Pathogenic roles of anti-C1q antibodies in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is often considered idiopathic, however excessive complement activation has been observed in pregnancy related manifestations. Anti-C1q antibodies (anti-C1q) are associated with the activation of complement pathway in lupus patients, while it remains unclear in RPL. Firstly, we showed that both the prevalence and titre of anti-C1q were significantly higher in unexplained RPL than in healthy parous individuals. Secondly, we established the murine model of anti-C1q induced pregnancy loss using a monoclonal anti-mouse C1q antibody, JL-1. In mice treated with JL-1, high ratio of pregnancy loss and fetal growth restriction were frequently observed and complement activation occurred. C5a receptor (C5aR) blockade cancelled these pathogenic changes in mice treated with JL-1. In conclusion, our study reveals an association between the prevalence of anti-C1q and RPL. Additionally, our murine model has indicated that anti-C1q can induce reproductive failure, which might be ameliorated by therapy targeting the C5-C5aR axis.

Basic Evaluation of the Newly Developed "Lias Auto P-FDP" Assay and the Influence of Plasmin-α2 Plasmin Inhibitor Complex Values on Discrepancy in the Comparison with "Lias Auto D-Dimer Neo" Assay.

BACKGROUND: Laboratory determination of fibrin/fibrinogen degradation products (FDP) levels, along with that of the D-dimer, is important for assessing the fibrinolytic situation. Recently, we developed a new FDP reagent "Lias Auto P-FDP", which can detect various FDP fragments. The purpose of this study was to evaluate the basic performance of the newly developed Lias Auto P-FDP and compare it with Lias Auto D-Dimer Neo assay. METHODS: The within-run precision of Lias Auto P-FDP and Lias Auto D-Dimer was determined 20 times in low and high value controls. The between-day precision was evaluated five times a day for five days. The linearity study was performed by diluting high value samples for 2 - 10-fold and 2 - 8-fold. The comparative study was performed using 172 patient samples with elevated FDP values. For the discrepancy analysis, the samples were divided into three groups by the discrepancy percentage between the FDP and D-dimer values. The groups were defined as follows: lower discrepancy group, less than -20%; no discrepancy group, -20% to 20%; upper discrepancy group, more than 20%. RESULTS: The coefficient of variation % (CV%) in within-run and between-day precision were within 3.8% for both FDP and the D-dimer. The correlation coefficients were more than 0.999 and the linearity was high. In the comparative study, the values of FDP were higher than that of the D-dimer in all samples. The median FDP and D-dimer values of lower discrepancy, no discrepancy, and upper discrepancy groups were 11.8, 20.3, and 51.4, and 8.0, 11.3, and 13.1, respectively. FDP showed an increasing tendency but D-Dimer showed constant values. Thus, the possible cause of discrepancy between FDP and D-dimer values were the elevated FDP values. In addition, the values of plasmin-α2 plasmin inhibitor complex (PIC) in the upper discrepancy group were higher than that of the lower and no discrepancy groups, indicating progression of fibrinolysis. CONCLUSIONS: In this study, we evaluated the newly developed Lias Auto P-FDP reagent and confirmed that the basic performance was acceptable. FDP was elevated in samples with high PIC values, which indicated progression of fibrinolysis. Determination of fibrinolysis conditions by FDP measurement is important.

Interferon-inducible Mx1 protein is highly expressed in renal tissues from treatment-naïve lupus nephritis, but not in those under immunosuppressive treatment.

OBJECTIVES: The aim of this study was to clarify the consequences of Mx1, one of the IFN-inducible proteins, in the peripheral blood as well as in renal tissues in patients with systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Mx1 protein concentrations in (PBMCs) from 18 SLE patients mostly in their stable disease status, 11 IgA nephropathy (IgAN) patients, 5 ANCA-associated vasculitis (AAV) patients and 16 healthy controls were measured using enzyme-linked immunosorbent assay (ELISA). Mx1 expression in renal specimens from 18 patients with lupus nephritis (LN), 18 with IgAN and 10 with AAV were evaluated using immunohistochemistry. RESULTS: Mx1 protein concentrations in lysates of PBMCs were significantly higher in SLE patients compared with those in other three groups. Mx1-positive area in renal tissues was significantly dominant in both glomeruli and renal tubules of LN compared with other renal diseases. Renal Mx1 protein levels were lower in LN after immunosuppressive treatment, compared with those from immunosuppressant-naïve patients. CONCLUSION: Mx1 levels were upregulated in lupus peripheral blood even when their disease activities were stable. On the other hand, Mx1 was highly expressed in kidneys from patients with LN before treatment, which was decreased after immunosuppressive treatment. These results suggest that Mx1 is a potential marker for the diagnosis of SLE in the peripheral blood and also for the activity of lupus nephritis in the kidney.

