Anti-survival motor neuron complex antibodies as a novel biomarker for pulmonary arterial hypertension and interstitial lung disease in mixed connective tissue disease.

OBJECTIVE: The presence of anti-U1 RNP antibodies (Abs) is critical for diagnosing MCTD. The aim of this study is to evaluate the clinical relevance of anti-survival motor neuron (SMN) complex Abs, which often coexist with anti-U1 RNP Abs. METHODS: A total of 158 newly diagnosed consecutive cases of SLE, SSc or MCTD with anti-U1 RNP Abs were enrolled in this multicentre observational study between April 2014 and August 2022. Serum anti-SMN complex Abs were screened by immunoprecipitation of 35S-methionine-labelled cell extracts, and associations between anti-SMN complex Abs positivity and clinical characteristics were analysed. RESULTS: Anti-SMN complex Abs were detected in 36% of MCTD patients, which was significantly higher than that in SLE (8%) or SSc (12%). Among MCTD patients classified based on the combination of the clinical features of SLE, SSc and idiopathic inflammatory myopathies, anti-SMN complex Abs showed the highest prevalence in a subset with clinical features of all three components. Anti-SMN complex Abs-positive MCTD had a higher prevalence of pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD), which are related to poor prognosis, than negative patients. Moreover, all three cases of death within 1 year of the treatment were positive for anti-SMN complex Abs. CONCLUSIONS: Anti-SMN complex Abs is the first biomarker of a typical subset of MCTD which bears organ damages such as PAH and ILD.

Analytic and Clinical Validity of Myositis-Specific Antibodies by Line-Blot Immunoassay Is Essential.

OBJECTIVES: This study assessed the concordance between line blot (LB) and immunoprecipitation (IP) assays for detecting myositis-specific antibodies (MSAs) in idiopathic inflammatory myopathies (IIMs) and their association with IIM subtypes. METHODS: One hundred patients with IIM were enrolled, and MSA was detected using LB and IP. The IIM subtypes, including immune-mediated necrotizing myopathy-like, anti-tRNA synthetase syndrome-like, and clinically amyopathic dermatomyositis-like, were clinically diagnosed. The validity and reliability of the LB compared with the IP were evaluated. Optimal cutoff levels for LB were determined using various statistical methods including Cohen κ, Gwet's AC, diagnostic odds ratios, and receiver operating characteristic analysis. RESULTS: Line blot exhibited lower specificity and accuracy than IP in predicting IIM subtypes. Some MSAs performed better at higher LB cutoff values. Anti-signal recognition particle antibodies showed poor performance in predicting the immune-mediated necrotizing myopathy-like subtype using LB. Raising the cutoffs improved the reliability of anti-threonyl-tRNA synthetase and anti-signal recognition particle antibodies. Anti-histidyl-tRNA synthetase antibodies performed well at lower positivity, whereas diagnostic odds ratios increased for anti-transcription intermediary factor 1γ and anti-nuclear matrix protein 2 with higher cutoffs. CONCLUSIONS: Inconsistencies between LB and IP have been observed in patients with IIM. Individual optimal cutoffs for MSA by LB correlating with IP were determined. Rheumatologists should consider the differences between LB and IP results when classifying IIM subtypes.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Clinical significance of serum autoantibodies in patients with sarcoidosis.

BACKGROUND: The association between sarcoidosis and autoimmunity has been reported for years. However, the significance of autoantibodies in the pathophysiology and clinical management of sarcoidosis is not well understood. No autoantibodies that can be used as serologic biomarkers to diagnose the disease, monitor the state of the disease, and predict the prognosis of patients are established. METHODS: We performed a comprehensive analysis of serum autoantibodies and analyzed their associations with clinical features of sarcoidosis. RESULTS: Patients with systemic autoimmune rheumatic diseases-associated autoantibodies had a higher prevalence of advanced radiographic stage and consolidations in high-resolution computed tomography than patients without autoantibodies (p < .05). Age, sex, clinical history, pulmonary function tests, serum angiotensin-converting enzyme levels, rheumatoid factor, and the number of involved organs were not significantly different between the two groups. CONCLUSIONS: There may be an association between autoantibodies and more advanced pulmonary lesions in patients with sarcoidosis. Further investigations are needed to establish the significance of autoantibodies.

