Humoral and cellular response after BNT162b2 vaccine booster in hemodialysis patients and kidney transplant recipients.

BACKGROUND: Vulnerable populations, such as hemodialysis (HD) patients and kidney transplant (RTx) recipients, have priority for anti-COVID-19 vaccination, because of their impaired immune status. Here, we investigated the immune response after vaccination with BNT162b2 (two doses plus booster) in HD and RTx patients. METHODS: A prospective, observational study was started in two homogeneous groups of 55 HD and 51 RTx patients previously matched from a cohort of 336 patients. Anti-RBD IgG levels, assayed after the second dose with BNT162b2 mRNA, were used to stratify subjects into quintiles. After the second dose and after booster, anti-RBD and IGRA test were evaluated in RTx and HD, belonging to the first and fifth quintiles. RESULTS: After the second dose of vaccine, the median circulating levels of anti-RBD IgG were significantly higher in HD (1456 AU/mL) compared to RTx (27.30 AU/mL). IGRA test showed significantly higher values in the HD (382 mIU/mL) compared with the RTx (73 mIU/mL). After the booster, humoral response increased significantly in both HD (p = 0.0002) and RTx groups (p = 0.009), whereas the T-cellular immunity remained essentially stable in most patients. In RTx patients with a low humoral response after the second dose, the third dose did not significantly strengthen either humoral or cellular immunity. CONCLUSIONS: For HD and RTx, there is great variability in the humoral response to anti-COVID-19 vaccination, with a stronger response in the HD group. The booster dose was ineffective at reinforcing the humoral and cellular immune response in most RTx patients hyporesponsive to the second dose.

Increased concentration of soluble leukocyte-associated immunoglobulin-like receptor in sera from patients with blistering diseases: possible pathophysiological implications?

BACKGROUND: Autoimmune blistering diseases (AIBD), are a heterogeneous group. Despite their pathogenesis is not completely understood, autoantibodies against directed adhesion molecules of the skin and adjacent mucous membranes could play a key role. The leukocyte-associated-Ig-like-receptor (LAIR) family is a small group of immunoreceptor-tyrosine-based-inhibition-motif-containing inhibitory receptors, recognizing collagens. LAIR-1 is a transmembrane glycoprotein expressed on human-peripheral-blood-leukocytes. LAIR-2 is a secreted receptor mainly produced by CD4+ T-lymphocytes, and is able to regulate the inhibitory potential of LAIR-1. Both LAIRs have been associated with several autoimmune diseases and inflammatory responses. METHODS: We evaluated circulating LAIRs in patients with different blistering skin diseases by ELISA. RESULTS: A significant increase of serum LAIR-2, and to a lesser extent of sLAIR-1 (with the exception of Pemphigus vulgaris), in the whole group of patients with bullous diseases, irrespective of the pathogenesis, compared to healthy controls was evident. CONCLUSIONS: Although the pathophysiological meaning of LAIR is not completely elucidated, the presence of increased concentration of LAIR proteins can somehow modulate the cascade of inflammatory phenomenon occurring in bullous skin diseases, in different way depending upon specific skin disease considered.

Multiparametric autoantibody analysis: a new paradigm for the diagnosis of connective tissue diseases.

BACKGROUND: In patients affected by connective tissue diseases (CTDs), the identification of wide autoantibody profiles may prove useful in early diagnosis, in the evaluation of prognosis (risk stratification), and in predicting response to therapy. The aim of the present study was to evaluate the utility of multiparametric autoantibody analysis performed by a new fully automated particle-based multi-analyte technology (PMAT) digital system in a large multicenter cohort of CTD patients and controls. METHODS: Serum samples from 787 patients with CTD (166 systemic lupus erythematosus; 133 systemic sclerosis; 279 Sjögren's syndrome; 106 idiopathic inflammatory myopathies; 103 undifferentiated CTD), 339 patients with other disorders (disease controls) (118 infectious diseases, 110 organ-specific autoimmune diseases, 111 other rheumatic diseases), and 121 healthy subjects were collected in 13 rheumatologic centers of the FIRMA group. Sera were analyzed with the Aptiva-PMAT instrument (Inova Diagnostics) for a panel of 29 autoantibodies. RESULTS: Multiparametric logistic regression showed that enlarged antibody profiles have a higher diagnostic efficiency than that of individual antibodies or of antibodies that constitute classification criteria for a given disease and that probability of disease increases with multiple positive autoantibodies. CONCLUSIONS: This is the first study that analyzes the clinical and diagnostic impact of autoantibody profiling in CTD. The results obtained with the new Aptiva-PMAT method may open interesting perspectives in the diagnosis and sub-classification of patients with autoimmune rheumatic diseases.

