a clear function. The expression of inflammatory genes in response to inflammatory stimuli was suppressed in MH7A transduced with miR-766-3p. We showed that miR-766-3p indirectly reduced the activation of NF-κB and clarified that this mechanism was partially involved in the reduction of the mineralocorticoid receptor expression. In addition, the inflammatory responses were suppressed in other types of cells. These results indicate the novel function of miR-766-3p, findings that may aid in the development of therapies to suppress inflammation, not only in RA but also in other diseases.

Aortic Aneurysm as a Complication of Granulomatosis with Polyangiitis Successfully Treated with Prednisolone and Cyclophosphamide: A Case Report and Review of the Literature.

A 57-year-old Japanese man was admitted to the hospital with back pain and fever, multiple lung nodules, and abdominal aortic aneurysm (AAA). Laboratory tests performed at admission showed an increased proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) level. Video-associated thoracoscopic lung biopsy was performed; pathologic examination showed granulation tissue with necrosis and multinucleated giant cells. The diagnosis of granulomatosis with polyangiitis (GPA) was confirmed on the basis of the clinical presentation, laboratory findings, and lung biopsy. All symptoms were ameliorated, and the serum level of PR3-ANCA declined following treatment with prednisolone and cyclophosphamide. Although the association of GPA with AAA is rare, GPA may be included among the large vessel vasculitides that can give rise to aortic aneurysm.

Kinase inhibitors of the IGF-1R as a potential therapeutic agent for rheumatoid arthritis.

We have previously shown that the inhibition of connective tissue growth factor (CTGF) is a potential therapeutic strategy against rheumatoid arthritis (RA). CTGF consists of four distinct modules, including the insulin-like growth factor binding protein (IGFBP). In serum, insulin-like growth factors (IGFs) bind IGFBPs, interact with the IGF-1 receptor (IGF-1 R), and regulate anabolic effects and bone metabolism. We investigated the correlation between IGF-1 and the pathogenesis of RA, and the inhibitory effect on osteoclastogenesis and angiogenesis of the small molecular weight kinase inhibitor of the IGF-1 R, NVP-AEW541, against pathogenesis of RA in vitro. Cell proliferation was evaluated by cell count and immunoblotting. The expression of IGF-1 and IGF-1 R was evaluated by RT-PCR. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay, and osteoclast-specific enzyme production. Angiogenesis was evaluated by a tube formation assay using human umbilical vein endothelial cells (HUVECs). The proliferation of MH7A cells was found to be inhibited in the presence of NVP-AEW541, and the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was downregulated in MH7A cells. IGF-1 and IGF-1 R mRNA expression levels were upregulated during formation of M-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL)-mediated osteoclast formation. Moreover, osteoclastogenesis was suppressed in the presence of NVP-AEW541. The formation of the tubular network was enhanced by IGF-1, and this effect was neutralized by NVP-ARE541. Our findings suggest that NVP-AEW541 may be utilized as a potential therapeutic agent in the treatment of RA.

Intestinal perforation due to hemorrhagic Cytomegalovirus enteritis in a patient with severe uncontrolled lupus nephritis: a case and review of the literature.

A 31-year-old woman with systemic lupus erythematosus and lupus nephritis was treated with prednisone and immunosuppressants. After her lupus nephritis symptoms worsened, both high-dose steroid and cyclophosphamide pulse therapy were administered. The patient developed an intestinal perforation, and laparoscopic Hartmann's surgery was performed on the sigmoid colon. Serum Cytomegalovirus (CMV) antigen C7HRP was detected, and the patient was diagnosed with CMV colitis and underwent a colon resection. Severe hematochezia continued despite ganciclovir administration, and the patient underwent laparoscopic total colectomy and partial ileostomy. CMV enteritis should be considered in patients treated with prednisone and immunosuppressants and those who have abdominal pain and hematochezia. Immunocompromised patients with intestinal perforation due to CMV enteritis have a poor prognosis. We report a case with along with the results of a literature review.

Inhibition of each module of connective tissue growth factor as a potential therapeutic target for rheumatoid arthritis.

We previously reported the importance of connective tissue growth factor (CTGF) in rheumatoid arthritis (RA). CTGF contains four distinct modules connected in tandem, namely insulin-like growth factor-binding protein (IGFBP)-like, von Willebrand factor (vWF) type C repeat, thrombospondin type 1 (TSP-1) repeat, and carboxyl-terminal (CT) modules. The relationships between each of these modules of CTGF and RA remain unknown. Here, we analyzed how inhibition of each CTGF module affects the pathophysiology of RA. We conducted stimulation and suppression experiments on synovial cells (MH7A) obtained from patients with RA. Moreover, we examined angiogenesis by means of a tube-formation assay performed using human umbilical vein endothelial cells (HUVECs), and we used tartrate-resistant acid phosphatase (TRAP) staining to analyze osteoclastogenesis. Our results showed that M-CSF/RANKL-mediated osteoclastogenesis was enhanced when CTGF was added, but the effect of CTGF was neutralized by mAbs against CTGF modules 1-4. Furthermore, CTGF treatment of HUVECs induced formation of tubular networks, which resulted in acceleration of the angiogenesis of RA synoviocytes, and quantification showed that this tubular-network formation was also disrupted by anti-CTGF module 1-4 mAbs. Lastly, TNF-α enhanced the expression of CTGF and matrix metalloproteinase-3 (MMP3) in MH7A cells, and this enhancement was potently neutralized by mAbs against CTGF modules 1, 3 and 4. Thus, our results indicate that not only a mAb against CTGF but also mAbs against each specific module of CTGF might serve as potential therapeutic agents in the treatment of RA.