Capillary pathology with prominent basement membrane reduplication is the hallmark histopathological feature of scleromyositis.

AIMS: We aim to perform ultrastructural and histopathological analysis of muscle biopsies from a large group of systemic sclerosis (SSc) patients, including some with early/mild SSc features, and examine whether capillary pathology differentiates 'scleromyositis' (SM) from other auto-immune myositis (AIM) subsets. METHODS: Muscle biopsies from a total of 60 SM patients and 43 AIM controls from two independent cohorts were examined by electron microscopy, collagen-4 immunofluorescence (Col4IF) and routine light microscopy. RESULTS: Ultrastructural examination revealed prominent capillary basement membrane (BM) reduplication (4+ layers in >50% of capillaries) in 65% of SM vs 0% of AIM controls (p < 0.001). In SM cases without prominent BM reduplication, capillary dilation was the most distinctive feature, present in 8% of capillaries in SM vs 2% in controls (p = 0.001). Accumulation of ensheathed pericyte processes was another characteristic feature of SM and closely correlated with the degree of BM reduplication (r = 0.833, p < 0.001). On light microscopy, BM marker Col4IF revealed more frequent capillary enlargement in SM than in controls (84% vs 21%, p < 0.001). SM cases were classified as non-inflammatory myopathy (36%), non-specific myositis (33%) or immune-mediated necrotizing myopathy (31%), but despite this histopathological heterogeneity, prominent BM reduplication remained a constant finding. In the 16 SM patients with early/mild SSc features, 63% showed prominent BM reduplication. CONCLUSIONS: These results show that capillary pathology, and in particular prominent capillary BM reduplication, is the hallmark histopathological feature of SM even in patients with early/mild SSc and support the concept of SM as an organ manifestation of SSc and a distinct subset of AIM.

Autoantibody profiles delineate distinct subsets of scleromyositis.

OBJECTIVE: Scleromyositis remains incompletely characterized owing in part to its heterogeneity. The purpose of this study was to explore the role of autoantibody profiles to define subsets of scleromyositis. METHODS: Subjects with scleromyositis from a prospective cohort were divided into three groups based on autoantibody profiles: subjects with SSc-specific autoantibodies (anti-centromere, -topoisomerase 1, -RNA polymerase III, -Th/To, -fibrillarin), subjects with SSc-overlap autoantibodies (anti-PM/Scl, -U1RNP, -Ku) and subjects without SSc-related autoantibodies. Clinical features, laboratory tests and histopathological findings were retrieved and compared between groups. RESULTS: Of 42 scleromyositis subjects (79% female, mean age at diagnosis 55 years, mean disease duration 3.5 years), 8 (19%) subjects had SSc-specific autoantibodies, 14 (33%) SSc-overlap autoantibodies and 20 (48%) had no SSc-related autoantibodies. One-third had no skin involvement, a finding more frequent in the SSc-overlap subjects and those without SSc-related autoantibodies. Proximal and distal weakness was common and head drop/bent spine was found in 50% of the SSc-specific and 35% of the subjects without SSc-related autoantibodies. Of note, the group without SSc-related autoantibodies had the only cases of severe cardiac systolic dysfunction (n = 1) and scleroderma renal crisis (n = 1), as well as three out of the four cancers and three out of the four deaths. CONCLUSION: In this carefully phenotyped series of scleromyositis subjects, absence of SSc-related autoantibodies was common and associated with distinct features and poor prognosis. Future studies are needed to validate these results and possibly identify novel autoantibodies or other biomarkers associated with scleromyositis.

Autoimmune pulmonary alveolar proteinosis exacerbated by steroid therapy due to misdiagnosis as anti-aminoacyl-tRNA synthetase (ARS) antibody positive- interstitial pneumonia: a case report.