Comparison of current methods for anti-dsDNA antibody detection and reshaping diagnostic strategies.

Anti-double-stranded DNA antibodies (anti-dsDNA) are considered a specific marker for systemic lupus erythematosus (SLE). Though the Farr technique was once the reference method for their detection, it has been almost entirely replaced by more recently developed assays. However, there is still no solid evidence of the commutability of these methods in terms of diagnostic accuracy and their correlation with the Crithidia luciliae immunofluorescence test (CLIFT). Anti-dsDNA antibody levels were measured in 80 subjects: 24 patients with SLE, 36 disease controls drawn from different autoimmune rheumatic diseases (14 systemic sclerosis, 10 Sjögren's syndrome, nine autoimmune myositis, three mixed connective tissue disease), 10 inflammatory arthritis and 10 apparently healthy blood donors by eight different methods: fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay (two assays), multiplex flow immunoassay, particle multi-analyte technology immunoassay and two CLIFT. At the recommended manufacturer cut-off, the sensitivity varied from 67% to 92%, while the specificity ranged from 84% to 98%. Positive agreement among CLIFT and the other assays was higher than negative agreement. Mean agreement among methods assessed by the Cohen's kappa was 0.715, ranging from moderate (0.588) to almost perfect (0.888). Evaluation of the concordance among quantitative values by regression analysis showed a poor correlation index (mean r2, 0.66). The present study shows that current technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high intermethod variability, harmonization and commutability of anti-dsDNA antibody testing remains an unachieved goal.

Current technologies for anti-ENA antibody detection: State-of-the-art of diagnostic immunoassays.

BACKGROUND: Autoantibodies against extractable nuclear antigens (ENA) play a pivotal role in the diagnosis and classification of systemic autoimmune rheumatic diseases (SARD). In recent years, newly developed methods have enabled the simultaneous and quantitative detection of multiple anti-ENA reactivities. However, data regarding the comparability of results obtained using different technologies across different platforms are scarce. In this study we compared eight different immunoassays, commonly used in current laboratory practice for detection of anti-ENA antibodies. METHODS: Sixty patients suffering from different SARD, 10 inflammatory arthritis patients (disease controls) and 10 healthy blood donors were included in this comparative study. Sera were collected in 15 centers belonging to the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine. We evaluated the analytical sensitivity, specificity and diagnostic accuracy of each method for antibodies to Sm, RNP, Ro60, Ro52, Scl70, CENP-B and Jo1. Cohen's kappa was used to analyze the agreement among methods. RESULTS: Average agreement among methods was 0.82, ranging from substantial (k = 0.72) to almost perfect (k = 0.92). However, while the specificity was very good for all methods, some differences emerged regarding the analytical sensitivity. CONCLUSIONS: Diagnostic performance of current technologies for anti-ENA antibody detection showed good comparability. However, as some differences exist among methods, laboratory scientists and clinicians must be aware of the diagnostic accuracy of the testing method in use.

An Italian Multicenter Study on Anti-NXP2 Antibodies: Clinical and Serological Associations.