Clinical significance of plasma presepsin levels in patients with systemic lupus erythematosus.

OBJECTIVES: Presepsin (PSEP: soluble CD14 subtype) is produced from bacteria-stimulated monocytes or neutrophils, thus recognized as a biomarker of sepsis. Aberrant functions in monocyte or neutrophils are increasingly recognized in systemic lupus erythematosus (SLE). We investigated whether plasma PSEP reflects disease activity in patients with SLE. METHODS: This retrospective study comprised 35 patients with SLE and 72 with non-SLE autoimmune diseases who visited our facility during the period from August 2012 to September 2015. Plasma PSEP levels and laboratory data were compared between SLE and non-SLE. Clinical markers of SLE disease activity, including SLE disease activity index 2000 (SLEDAI-2K), serum complement concentrations and serum anti-ds-DNA antibodies were assessed in correlation with plasma PSEP levels. RESULTS: Plasma PSEP levels in SLE were higher than those in non-SLE. This phenomenon holds true when comparing SLE and non-SLE patients in the absence of infection (p = .0008). Plasma PSEP levels in SLE patients negatively correlated with C3 (r = -0.4454, p = .0430), CH50 (r = -0.4502, p = .0406) and positively with SLEDAI-2K (r = 0.4801, p = .0237). CONCLUSION: Elevated plasma PSEP levels were correlated with disease activity of SLE, suggesting inappropriate monocyte or neutrophil activation in the pathophysiology of SLE exacerbation.

Evaluation of the alternative classification criteria of systemic lupus erythematosus established by Systemic Lupus International Collaborating Clinics (SLICC).

OBJECTIVE: To evaluate the performance of the 2012 Systemic Lupus International Collaborating Clinics criteria (SLICC-12) on classifying systemic lupus erythematosus (SLE) in an uncontrolled multi-centered study with real-life scenario of the patients in Japan. METHODS: This study comprised 495 patients with SLE or non-SLE rheumatic diseases and allied conditions from 12 institutes in Japan. Chart review of each patient was performed by the 27 expert rheumatologists and diagnosis of 487 cases reached to the consensus. Value of the SLICC-12 on SLE classification was analyzed comparing with the 1997 revised American College of Rheumatology SLE classification criteria (ACR-97) employing the expert-consented diagnoses. RESULTS: Compared to the ACR-97, the SLICC-12 had a higher sensitivity (ACR-97 vs. SLICC-12: 0.88 vs. 0.99, p < .01) and comparable specificity (0.85 vs. 0.80). The rate of misclassification (0.14 vs. 0.11) or the area under the receiver operating characteristic curves (0.863 vs. 0.894) was not statistically different. In the cases that diagnoses corresponded in high rates among experts, both criteria showed high accordance of SLE classification over 85% with the expert diagnoses. CONCLUSION: Although employment of SLICC-12 for the classification for SLE should be carefully considered, the SLICC-12 showed the higher sensitivity on classifying SLE in Japanese population.

Bi-ventricular interplay in patients with systemic sclerosis-associated pulmonary arterial hypertension: Detection by cardiac magnetic resonance.

OBJECTIVES: Pulmonary arterial hypertension (PAH) associated with systemic sclerosis (SSc) has a poor prognosis compared to PAH associated with other connective tissue diseases (CTD). The objective of this study was to examine the difference in hemodynamic state between SSc-PAH and other CTD-PAH by performing cardiac magnetic resonance (CMR) imaging. METHODS: A single center retrospective analysis was conducted comprising 40 consecutive CTD patients who underwent right heart catheterization and CMR at the same period from January 2010 to October 2015. RESULTS: Thirty-two patients had pre-capillary pulmonary hypertension. Of these, 15 had SSc and 17 had other CTD. CMR measurements, particularly the ratio of right to left end-diastolic volume (RVEDV/LVEDV), correlated well with mean pulmonary arterial pressure (mPAP). Conversely, RVEDV/LVEDV and mPAP correlated differently in SSc and non-SSc patients. In SSc patients, the ratio of RVEDV/LVEDV to mPAP was significantly higher compared to non-SSc patients. In the follow-up study, 2 SSc patients exhibited increased RVEDV/LVEDV in spite of decreased mPAP following treatment. Kaplan-Meier analysis revealed poor prognosis of patients with increased RVEDV/LVEDV following treatment. CONCLUSIONS: Our data indicated that altered bi-ventricular interplay detected at CMR may represent SSc-related cardiac involvement and reflect poor prognosis of SSc-PAH.