BACKGROUND: Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are myositis-specific autoantibodies that have been identified in a subset of patients with interstitial pneumonia who do not present with dermatomyositis or polymyositis. Anti-ARS antibody-positive interstitial pneumonia is commonly treated with steroids or immunosuppressive agents and is usually responsive to these therapies. Here, we present in detail a case in which respiratory failure of a patient diagnosed with anti-ARS antibody-positive interstitial pneumonia was exacerbated by treatment with steroids and immunosuppressive agents. Further examination revealed misdiagnosis of this patient and a subsequent diagnosis of autoimmune pulmonary alveolar proteinosis. CASE PRESENTATION: A 66-year-old man presented to the hospital with dyspnea on exertion, which resulted in the detection of interstitial pneumonia. Serum anti-ARS antibodies were detected; however, there were no other findings suggestive of myositis. Pulmonary alveolar proteinosis (PAP) was suspected based on the marked increase in serum KL-6 and chest computed tomography findings. The bronchoalveolar lavage revealed no milky changes in the lavage fluid. After treatment with steroids and initiation of immunosuppressive agents for anti-ARS antibody-positive interstitial pneumonia, respiratory failure and chest imaging findings showed worsening of the condition. Bronchoscopy was repeated, and milk-like alveolar lavage fluid was collected; serum anti-granulocyte macrophage colony-stimulating factor antibody was identified. Steroids and immunosuppressive agents were gradually tapered and discontinued, and the patient's condition stabilized after repeated alveolar lavage under general anesthesia. CONCLUSION: Due to similar presentation, PAP can be misdiagnosed as interstitial pneumonia. If pulmonary lesions due to interstitial pneumonia are exacerbated by immunosuppressive treatment, physicians should reconsider the diagnosis and include PAP in the differential diagnosis.

Immune recognition of lysyl-tRNA synthetase and isoleucyl-tRNA synthetase by anti-OJ antibody-positive sera.

OBJECTIVE: Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are useful for identifying a clinical subset of patients with idiopathic inflammatory myopathies (IIMs). Anti-OJ antibodies, which recognize multi-enzyme synthetase complexes including isoleucyl-tRNA synthetase (IARS) and lysyl-tRNA synthetase (KARS), are among the anti-ARS antibodies. Although testing antibodies to other ARSs have been used clinically, no validated immunoassays for detecting anti-OJ antibodies are available. We aimed to establish an anti-OJ ELISA. METHODS: Serum samples were collected from 279 patients with IIMs and 22 patients with idiopathic interstitial pneumonia. Sixty-four of the samples that had been confirmed to be negative for anti-OJ by standard immunoprecipitation were used as the negative control, and 12 anti-OJ-positive reference sera were used as the positive control. Antibodies to IARS and KARS were assayed by ELISA using biotinylated recombinant proteins generated by in vitro transcription/translation. RESULTS: The anti-OJ-positive sera strongly reacted with the KARS and IARS recombinant proteins in ELISA. Although all 12 reference sera were positive in the anti-KARS ELISA, 4 of the 64 anti-OJ-negative sera were also weakly positive. The sensitivity and the specificity were 100% and 93.8%, respectively. Since our anti-KARS ELISA performed well, showing a high agreement with the results for immunoprecipitation (Cohen's κ > 0.8), the remaining 237 samples were also tested. Thirteen anti-KARS-positive sera were newly found by ELISA, all of which were anti-OJ positive by immunoprecipitation. CONCLUSION: Immunoassays for detecting anti-OJ antibodies using KARS and IARS recombinant proteins were developed. Our ELISAs performed well, with very high agreement of the results by immunoprecipitation and can be applied to the first reliable, easy-to-use measurement assays for anti-OJ antibodies.

Presence of anti-TIF-1γ, anti-Ro52, anti-SSA/Ro60 and anti-Su/Ago2 antibodies in breast cancer: a cross-sectional study.