The identification of anti-NXP2 antibodies is considered a serological marker of dermatomyositis (DM), with calcinosis, severe myositis and, in some reports, with cancer. Historically, these associations with anti-NXP2 antibodies have been detected by immunoprecipitation (IP), but in the last few years commercial immunoblotting assays have been released. The aim of this collaborative project was to analyse the clinical features associated to anti-NXP2 antibodies, both with commercial line blot (LB) and IP. Myositis-specific and myositis-associated autoantibodies were detected in single centres by commercial line blot (LB); available sera were evaluated in a single centre by protein and RNA immunoprecipitation (IP), and IP-Western blot. Sixty patients anti-NXP2+ (NXP2+) positive by LB were compared with 211 patients anti-NXP2 negative with idiopathic inflammatory myositis (IIM). NXP2+ showed a younger age at IIM onset (p = 0.0014), more frequent diagnosis of dermatomyositis (p = 0.026) and inclusion-body myositis (p = 0.009), and lower rate of anti-synthetase syndrome (p < 0.0001). As for clinical features, NXP2+ more frequently develop specific skin manifestations and less frequently features related with overlap myositis and anti-synthetase syndrome. IP confirmed NXP2 positivity in 31 of 52 available sera (62%). Most clinical associations were confirmed comparing NXP2 LB+/IP+ versus NXP2-negative myositis, with the following exceptions: inclusion-body myositis diagnosis was not detected, whilst dysphagia and myositis were found more frequently in NXP2 LB+/IP+ patients. The 21 LB+ /IP-myositis patients did not show differences in clinical features when compared with the NXP2-myositis patients and more frequently displayed multiple positivity at LB. Risk of developing cancer-associated myositis was similar between NXP2-positive and NXP2-negative myositis patients, either when detected by LB or IP. Protein-IP confirmed NXP2 antibodies in nearly 60% of sera positive for the same specificity with commercial assay. Double-positive cases rarely occurred in myositis patients with a clinical diagnosis other than dermatomyositis. Patients only positive by LB (LB+/IP-) did not display clinical features typical of NXP2. NXP2 positivity by LB should be confirmed by other methods in order to correctly diagnose and characterize patients affected by idiopathic inflammatory myositis.

Perivascular and endomysial macrophages expressing VEGF and CXCL12 promote angiogenesis in anti-HMGCR immune-mediated necrotizing myopathy.

OBJECTIVES: To study the phenotype of macrophage infiltrates and their role in angiogenesis in different idiopathic inflammatory myopathies (IIMs). METHODS: The density and distribution of the subpopulations of macrophages subsets (M1, inducible nitric oxide+, CD11c+; M2, arginase-1+), endomysial capillaries (CD31+, FLK1+), degenerating (C5b-9+) and regenerating (NCAM+) myofibres were investigated by immunohistochemistry in human muscle samples of diagnostic biopsies from a large cohort of untreated patients (n: 81) suffering from anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (anti-HMGCR)+ immune mediated necrotizing myopathy (IMNM), anti-signal recognition particle (anti-SRP)+ IMNM, seronegative IMNM, DM, PM, PM with mitochondrial pathology, sporadic IBM, scleromyositis, and anti-synthetase syndrome. The samples were compared with mitochondrial myopathy and control muscle samples. RESULTS: Compared with the other IIMs and controls, endomysial capillary density (CD) was higher in anti-HMGCR+ IMNM, where M1 and M2 macrophages, detected by confocal microscopy, infiltrated perivascular endomysium and expressed angiogenic molecules such as VEGF-A and CXCL12. These angiogenic macrophages were preferentially associated with CD31+ FLK1+ microvessels in anti-HMGCR+ IMNM. The VEGF-A+ M2 macrophage density was significantly correlated with CD (rS: 0.98; P: 0.0004). Western blot analyses revealed increased expression levels of VEGF-A, FLK1, HIF-1α and CXCL12 in anti-HMGCR+ IMNM. CD and expression levels of these angiogenic molecules were not increased in anti-SRP+ and seronegative IMNM, offering additional, useful information for differential diagnosis among these IIM subtypes. CONCLUSION: Our findings suggest that in IIMs, infiltrating macrophages and microvascular cells interactions play a pivotal role in coordinating myogenesis and angiogenesis. This reciprocal crosstalk seems to distinguish anti-HMGCR associated IMNM.

An Alternative "Trojan Horse" Hypothesis for COVID-19: Immune Deficiency of IL-10 and SARS-CoV-2 Biology.