Maintenance treatment using abatacept with dose reduction after achievement of low disease activity in patients with rheumatoid arthritis (MATADOR) - A prospective, multicenter, single arm pilot clinical trial.

OBJECTIVES: To preliminarily evaluate the feasibility of maintenance therapy with reduced dose of intravenous abatacept (ABT) to 250 mg/body/month after achieving remission or low disease activity (LDA). PATIENTS AND METHODS: RA patients treated with ABT at 13 sites were enrolled in this prospective interventional pilot study during the period between March 2013 and March 2015. Inclusion criteria were (1) age at 20 years or older, (2) under treatment with monthly intravenous ABT at approved doses, (3) DAS28-CRP lower than 2.7 at least for 6 months, (4) agreed to join this trial with written informed consent and (5) body weight under 125 kg. Enrolled patients were maintained with intravenous monthly ABT at a reduced dose of 250 mg/body (MATADOR protocol). The primary end point was the proportion of the patients continued with MATADOR protocol at week 48. MATADOR protocol was discontinued upon disease flare or other reasons such as patients' request or severe adverse event (AE). Disease activities and structural changes were also evaluated. RESULTS: Fifty-three patients fulfilled the entry criteria and were followed for 1-year. MATADOR protocol was continued for 1-year in 43 (81%) of the evaluated patients. Three patients experienced severe AEs. Mean DAS28-CRP and remission rate were 1.56 and 88% when ABT reduced and 1.80 and 81% at 1-year, respectively. Structural remission was achieved in 34 out of 42 evaluated patients. CONCLUSIONS: Reduced dose of intravenous ABT was proposed as a feasible choice for maintenance therapy for RA after achievement of remission/LDA, although further randomized trials would be awaited.

Autoantibodies against a complement component 1 q subcomponent contribute to complement activation and recurrent thrombosis/pregnancy morbidity in anti-phospholipid syndrome.

OBJECTIVE: To investigate the prevalence and significance of the autoantibodies against complement component 1 q subcomponent (C1q) in patients with APS. METHODS: In all, 40 consecutive primary APS patients, 42 patients with non-SLE CTDs and 20 SLE patients negative for aPL were enrolled in this retrospective analysis. Refractory APS was defined as a clinical status of recurring thrombosis or pregnancy morbidity during adequate secondary prophylaxis. An ELISA was used to measure serum levels of anti-C1q antibodies and anaphylatoxins (C3a, C4a). RESULTS: Anti-C1q antibodies were found in 36% (15/42) and 2.5% (1/40) of primary APS patients and controls, respectively. Among primary APS patients, anti-C1q antibody titres were significantly correlated with serum C4a levels (P = 0.013). Neither the prevalence nor the titre of anti-C1q antibodies was associated with any specific clinical manifestations of APS, nor titres of aPL. Refractory APS patients (n = 10) had a higher prevalence of anti-C1q antibodies (9/10 vs 6/32, P = 0.01) than APS patients without recurrence (n = 32). CONCLUSION: Anti-C1q antibodies are associated with complement activation in APS and may contribute to the pathogenesis, particularly in refractory cases.

[The pathogenesis, diagnosis and treatment of antiphospholipid syndrome].

Antiphospholipid syndrome (APS) is a clinical condition characterized by recurrent thrombotic events and/or pregnancy morbidity associated with the persistence of antiphospholipid antibodies (aPLs). It is the one of the most common diseases in acquired thrombophilia. In approximately 50 % of APS patients, systemic lupus erythematosus coexist. APS is diagnosed by the 2006 Sydney revised Sapporo criteria. The pathogenic processes of APS have not been fully elucidated. Multiple mechanisms have been proposed, including interference with hemostatic reactions, activation of endothelial cells and complement activation. The treatment of APS is virtually the preventive therapy such as long-term anticoagulation and the role of corticosteroid or immunosuppressive agents is limited.