Rapid onset of small intestinal perforation after successful steroid treatment in eosinophilic granulomatosis with polyangiitis.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a disorder characterized by extravascular granulomas, hypereosinophilia, and pulmonary and systemic small-vessel vasculitis. Bowel perforation is a rare but often fatal complication of EGPA. In the present report, we describe a case of small intestinal perforation in a patient with EGPA. Through various examinations, we confirmed the presence of EGPA, and the patient responded well to steroid therapy. However, as the patient's condition subsequently worsened, the small intestine was consequently resected. The patient's overall condition improved thereafter. Thus, we believe that careful attention should be paid to intestinal symptoms and perforation in patients with EGPA.

Epstein-Barr virus-positive multiple myeloma developing after immunosuppressant therapy for rheumatoid arthritis: a case report and review of literature.

A 61-year-old woman was diagnosed as having rheumatoid arthritis (RA) and began treatment with salazosulfapyridine (SASP) and methotrexate (MTX) in 2008; the administration of concomitant tacrolimus (TAC) was initiated in 2010. She subsequently developed concurrent multiple myeloma (MM), immunoglobulin G (IgG)-κ type, in 2012. A portion of the tumor cells tested positive for Epstein-Barr virus-encoded small RNA (EBER). MTX treatment was discontinued in 2014, and the exacerbation of MM ensued. The patient received two cycles of bortezomib plus dexamethasone (BD) therapy and attained a complete response (CR). She then underwent an autologous peripheral blood stem cell transplantation. The Epstein-Barr (EB) virus infection arising from the increased RA disease activity and immunosuppressant medication might have influenced the development of MM in this case. Most reported patients with EB virus-positive plasmacytoma are in a state of immunosuppression, and this condition may fall within the category of other iatrogenic immunodeficiency-associated lymphoproliferative disorders. No other reports of plasmacytoma occurring in a background of RA or after TAC or MTX therapy have been made, and the present case is the first such report.

Inhibition of the insulin-like growth factor system is a potential therapy for rheumatoid arthritis.

OBJECTIVE: We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). Insulin-like growth factor binding proteins (IGFBPs) are modules of CTGF. IGFBPs bind IGF-I and IGF-II. IGF-I plays a role in the regulation of immunity, bone metabolism and inflammation. Therefore, we investigated how the IGF system is associated with RA disease progression. METHODS: Serum samples were collected from RA patients. IGF-I and IGFBP-3 production were evaluated by enzyme-linked immunosorbent assay, real-time RT-PCR and indirect immunofluorescence microscopy. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase staining, a bone resorption assay and osteoclast-specific enzyme production. Angiogenesis was examined by a tube formation assay using human umbilical vein endothelial cells. RESULTS: The serum concentrations of IGFBP-3 in RA patients were greater than those in normal controls. IGF-I and IGFBP-3 were produced primarily by macrophages in the RA synovium. Furthermore, tumor necrosis factor-α could induce aberrant IGF-I and IGFBP-3 production in synovial fibroblasts. IGF-I and IGFBP-3 promoted the induction of osteoclast generation and morphological changes, in combination with M-colony stimulating factor and the receptor activator of NF-κB ligand. In addition, IGF-I and IGFBP-3 induced angiogenesis, as determined by the tube formation assay. These effects were neutralized by anti-IGF-IR monoclonal antibody (mAb). CONCLUSIONS: These results indicate that aberrant IGF-I and IGFBP-3 production plays a role in abnormal osteoclastic activation and angiogenesis in RA. This work supports future clinical exploration of anti-IGF-IR mAb in drug repositioning as a new treatment for RA.

Serum proteome analysis in patients with rheumatoid arthritis receiving therapy with etanercept, a chimeric tumor necrosis factor-alpha receptor.