The coronavirus disease 2019 (COVID-19) pandemic has been a challenge for emergency care units worldwide due to the large numbers of patients, the scarcity of information, the medical resources, and the uncertainty regarding the disease's etiology and pathogenesis. The transmission of the virus and a probable post-pandemic of SARS-CoV-2 will depend on how deep this disease, the duration of immunity and the degree of cross immunity between SARS-CoV-2 and other pathogens either bacteria or fungi can be understood. Most mortalities have been related to an atypical pneumonia consisted of a sudden worsening of general condition of the admitted positive COVID-19 patients. The severe thromboembolism, often characterized by violent pulmonary and systemic complications, have been described with a blend of inflammatory-infectious patterns that rapidly shifted into a typical systemic inflammatory response syndrome (SIRS) or into an acute respiratory distress syndrome (ARDS) that eventually concluded into a multi-organ failure (MOF) and death. The fatality rate reported in our Covid-19 structure, SG Moscati Hospital of Taranto province in Italy, was higher in elderly male people with preexisting chronic pulmonary disease (COPD), patients with cancer and preexisting cardio-vascular diseases (CVD). We assumed a different theoretical position to clarify the higher mortality seen among those patients that was not as obvious as it appeared, we thus offered different pathophysiological picture that could help to recent solutions in therapy and prevention.

ELISA detection of anti-desmoglein 1 and anti-desmoglein 3 and indirect immunofluorescence in oral pemphigus: A retrospective study.

OBJECTIVE: The aim of this study was to test the efficacy of autoantibodies to desmoglein 1 and desmoglein 3 detected by ELISA and indirect immunofluorescence in the diagnosis of oral pemphigus and to correlate the antibody titres with the severity of the disease. MATERIALS AND METHODS: We report a retrospective cohort study of 22 patients with oral pemphigus and 64 controls from a single tertiary centre. Data about histopathological examination, direct immunofluorescence, indirect immunofluorescence and ELISA were analysed. Global validation of ELISA and IIF both alone and combined was established by calculating sensitivity, specificity, accuracy and both positive predictive value and negative predictive value. The relationship between Oral Disease Severity Score values and ELISA titres was analysed using Pearson's coefficient. RESULTS: The best diagnostic performance was observed for anti-desmoglein 3 ELISA. The sensitivity was 75% and specificity 100% and positive predictive value and negative predictive value were 92.5% and accuracy 93.9%. The level of agreement with histopathology + direct immunofluorescence was substantial (k = .758). Anti-desmoglein 3 titres showed a significant correlation with Oral Disease Severity Score (p < .05). CONCLUSIONS: Serological tests are commonly employed during clinical practice as adjunctive tools. Anti-desmoglein 3 ELISA should be considered as a first-instance diagnostic test for oral pemphigus early detection.

Diagnosing and Monitoring Celiac Patients with Selective IgA Deficiency: Still an Open Issue.

Although, the association between celiac disease (CD) and selective immunoglobulin A deficiency (SIgAD) has been known for more than fifty years, the procedures for diagnosing and monitoring patients with both conditions are still far from definitive. When serological markers were introduced as pre-bioptic investigations, it was immediately clear that searching for specific IgA antibodies without checking total serum IgA could lead to a failure in diagnosing IgA-deficient CD patients, while specific IgG antibodies could be useful as additional tests, because they are frequently found in the serum of affected patients. Nonetheless, until recently the diagnosis of CD in IgA-deficient patients was based on the few, fragmentary and often contradictory data available in literature. The introduction of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines in 2012 provided the current criteria for diagnosing CD in IgA-deficient patients, although some issues remained open, such as the selection of patients who should undergo specific IgG antibody testing and the choice of the most reliable IgG-based test for both diagnosis and follow-up. A real-life study recently assessed the impact of the 2012 ESPGHAN guidelines in diagnosing and monitoring CD in SIgAD patients, highlighting several pitfalls that can lead to operational uncertainties and difficulties in patient management. In the present report, the evolution of diagnostic tools and criteria for CD in SIgAD patients has been critically assessed, both strengths and open issues have been highlighted, and future perspectives for improving the current diagnostic protocols have been suggested.

Cytogenetic and Array-CGH Characterization of a Simple Case of Reciprocal t(3;10) Translocation Reveals a Hidden Deletion at 5q12.