Imeglimin modulates mitochondria biology and facilitates mitokine secretion in 3T3-L1 adipocytes.

AIMS: Imeglimin, a novel antidiabetic drug, has recently been reported to affect pancreatic ß-cells and hepatocytes. Adipose tissue plays a crucial role in systemic metabolism. However, its effect on adipocytes remains unexplored. Herein, we investigated the effects of imeglimin on adipocytes, particularly in the mitochondria. MAIN METHODS: The 3T3-L1 adipocytes were treated with imeglimin. Mitochondrial respiratory complex I activity and NAD+, NADH, and AMP levels were measured. Protein expression levels were determined by western blotting, mitochondrial DNA and mRNA expression levels were determined using quantitative polymerase chain reaction, and secreted adipocytokine and mitokine levels were determined using adipokine array and enzyme-linked immunosorbent assay. KEY FINDINGS: Imeglimin inhibited complex I activity, decreased the NAD+/NADH ratio, and increased AMP levels, which were associated with the enhanced phosphorylation of AMP-activated protein kinase. In addition, imeglimin increased the mitochondrial DNA content and levels of mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator 1-α mRNA, which were abolished by Ly294002, a phosphoinositide 3-kinase inhibitor. Furthermore, imeglimin facilitated the expression levels of markers of the mitochondrial unfolded protein response, and the gene expression and secretion of two mitokines, fibroblast growth factor 21 and growth differentiation factor 15. The production of both mitokines was transcriptionally regulated and abolished by phosphoinositide 3-kinase and Akt inhibitors. SIGNIFICANCE: Imeglimin modulates mitochondrial biology in adipocytes and may exert a mitohormetic effect through mitokine secretion.

Cytokine and chemokine multiplex analysis-based exploration for potential treatment and prognostic prediction in large-vessel vasculitis: A preliminary observational study.

Large-vessel vasculitis (LVV) is subclassified into two phenotypes; Takayasu arteritis and giant cell arteritis. Although the pathogenesis of LVV is not fully established, IL-6-IL-17 axis and IL-12-IFN-γ axis play critical roles in the disease development. We aimed to clarify the association between the disease state and cytokine/chemokine levels, to assess disease course as prognosis and to predict regulators in patients with LVV using the blood profiles of multiple cytokines/chemokines. This retrospective analysis comprised 35 LVV patients whose blood were collected, and multiplex cytokine/chemokine analysis with 28 analytes was performed. The differences of cytokines/chemokines corresponding disease status, upstream regulator analysis, pathway analysis and cluster analysis were conducted using the cytokines/chemokines profile. Relapse-free survival rate was calculated with Kaplan-Meier analysis in the classified clusters. In the robust analysis, IL-4, CCL2/MCP-1, TNFSF13/APRIL, TNFSF13B/BAFF, CHI3L1 and VEGF-A levels were significantly changed after treatment. Untreated LVV patients demonstrated activation of NFκB-related molecules and these patients are potentially treated with JAK/STAT inhibitors, anti-TNF-α inhibitors and IL-6 inhibitors. Cluster analysis in active LVV patients revealed two clusters including one with high blood levels of IL-1ß, IL-6, IL-17, IL-23 and CCL20/MIP-3. A subgroup of the LVV patients showed activated IL-17 signature with high relapse frequency, and JAK/TyK2 inhibitors and IFN-γ inhibitors were detected as potentially upstream inhibitors. Blood cytokine/chemokine profiles would be useful for prediction of relapse and potentially contributes to establish therapeutic strategy as precision medicine in LVV patients.

Measurement of coagulation factor antibody levels is useful for diagnosis and determining therapeutic efficacy in hemorrhagic patients with autoantibodies to coagulation factor VIII and factor V: results from a single center in Japan.

Coagulation factor inhibitors (CFIs) sometimes cause fatal bleeding conditions. Determination of an inhibitor titer (INH-titer) using the Bethesda method is essential for diagnosing diseases associated with CFIs and examining the effects of immunosuppressive therapy. We reviewed 17 cases with CFIs (acquired hemophilia A, n = 11; FV inhibitor, n = 6) to examine the usefulness of determining quantities of an autoantibody to a coagulation factor (CF-IgG) by ELISA for diagnosis and therapeutic efficacy, as compared with INH-titer. One patient with an INH-titer and no evidence of CF-IgG was lupus anticoagulant (LA)-positive, and thus the positive INH-titer may have been a false positive caused by LA. Although INH-titer alone was insufficient to correctly identify patients with CFI, determination of CF-IgG appeared to be useful. In addition, even after INH-titer disappearance, hemorrhagic conditions recurred when CF-IgG was detected. These findings suggest that the presence of a clearance antibody against the coagulation factor might reduce the activity of that coagulation factor even after disappearance of the corresponding neutralizing antibody. Although the diagnosis and therapeutic efficacy can also be determined by INH-titer disappearance and improvement of corresponding coagulation factor activity, determination of CF-IgG by ELISA can improve the accuracy of these assessments.