OBJECTIVES: The presence of myositis-specific antibodies (MSA), was recently reported in healthy individuals, cancer patients without myopathy and paraneoplastic rheumatic syndromes. We sought to analyze the frequency of MSA, myositis-associated antibodies (MAA) and autoantibodies related to systemic autoimmune rheumatic diseases (SARD) in breast cancer patients. METHODS: One hundred fifty-two breast cancer patients were enrolled in a cross-sectional study. Clinical information was collected, and autoantibodies tested by immunoprecipitation of an 35S-methionine-labeled K562 cell extract, enzyme-linked immunosorbent assay (ELISA) and Western blot when indicated. All statistical tests were performed using the software statistical package for the social science (SPSS) ver. 19.0 (IBM Inc., NYSE, USA). RESULTS: Autoantibodies associated with SARD: anti-52 kD ribonucleoprotein/tripartite motif-containing 21 (anti-Ro52/TRIM21) was found in 5.9% (9/152), anti-Sjögren syndrome-related antigen A/60 kD ribonucleoprotein antibody (anti-SSA/Ro60) in 3.9% (6/152) and anti-Su antigen/Argonaute 2 antibody (anti-Su/Ago2) in 2.6% (4/152). Meanwhile, anti-transcription intermediary factor-1γ (anti-TIF-1γ, p155/140) antibody was positive in 2 cases and anti-polymyositis/scleroderma antibody was detected in one case. As a whole, 14.47% (22/152) of breast cancer patients showed autoantibodies associated with SARD. These specific autoantibodies were not associated with the presence of rheumatic diseases except one rheumatoid arthritis patient positive for anti-Ro52/TRIM21. CONCLUSIONS: Autoantibodies to TIF-1γ were found in two patients with breast cancer without dermatomyositis (DM). More common specificities were autoantibodies anti-SSA/Ro60, anti-Ro52/TRIM21 and anti-Su/Ago2. More studies are needed in order to establish the biological meaning of the presence of SARD-associated autoantibodies in breast cancer.

Evaluation of Glomerular Hemodynamic Function by Empagliflozin in Diabetic Mice Using In Vivo Imaging.

BACKGROUND: Sodium glucose cotransporter 2 inhibitors may reduce kidney hyperfiltration, thereby preventing diabetic kidney disease progression, which may in turn reduce cardiovascular risk, including heart failure. However, the mechanisms that regulate renal function responses to sodium glucose cotransporter 2 inhibition are not yet fully understood. We explored the renal protective effects of sodium glucose cotransporter 2 inhibition with empagliflozin, with a focus on glomerular hemodynamic effects and tubuloglomerular feedback using in vivo multiphoton microscopy imaging techniques. METHODS: C57BL/6 mice and spontaneously diabetic Ins2+/Akita mice were studied. The mice were treated with empagliflozin (20 mg·kg-1·d-1) and insulin for 4 weeks, and the single-nephron glomerular filtration rate was measured using multiphoton microscope. A neuronal nitric oxide synthase inhibitor (7-nitroindazole, 20 mg·kg-1·d-1) or a cyclooxygenase-2 inhibitor (SC58236, 6 mg/L), or an A1 adenosine receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, 1 mg·kg-1·d-1) was administered to elucidate the mechanisms of tubuloglomerular feedback signaling and single-nephron glomerular filtration rate regulation. RESULTS: The urinary excretion of adenosine, nitric oxide metabolites, and the prostanoid prostaglandin E2 was also quantified. The single-nephron glomerular filtration rate in the Ins2+/Akita group was higher than in controls (C57BL/6; 4.9±1.3 nL/min versus Ins2+/Akita; 15.8±6.8 nL/min) and lower in Ins2+/Akita /empagliflozin to 8.0±3.3 nL/min (P<0.01). In vivo imaging also revealed concomitant afferent arteriolar dilation (P<0.01) and increased glomerular permeability of albumin in the Ins2+/Akita group. Empagliflozin ameliorated these changes (P<0.01). Urinary adenosine excretion in the Ins2+/Akita/empagliflozin group was higher than in Ins2+/Akita (Ins2+/Akita; 3.4±1.4 nmol/d, Ins2+/Akita/empagliflozin; 11.2±3.0 nmol/d, P<0.05), whereas nitric oxide metabolites and prostaglandin E2 did not differ. A1 adenosine receptor antagonism, but not neuronal nitric oxide synthase or cyclooxygenase-2 inhibition, blocked the effect of empagliflozin on renal function. Empagliflozin increased urinary adenosine excretion and reduced hyperfiltration via afferent arteriolar constriction, effects that were abolished by A1 adenosine receptor blockade. CONCLUSIONS: Adenosine/A1 adenosine receptor pathways play a pivotal role in the regulation of the single-nephron glomerular filtration rate via tubuloglomerular feedback mechanisms in response to sodium glucose cotransporter 2 inhibition, which may contribute to renal and cardiovascular protective effects reported in clinical trials.