Chromosome deletions, including band 5q12, have rarely been reported and have been associated with a wide range of clinical manifestations, such as postnatal growth retardation, intellectual disability, hyperactivity, nonspecific ocular defects, facial dysmorphism, and epilepsy. In this study, we describe for the first time a child with growth retardation in which we identified a balanced t(3;10) translocation by conventional cytogenetic analysis in addition to an 8.6 Mb 5q12 deletion through array-CGH. Our results show that the phenotypic abnormalities of a case that had been interpreted as "balanced" by conventional cytogenetics are mainly due to a cryptic deletion, highlighting the need for molecular investigation in subjects with an abnormal phenotype before assuming the cause is an apparently simple cytogenetic rearrangement. Finally, we identify PDE4D and PIK3R1 genes as the two major candidates responsible for the clinical features expressed in our patient.

Diagnostic accuracy of anti-phospholipase A2 receptor (PLA2R) antibodies in idiopathic membranous nephropathy: an Italian experience.

BACKGROUND: Autoantibodies against-phospholipase A2 receptor (PLA2R) are specific markers of idiopathic membranous nephropathy (iMN). Enzyme-linked immunosorbent assay (ELISA) is becoming the preferred method in many laboratories for the determination of anti-PLA2R antibodies, because it provides quantitative results, and is not prone to subjective interpretation, as is the case with indirect immunofluorescence assay. METHODS: The purpose of our study was to determine the diagnostic performance of serum PLA2R antibodies detected by commercially available ELISA in a large Italian multicenter cohort of patients with biopsy-proven iMN and in patients with other renal diseases, with special focus on evaluating the optimal cut-off value to discriminate positive and negative results. A total of 495 consecutive patients were recruited. Renal biopsies were performed in all patients, and blood samples were taken before the initiation of immunosuppressive treatment. RESULTS: According to the clinical diagnosis and to kidney biopsy, 126 patients were diagnosed with iMN and 369 had other non-membranous nephropathies. Anti-PLA2R autoantibodies were detected using a commercial anti-PLA2R ELISA. At a cut-off value of 20 relative units (RU)/ml indicated by the manufacturer for positive classification, sensitivity was 61.1% and specificity 99.7%. At a cut-off value of 14 RU/ml indicated by the manufacturer for borderline results, sensitivity was 63.5% and specificity remained the same (99.7%). At a cut-off of 2.7 RU/ml, selected as the optimal cut-off on the basis of ROC curve analysis, sensitivity was 83.3% and specificity 95.1%. The best overall efficiency of the test was observed at 2.7 RU/ml; however, the highest positive likelihood ratio and diagnostic odds ratio were achieved at 14 RU/ml. A cut-off threshold higher than 14 RU/ml or lower than 2.7 RU/ml entailed worse test performance. CONCLUSION: Depending on the clinical use (early diagnosis or as a support to confirm clinical diagnosis), nephrologists may take advantage of this evidence by choosing the most convenient cut-off. However, renal biopsy remains mandatory for the definitive diagnosis of iMN and for the assessment of disease severity.

Evaluation of BP180-NC16A ELISA in exclusive oral pemphigoid diagnosis. A comparative study.

OBJECTIVE: Aims of this study were to test the efficacy of anti-BP180-NC160 ELISA in the diagnosis of oral pemphigoid compared to the gold standard, represented by direct immunofluorescence and pathological examination, to correlate the antibody titers with the severity of the disease and the demographical data. MATERIALS AND METHODS: Patients with a suspect of oral pemphigoid were enrolled and underwent biopsy and sera collection both, in order to perform histopathological examination, direct immunofluorescence and ELISA. The test outcomes were compared, and ELISA sensitivity, specificity, accuracy, and negative and positive predictive values were calculated. RESULTS: ELISA showed good specificity (83.3%), while sensitivity was only 50%. A moderate correlation between antibody titers and disease severity was recorded. CONCLUSIONS: Mucomembranous Pemphigoid is an autoimmune autoantibody-mediated blistering disease, often affecting exclusively the oral mucosa. Currently, the biopsy is required to diagnose this disease, but serological tests are also commonly employed during clinical practice as adjunctive tools. BP180-NC160 ELISA should be considered an ancillary diagnostic test in course of oral pemphigoid; direct immunofluorescence + histologic examination remains the diagnostic gold standard.

Long-term efficacy of adding intravenous immunoglobulins as treatment of refractory dysphagia related to myositis: a retrospective analysis.