Characterization of fibrin/fibrinogen degradation products reagents and their utility in critical care patients with enhanced fibrinolysis.

INTRODUCTION: Fibrin/fibrinogen degradation products (FDP) values reflect coagulation and fibrinolysis status, and FDP levels are helpful for diagnosis and classification of disseminated intravascular coagulation (DIC). FDP measurement has always played a key role in diagnosing DIC, a phenomenon that has recently gained renewed attention because of its occurrence in coronavirus disease 2019 (COVID-19) patients. Although the evaluation of FDP is crucial for the management of critical care, the variability among FDP reagents is unclear. In this study, we aimed to compare LIASAUTO P-FDP with three FDP reagents and investigate their characteristics. METHODS: In total, 172 plasmas samples were used in the correlation. The sample data were divided into three groups including negative, no and positive discrepancy based on the discrepancy percentages calculated from each correlation between LIASAUTO P-FDP and other three reagents. D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), fibrinogen (Fbg) and Plasmin-α2 Plasmin Inhibitor (α2 PI) were measured and included in data analysis. RESULTS: The positive discrepancy groups showed higher D-dimer, PIC and FMC values than the negative discrepancy groups. The data indicated that LIASAUTO P-FDP had higher reactivity to D-dimer than other reagents and the values were elevated in the fibrinolysis-enhanced samples with various FDP fragments. CONCLUSION: LIASAUTO P-FDP displayed the reactivity towards various fibrin/fibrinogen degradation products, and it might be useful for DIC diagnosis because the fibrinolytic status differed in the DIC types and stages.

Comparative evaluation of reagents for measuring protein S activity: possibility of harmonization.

Patients with congenital protein S (PS) deficiency show a hereditary predisposition for thrombosis, and PS deficiency is prevalent among Japanese populations. Diagnosis is based on symptoms of thrombosis and reduced PS activity. Three reagents that use different measurement principles for determining PS activity are available in Japan. This study aimed to confirm the possibility of harmonization of these three reagents to establish a universal standard for PS activity in Japanese populations. Commercial normal plasma and plasma samples obtained from healthy individuals and healthy pregnant women were tested at three facilities using three reagents for measuring PS: STA-Staclot Protein S (STA-PS), HemosIL Protein S (Clotting) (IL-PS), and a total PS assay (SNT-PS). The within-run precision of each reagent was good, as each had a coefficient of variation of ≤ 3.8%. The dilution linearity for each reagent was also good. The correlation coefficient was 0.94 for STA-PS vs. IL-PS, 0.93 for SNT-PS vs. STA-PS, and 0.90 for SNT-PS vs. IL-PS, indicating a good correlation. Although the three reagents available in Japan for measuring PS activity use different measurement methods, each showed good performance, and large differences were not observed between the obtained values. Harmonization among them appears possible.

Knockdown of long noncoding RNA dreh facilitates cell surface GLUT4 expression and glucose uptake through the involvement of vimentin in 3T3-L1 adipocytes.

Glucose uptake in adipocytes is crucial for regulating systemic metabolism. Long noncoding RNAs (lncRNAs), defined as being transcripts with lengths exceeding 200 nucleotides that are not translated, are recently identified regulators of cellular functions. Previously, we have shown that an lncRNA, "down-regulated expression by hepatitis B virus X" (dreh), is involved in glucose transport in skeletal muscle cells. Here, we aimed to examine the involvement of dreh in glucose transport in 3T3-L1 adipocytes. Expression analysis showed that dreh was expressed in 3T3-L1 fibroblasts and adipocytes. Knockdown of dreh expression using its specific siRNAs lowered the glucose concentration of the medium and facilitated [3H]-2-deoxyglucose transport in adipocytes. Additionally, dreh silencing enhanced the protein expression of glucose transporter (GLUT4) in the plasma membrane of adipocytes. Treatment with siRNA against vimentin attenuated the glucose-lowering effect of dreh depletion. These results suggest that the repression of dreh facilitates glucose transport via increased GLUT4 expression in the plasma membrane through the involvement of vimentin in 3T3-L1 adipocytes. In conclusion, dreh is the first observed lncRNA that regulates glucose transport in adipocytes and could serve as a novel therapeutic target for diabetes by modulating adipocyte function. Considering the new function of dreh, we propose that dreh be renamed "down-regulated expression-related hexose/glucose transport enhancer."