Non-purine selective xanthine oxidase inhibitor ameliorates glomerular endothelial injury in InsAkita diabetic mice.

Endothelial dysfunction represents a predominant early feature of diabetes, rendering patients with diabetes prone to renal complications, e.g., proteinuria. Recent studies have indicated a possible role for xanthine oxidase (XO) in the pathogenesis of vascular dysfunctions associated with diabetes. In the present study, we investigated the contribution of XO activation on the progression of diabetic nephropathy in a mouse model using selective XO inhibitors. Male Ins2Akita heterozygous mice were used with wild-type mice as controls. Akita mice were treated with topiroxostat (Topi) or vehicle for 4 wk. Serum uric acid levels were significantly reduced in Akita + Topi mice compared with Akita + vehicle mice. The Akita + Topi group had a significant reduction in urinary albumin excretion compared with the Akita + vehicle group. Mesangial expansion, glomerular collagen type IV deposition, and glomerular endothelial injury (assessed by lectin staining and transmission electron microscopy) were considerably reduced in the Akita + topi group compared with the Akita + vehicle group. Furthermore, glomerular permeability was significantly higher in the Akita + vehicle group compared with the wild-type group. These changes were reduced with the administration of Topi. We conclude that XO inhibitors preserve glomerular endothelial functions and rescue compromised glomerular permeability, suggesting that XO activation plays a vital role in the pathogenesis of diabetic nephropathy.

Bardoxolone methyl analog attenuates proteinuria-induced tubular damage by modulating mitochondrial function.

Multiple clinical studies have shown that bardoxolone methyl, a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), is effective in increasing glomerular filtration rate in patients with chronic kidney disease. However, whether an Nrf2 activator can protect tubules from proteinuria-induced tubular damage via anti-inflammatory and antioxidative stress mechanisms is unknown. Using an Institute of Cancer Research-derived glomerulonephritis (ICGN) mouse model of nephrosis, we examined the effects of dihydro-CDDO-trifluoroethyl amide (dh404), a rodent-tolerable bardoxolone methyl analog, in protecting the tubulointerstitium; dh404 markedly suppressed tubular epithelial cell damage in the renal interstitium of ICGN mice. The tubular epithelial cells of ICGN mice showed a decrease in the size and number of mitochondria, as well as the breakdown of the crista structure, whereas the number and ultrastructure of mitochondria were maintained by the dh404 treatment. To further determine the effect of dh404 on mitochondrial function, we used human proximal tubular cells in vitro. Stimulation with albumin and free fatty acid increased mitochondrial reactive oxygen species (ROS). However, dh404 administration diminished mitochondrial ROS. Our data show that dh404 significantly reduced proteinuria-induced tubular cell mitochondrial damage, suggesting that improved redox balance and mitochondrial function and suppression of inflammation underlie the cytoprotective mechanism of Nrf2 activators, including bardoxolone methyl, in diabetic kidney disease.-Nagasu, H., Sogawa, Y., Kidokoro, K., Itano, S., Yamamoto, T., Satoh, M., Sasaki, T., Suzuki, T., Yamamoto, M., Wigley, W. C., Proksch, J. W., Meyer, C. J., Kashihara, N. Bardoxolone methyl analog attenuates proteinuria-induced tubular damage by modulating mitochondrial function.