AIM: Rheumatoid arthritis (RA) is a chronic inflammatory disorder of the synovium resulting in the destruction of affected joint cartilage and bone structures. Etanercept is a biological agent that blocks the tumor necrosis factor-α (TNF-α)-mediated inflammatory processes in RA patients, and has a regenerative effect on cartilage. In order to identify novel disease-related proteins and candidate biomarkers, we performed proteomic profiling of the serum in patients with RA who were treated with etanercept. METHOD: Serum samples were obtained from eight RA patients before and after etanercept treatment. The low molecular weight proteins in the serum were concentrated and analyzed by liquid chromatography-tandem mass spectrometry. The results before and after etanercept treatment were compared by the spectrum count method. RESULTS: Among a total of 477 proteins identified, 12 were found to be decreased and five were increased by etanercept treatment. Some of the changed proteins were known to be related to RA, and most of the other changed proteins may play possible roles in the TNF-α signaling pathway or the state of cartilage and extracellular matrix. CONCLUSION: The present proteomic study identified several proteins that could be involved in the pathogenesis of RA. These findings could thus lead to the identification of novel candidate disease-related protein biomarkers for RA, or indicate new targets for therapy.

Differential anti-Golgi complex autoantibody production following murine lactate dehydrogenase-elevating virus infection.

Lactate dehydrogenase-elevating virus (LDV) causes asymptomatic infection and persistent viremia in mice with unique infectious specificity directed to a certain subpopulation of macrophages leading to chronic infection and an immunological disorder that includes hyperimmunoglobulinemia and production of autoantibodies. Infection with a species of LDV originally isolated from mice carrying an LDV-contaminated transplantable tumor (LDV-W) was reported to induce anti-Golgi complex antibody (AGA) production. In contrast, infection with the most common LDV species (LDV-P) was not associated with AGA production. Here we performed the first independent side by side comparison of the effects of the two LDV strains on their hosts as an initial approach to investigating the production of AGA. After viral inoculation, both LDV-W and LDV-P infected mice exhibited similar changes in lactate dehydrogenase in plasma suggesting similar viral activity. However, AGA production was observed in only the LDV-W infected mice and these mice exhibited plasma IgG elevation and immune complex formation. These data validated the differential potential of LDV-W and LDV-P in the production of AGA. Future comparative characterizations in the immune processing of Golgi complex autoantigens using these viral strains may be useful in obtaining specific insights in the specific anti-Golgi complex autoimmune responses.

Nucleolar staining cannot be used as a screening test for the scleroderma marker anti-RNA polymerase I/III antibodies.

OBJECTIVE: Anti-RNA polymerase I/III (anti-RNAP I/III) antibodies are clinically useful markers of scleroderma, and their presence is associated with diffuse skin disease and an increased risk of cardiac and kidney involvement. Although RNAP I antibodies localize to the nucleolus, nucleolar staining by many anti-RNAP antibody-positive sera is not always observed. Nucleolar staining by anti-RNAP antibody-positive sera was examined by double staining with antifibrillarin antibodies to evaluate whether nucleolar staining can be used as a screening test for anti-RNAP I/III antibodies. In addition, the relationships between nucleolar staining and levels of anti-RNAP III antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) assay. METHODS: Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides, by anti-RNAP III ELISA, and by IP assay using (35)S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and by IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed. RESULTS: All 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44% of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining. CONCLUSION: Although some sera positive for anti-RNAP I/III antibodies clearly stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.

Autoimmune targeting of key components of RNA interference.

RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.

Autoimmune responses to proliferating cell nuclear antigen multiprotein complexes involved in cell proliferation are strongly associated with their structure and biologic function in patients with systemic lupus erythematosus.

OBJECTIVE: To analyze the reaction of lupus sera with proliferating cell nuclear antigen (PCNA) multiprotein complexes (PCNA complexes), which are part of the protein machinery involved in cell proliferation. METHODS: PCNA complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA monoclonal antibodies (TOB7, TO17, and TO30); monomeric and trimeric PCNA forms (AK-PCNA) were purified using anti-PCNA serum AK. The reactions to these antigens of 10 anti-PCNA-positive and 40 anti-PCNA-negative sera selected from 560 lupus patients were tested by immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISAs). RESULTS: With one exception (serum OK), anti-PCNA-positive sera reacted exclusively with only the 34-kd polypeptide. In contrast, 14 of 40 anti-PCNA-negative sera reacted with multiple proteins within PCNA complexes. Most anti-PCNA-positive sera probably recognize as epitopes the binding sites for other proteins on PCNA, which are likely hidden when PCNA is complexed with other proteins. As a consequence, only serum OK reacted with the PCNA complex in a series of ELISAs. Using AK-PCNA as a competitive inhibitor, it was determined that serum OK reacts with both the 58-kd polypeptide and the 34-kd PCNA within complexes. Together with the results of a longitudinal analysis, these results suggest that the immune system of patient OK likely recognized the complexed PCNA protein, after which the autoimmune response spread to other elements of the complexes. CONCLUSION: Intermolecular-intrastructural help, leading to the spread of autoimmune response from PCNA to other proteins associated with its biologic function, plays a crucial role in the induction of the autoimmune response seen in lupus patients.