Novel assay based on diluted prothrombin time reflects anticoagulant effects of direct oral factor Xa inhibitors: Results of multicenter study in Japan.

BACKGROUND: Direct oral anticoagulants targeting factor Xa (DXaIs) are administered as prophylaxis for various venothrombotic diseases without routine monitoring required. However, assessment of their anticoagulant effects is necessary to prevent severe events, including major bleeding and/or refractory thrombosis. OBJECTIVES: We examined the correlation of ratio of inhibited thrombin generation (RITG), determined using a novel assay based on dilute prothrombin time (dPT), with coagulant markers and laboratory test results to show drug effects. In addition, RITG usefulness as a confirmation test for DXaI therapy was investigated. METHODS: Citrated plasma samples were obtained from patients treated with rivaroxaban (n = 882), apixaban (n = 1214), or edoxaban (n = 820) at 4 different institutions in Japan. Laboratory tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, and plasma concentrations of DXaIs, were conducted, with drug concentrations divided into peak and trough groups, within and after 5 h of administration. RESULTS: In each DXaI group, RITG was positively correlated with PT, APTT, and drug concentration, and negatively with D-dimer. RITG fluctuation during the peak and trough periods reflected the anticoagulant activity characteristic of each DXaI, which was different from blood concentration fluctuations. RITG showed a significant decrease in cases with thrombosis, while that was increased in those with hemorrhage. CONCLUSION: We developed RITG, a novel measurement method based on dPT. RITG represents residual coagulation ability in plasma samples, and is useful for assessment of bleeding and thrombotic tendencies in DXaI patients. RITG can be utilized to confirm the effectiveness of oral anticoagulation therapy with DXaI agents.

Lupus anticoagulant-hypoprothrombinemia syndrome and similar diseases: experiences at a single center in Japan.

Patients with lupus anticoagulant (LA), a thrombotic risk factor, along with decreased prothrombin (FII) activity are classified as lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) and occasionally show bleeding symptoms, although this is not essential for diagnosis. We treated 20 cases of LAHPS over a 3-year period. Median FII activity was 20.9% and the anti-prothrombin antibody (anti-II Ab), shown by ELISA findings, was detected in 55%. Bleeding symptoms were observed in 20%, although that finding was not correlated with FII activity or anti-FII Ab quantity. We also observed 21 LA cases with decreased activity of coagulation factors other than FII, which we have designated LAHPS-like syndrome (LLS). Among LLS patients, anti-FII Ab and bleeding symptoms were seen in 47.6% and 14.3%, respectively. Our findings suggest that bleeding in LAHPS and LLS cannot be explained only by FII activity decreased by anti-FII Ab. Low FVIII activity and the anti-FVIII antibody (anti-FVIII Ab) were detected in some LAHPS and LLS patients, making it difficult to distinguish those from acquired hemophilia A cases. Detection of anti-FVIII Ab quantity by ELISA may be useful for accurate determination, as that was not performed in our LAHPS or LLS patients.

Dreh, a long noncoding RNA repressed by metformin, regulates glucose transport in C2C12 skeletal muscle cells.

AIMS: The anti-hyperglycemic action of metformin on skeletal muscles is presently unclear. Long noncoding RNAs (lncRNAs) are implicated in multiple cellular functions. This study aims to explore the role of lncRNAs in the glucometabolic action of metformin on skeletal muscle cells. MAIN METHODS: Metformin accumulation was assessed using [14C]-metformin. A lncRNA array was used to investigate metformin-regulated lncRNAs in C2C12 skeletal muscle cells. Knockdown studies were applied to evaluate the function of lncRNA Dreh. A colorimetric assay was used for the measurement of medium glucose concentration; glucose transport was assessed using [3H]-2-deoxyglucose; real-time PCR was used for RNA expression analysis, and western blotting was used to assess protein expression in myotubes. A Dreh overexpression plasmid was transfected into the cells. KEY FINDINGS: Metformin accumulated in C2C12 myotubes. Metformin reduced medium glucose concentration and repressed lncRNA Dreh expression in the myotubes. Knockdown of Dreh in the myotubes resulted in reduced glucose concentration in the culture medium, increased glucose transport, and increased levels of GLUT4 protein in the plasma membrane. Overexpression of Dreh attenuated the glucose-lowering effect of metformin in myotubes. SIGNIFICANCE: The glucoregulatory actions of metformin are mediated in part by a lncRNA, Dreh, in the skeletal muscle cells. Dreh is a novel regulator for glucose transport and could be a therapeutic target for diabetes.