Klotho is a novel therapeutic target in peritoneal fibrosis via Wnt signaling inhibition.

BACKGROUND: Long-term exposure to bioincompatible peritoneal dialysate causes the loss of mesothelial cells and accumulation of matrix proteins, leading to an increase in the thickness of the submesothelial layer, thereby limiting the long-term effectiveness of peritoneal dialysis (PD). However, the detailed molecular mechanisms underlying the process of peritoneal fibrosis have not been clearly elucidated. Wnt/ß-catenin signaling pathway activation has been suggested to play a pivotal role in the development of organ fibrosis. Moreover, Klotho protein can regulate Wnt/ß-catenin signaling. We examined the role of Klotho protein in reducing peritoneal fibrosis by inhibiting Wnt/ß-catenin signaling. METHODS: The ß-catenin-activated transgenic (BAT) driving expression of nuclear ß-galactosidase reporter transgenic (BAT-LacZ) mice, the alpha-Klotho gene under control of human elongation factor 1 alpha promoter [Klotho transgenic (KLTG) and C57BL/6 background] and C57BL/6 mice [wild-type (WT)] were used. The mice received daily intraperitoneal (i.p.) injections of 4.25% glucose with lactate (PD solution) or saline as a control for 4 weeks. Other mice received daily i.p. injections of the same volume of saline (normal control). RESULTS: After exposure to PD, Wnt signal activation was observed on the peritoneal mesothelial cells in WT-PD mice. The peritoneal fibrosis was also accelerated in WT-PD mice. The protein expression of ß-catenin and Wnt-inducible genes were also remarkably increased in WT-PD mice. On the other hand, KLTG-PD mice attenuated activation of Wnt/ß-catenin signaling after exposure to PD and ameliorated the progression of peritoneal fibrosis. CONCLUSIONS: Overexpression of Klotho protein protects the peritoneal membrane through attenuation of the Wnt/ß-catenin signaling pathway. The availability of recombinant Klotho protein would provide a novel potential therapeutic target in peritoneal fibrosis.

Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein.

Objectives: Reference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA). Methods: Reference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation-mass spectrometry (IP-MS). Results: MND-REF stained 6-20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS. Conclusions: MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells is widely used for detection of antinuclear antibodies (ANA). The dichotomous outcome, negative or positive, is integrated in diagnostic and classification criteria for several systemic autoimmune diseases. However, the HEp-2 IIFA test has much more to offer: besides the titre or fluorescence intensity, it also provides fluorescence pattern(s). The latter include the nucleus and the cytoplasm of interphase cells as well as patterns associated with mitotic cells. The International Consensus on ANA Patterns (ICAP) initiative has previously reached consensus on the nomenclature and definitions of HEp-2 IIFA patterns. In the current paper, the ICAP consensus is presented on the clinical relevance of the 29 distinct HEp-2 IIFA patterns. This clinical relevance is primarily defined within the context of the suspected disease and includes recommendations for follow-up testing. The discussion includes how this information may benefit the clinicians in daily practice and how the knowledge can be used to further improve diagnostic and classification criteria.

Autoantibodies to a novel Rpp38 (Th/To) derived B-cell epitope are specific for systemic sclerosis and associate with a distinct clinical phenotype.