First-Line, Non-Criterial Antiphospholipid Antibody Testing for the Diagnosis of Antiphospholipid Syndrome in Clinical Practice: A Combination of Anti-ß2 -Glycoprotein I Domain I and Anti-Phosphatidylserine/Prothrombin Complex Antibodies Tests.

OBJECTIVE: To assess the value of a combination of anti-ß2 -glycoprotein I (anti-ß2 GPI) domain I antibody and anti-phosphatidylserine/prothrombin complex (anti-PS/PT) antibody tests for the diagnosis of antiphospholipid syndrome (APS). METHODS: This cross-sectional study involved a cohort of the patients who visited our clinic from April 2005 to March 2013. Tests for anti-ß2 GPI domain I antibodies, IgG anti-PS/PT antibodies, and IgM anti-PS/PT antibodies, together with tests for criteria-defined antiphospholipid antibodies (aPL), were performed in all patients. The total antiphospholipid score (aPL-S) was calculated for each patient according to titers of and positivity for aPL. RESULTS: The study enrolled 157 patients (51 patients with APS and 106 with non-APS autoimmune diseases). All 21 patients positive for both anti-ß2 GPI domain I antibodies and IgG and/or IgM (IgG/IgM) anti-PS/PT antibodies had APS with a high total aPL-S (median 46, range 26-76), as did all of the 10 patients who were positive for anti-ß2 GPI domain I antibodies but negative for IgG/IgM anti-PS/PT antibodies (median 22, range 4-39). Of the 14 patients who were positive for IgG/IgM anti-PS/PT antibodies but negative for anti-ß2 GPI domain I antibodies, 11 (79%) had APS; these individuals also had high total aPL-S values (median 23, range 11-60). In contrast, only 9 of the 112 patients (8%) with none of these antibodies had APS. CONCLUSION: The combination of the IgG anti-ß2 GPI domain I antibody and IgG/IgM anti-PS/PT antibody tests shows a high positive predictive value for the diagnosis of APS and a strong correlation with the aPL-S. This combination as the first-line test for aPL may contribute to the simple and definite identification of APS with a high risk of thrombosis in clinical practice.

Alternative pathway activation due to low level of complement factor H in primary antiphospholipid syndrome.

INTRODUCTION: Although complement activation has been proposed as a possible thrombophilic mechanism in antiphospholipid syndrome (APS), the origin of complement activation in APS remains unclear. Here, we focused on complement regulatory factors (CRF), which control the complement system to prevent damage to host tissue. We evaluated the function of two major CRF, membrane cofactor protein (MCP) and factor H (FH), in APS patients. MATERIALS AND METHODS: In this study, we analyzed preserved serum samples from 27 patients with primary APS (PAPS), 20 with APS complicated with SLE (APS + SLE), 24 with SLE (SLE), and 25 with other connective tissue diseases (Other CTD). Serum MCP and FH levels were tested by ELISA. Autoantibodies against FH were determined by both ELISA and western-blotting. RESULTS: Serum complement levels of PAPS were lower than those of other CTD (median C3: 82 vs 112 mg/dL, p < 0.01, C4: 15 vs 22 mg/dL, p < 0.05). Serum MCP levels did not significantly differ among the groups. Serum FH levels were significantly lower in PAPS patients compared with SLE or other CTD (median 204, 1275, and 1220 µg/mL, respectively, p < 0.01). In PAPS patients, serum FH levels were positively correlated with serum C3 levels (p < 0.01, R = 0.55), but no correlation was found with serum C4 levels (p = 0.22, R = 0.33). Autoantibodies against FH were not detected in any of our patients. CONCLUSIONS: Activation of the alternative complement pathway due to low level of FH is one of the possible thrombophilic mechanisms in PAPS.