OBJECTIVE: Detection of antinuclear antibodies and specific autoantibodies is important in the diagnosis and classification of SSc. Several proteins of the Th/To complex, including Rpp25, Rpp38 and hPop1 are the target of autoantibodies in SSc patients. However, very little is known about the epitope distribution of this autoantigen. Consequently, we screened Rpp25, Rpp38 and hPop1 for B cell epitopes and evaluated their clinical relevance. METHODS: Serum pools with (n = 2) and without (n = 1) anti-Th/To autoantibodies were generated and used for epitope discovery. Identified biomarker candidate sequences were then utilized to synthesize synthetic, biotinylated, soluble peptides. The peptides were tested to determine reactivity with sera from SSc cohorts (n = 202) and controls (n = 159) using a chemiluminescence immunoassay. Additionally, samples were also tested for antibodies to full-length recombinant Rpp25 antibodies by chemiluminescence immunoassay. RESULTS: Several immunodominant regions were found on the three proteins. The strongest reactivity was observed with an Rpp38 peptide (aa 229-243). Autoantibodies to the Rpp38 peptide were detected in 8/149 (5.4%) limited cutaneous SSc patients, but not in any of 159 controls (P = 0.003 by two-sided Fisher's exact probability test). Although reactivity to the novel antigenic peptide was correlated with the binding to Rpp25 (rho = 0.44; P < 0.0001), subsets of patient sera either reacted strongly with Rpp25 or with the novel Rpp38-derived peptide. CONCLUSION: A novel Rpp38 epitope holds promise to increase the sensitivity in the detection of anti-Th/To autoantibodies, thus enhancing the serological diagnosis of SSc.

Significance of nailfold videocapillaroscopy in patients with idiopathic inflammatory myopathies.

Objective: The aim of this study was to investigate the clinical and immunological significance of nailfold videocapillaroscopy (NVC) abnormalities in patients with idiopathic inflammatory myopathies (IIMs). Methods: Seventy consecutive Japanese patients with untreated IIMs, enrolled between April 2014 and August 2017, were prospectively studied. Clinical features, NVC findings, autoantibody profile by immunoprecipitation and ELISA, and histopathological findings of skin biopsies of DM rash were assessed at baseline and after 1-year of immunosuppressive therapy. Results: NVC abnormalities were found in 55.7% (39/70) of IIM patients, with significantly higher prevalence in DM (65.4%) compared with PM (27.8%) (P = 0.01). In subsets of patients classified by autoantibody specificities, the prevalence of NVC abnormalities was significantly higher in patients with anti-MDA5 (87.5%) and anti-transcriptional intermediary factor 1γ (88.9%) vs anti-aminoacyl-tRNA synthetase (26.9%, P < 0.001). Perivascular lymphocytic infiltration in the upper dermis of skin rash biopsy of DM was more severe in patients with NVC abnormalities (P < 0.05). Unexpectedly, NVC abnormalities disappeared in 75% of IIM patients after 1-year of immunosuppressive therapy in contrast to stable NVC changes seen in scleroderma patients. Conclusion: Nailfold microvascular abnormalities were common in DM patients, associated with anti-MDA5 and transcriptional intermediary factor 1γ antibodies, and perivascular inflammation in skin histology. NVC abnormalities in IIMs may become clinically useful markers for defining subsets of DM and understanding the pathogenesis of the clinical features seen in these patients.

Distinctive association of peripheral immune cell phenotypes with capillaroscopic microvascular patterns in systemic sclerosis.

OBJECTIVE: The pathological changes in SSc include immune system dysregulation and microvascular damage. However, the association of immune cell phenotype heterogeneity and microvascular abnormalities is unclear. The aim of this study is to elucidate this association in SSc. METHODS: Peripheral blood mononuclear cells obtained from 150 SSc patients were used for comprehensive flow cytometric analysis based on the Human Immunology Project. Hierarchical cluster analysis was used to classify SSc patients into subgroups and their association with microvascular abnormalities, as assessed by nailfold videocapillaroscopy (i.e. 'early', 'active' and 'late' patterns), was analysed. RESULTS: The proportions of activated CD4+ T cells, T cells re-expressing CD45RA, activated Th1 and Th17 cells and IgD-CD27- B cells were higher in SSc patients than in healthy individuals. Hierarchical cluster analysis stratified SSc patients into three groups: patients with few immune abnormalities (fewer abnormalities group), patients with high proportions of activated T and Treg cells (Treg-dominant group) and patients with high proportions of Tfh and plasmablasts (Tfh-dominant group). Age and disease duration were comparable among the groups. On the other hand, microvascular abnormalities, especially the 'late' nailfold videocapillaroscopy pattern, correlated with internal organ involvement. Among the groups stratified according to immune cell phenotype, the progression to the 'late' nailfold videocapillaroscopy pattern was more frequent in the Tfh-dominant group. CONCLUSION: Our study confirmed the presence of immunophenotypic abnormalities in SSc. Immunological abnormalities were not uniform but rather limited to subpopulations, particularly the Tfh-dominant group, where they were highly associated with microvascular abnormalities and organ involvement.

Establishment of an international autoantibody reference standard for human anti-DFS70 antibodies: proof-of-concept study for a novel Megapool strategy by pooling individual specific sera.

Background International autoantibody standards, traditionally based on material obtained from plasmapheresis of single subjects, represent individual immune response and may not comprehend the heterogeneity of the general population. The anti-DFS70 autoantibody yields a characteristic dense fine speckled (DFS) nuclear pattern on indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and speaks against autoimmunity. We propose a novel strategy for developing autoantibody reference standards, based on stepwise pooling of serum samples from hundreds of individuals with anti-DFS70 antibodies. Methods Within a 2-year period, serum samples were selected from routine HEp-2 IFA according to the following criteria: DFS HEp-2 IFA pattern at titer ≥1:640; anti-DFS70 reactivity in three analyte-specific tests (Western blot [WB], enzyme-linked immunosorbent assay [ELISA] and chemiluminescent immunoassay [CLIA]). Aliquots of individual samples were combined into progressively larger pools with stepwise validation of intermediary pools as for individual samples. Validated intermediary pools were merged into a final pool for lyophilization. Results A total of 741 validated samples yielded a 750 mL final pool that was lyophilized into thousands of 200 µL-aliquots. Reconstituted aliquots yielded the expected anti-DFS70 reactivity in ELISA, CLIA and WB, as well as high-titer DFS HEp-2 IFA pattern. The appropriate anti-DFS70 reactivity of the lyophilized pool was confirmed by seven international expert centers, using HEp-2 IFA, ELISA, WB and immunoprecipitation. Conclusions This proof-of-concept study provides an innovative and efficient strategy to build serum reference standards for autoantibody testing. The anti-DFS70 standard will integrate the panel of standards of Autoantibody Standardization Committee (ASC, www.autoab.org), contributing to education for proper assay validation and interpretation of the DFS pattern and other HEp-2 IFA patterns.

5-Aminolevulinic acid exerts renoprotective effect via Nrf2 activation in murine rhabdomyolysis-induced acute kidney injury.

AIM: Acute kidney injury (AKI) is associated with chronic kidney disease, as well as high mortality, but effective treatments for AKI are still lacking. A recent study reported the prevention of renal injury, such as ischemia-reperfusion injury, by 5-aminolevulinic acid (ALA), which induces an antioxidant effect. The current study aimed to investigate the effect of ALA in a rhabdomyolysis-induced mouse model of AKI created by intramuscular injection of 50% glycerol. METHODS: Rhabdomyolysis-induced AKI was induced by an intramuscular injection of glycerol (5 mL/kg body weight) into mice. Administration of ALA (30 mg/kg, by gavage) was started from 48 h before or 24 h after glycerol injection. The mice were sacrificed at 72 h after glycerol injection. The roles of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), which is one of the Nrf2-related antioxidants, were further investigated through in vivo and in vitro methods. RESULTS: 5-aminolevulinic acid markedly reduced renal dysfunction and tubular damage in mice with rhabdomyolysis-induced AKI. ALA administration decreased oxidative stress, macrophage infiltration, and inflammatory cytokines and apoptosis. The expression of Nrf2 was upregulated by ALA administration. However, administration of Zinc protoporphyrin-9 (ZnPPIX) to inhibit HO-1 activity did not abolish these improvements by ALA. The expression of Nrf2-associated antioxidant factors other than HO-1 was also increased. CONCLUSION: These findings indicate that ALA exerts its antioxidant activity via Nrf2-associated antioxidant factors to provide a renoprotective effect against rhabdomyolysis-induced AKI.