OBJECTIVE: Dysphagia is a life-threating manifestation of idiopathic inflammatory myopathies (IIM). However, we lack a univocal protocol for its treatment. The aim of this retrospective analysis was to evaluate the effectiveness of a step-up strategy by adding a 1-day pulse of IVIGs to immunosuppressants in IIM patients with refractory dysphagia diagnosed by Eating Assessment Tool (EAT)-10 and fibreoptic endoscopic evaluation of swallowing (FEES). METHODS: Dysphagia was defined as a pharyngo-oesophageal disturbance associated with EAT-10 score ≥3 and at least one FEES abnormality among propulsion failure, solid or liquid stasis. Eighteen out of 154 IIM patients had FEES-confirmed dysphagia and underwent 1 day IVIG 2 g/kg repeated 1 month apart for 3 months, because of dysphagia refractory to high-dose glucocorticoids with methotrexate and/or azathioprine. Clinical characteristics along with myositis-specific antibodies and muscle histopathological findings were studied in FEES-dysphagia IIM and IIM control patients. RESULTS: After three monthly doses of IVIG, EAT-10 score dropped with complete recover of defective propulsion and progressive decrease in percentage of both solid and liquid stasis. At 52-weeks' follow-up, reached in 12 patients, all these parameters were stable or further improved. An improvement in manual muscle strength test and a steroid-sparing effect of IVIG were also observed. Anti-PM/Scl 75/100 antibodies were much more frequent in the FEES-dysphagia group, while anti-Jo1 antibody was rarely detected. CONCLUSION: Our treatment schedule with 2 g/kg IVIG was effective for IIM-associated refractory dysphagia assessed by the combination of EAT-10 and FEES. These findings need to be prospectively tested in a larger cohort of IIM patients.

Severe muscle damage with myofiber necrosis and macrophage infiltrates characterize anti-Mi2 positive dermatomyositis.

OBJECTIVE: The aim of our study was to investigate clinical and histopathological findings in adult DM patients positive for anti-Mi2 (anti-Mi2+) antibodies compared with DM patients negative for anti-Mi2 (anti-Mi2-). METHODS: Clinical data of adult DM patients, who fulfilled EULAR/ACR 2017 classification criteria, were gathered from electronic medical records of three tertiary Rheumatology Units. Histopathological study was carried out on 12 anti-Mi2+ and 14 anti-Mi2- muscle biopsies performed for diagnostic purpose. Nine biopsies from immune mediated necrotizing myopathy (IMNM) patients were used as control group. RESULTS: Twenty-two anti-Mi2+ DM [90.9% female, mean age 56.5 (15.7) years] were compared with 69 anti-Mi2- DM patients [71% female, mean age 52.4 (17) years]. Anti-Mi2+ patients presented higher levels of serum muscle enzymes than anti-Mi2- patients [median (IQR) creatine-kinase fold increment: 16 (7-37)vs 3.5 (1-9.9), P <0.001] before treatment initiation. Moreover, a trend towards less pulmonary involvement was detected in anti-Mi2+ DM (9.1% vs 30.4%, P =0.05), without any case of rapidly progressive interstitial lung disease. At muscle histology, anti-Mi2+ patients showed more necrotic/degenerative fibres than anti-Mi2- patients [mean 5.3% (5) vs 0.8% (1), P <0.01], but similar to IMNM [5.9% (6), P >0.05]. In addition, the endomysial macrophage score was similar between anti-Mi2+ and IMNM patients [mean 1.2 (0.9) vs 1.3 (0.5), P >0.05], whereas lower macrophage infiltration was found in anti-Mi2- DM [mean 0.4 (0.5), <0.01]. CONCLUSIONS: Anti-Mi2+ patients represent a specific DM subset with high muscle damage. Histological hallmarks were a higher prevalence of myofiber necrosis, endomysial involvement and macrophage infiltrates at muscle biopsy.

A new diagnostic algorithm for pattern-oriented autoantibody testing according to the ICAP nomenclature: A pilot study.

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart. 710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens. ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern. This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed.

Anti-DFS70 antibodies detected by specific methods in patients with thrombosis or recurrent pregnancy loss: no evidence of an association.

A dense fine speckled pattern (DFS) caused by antibodies to the DFS70 kDa nuclear protein is a relatively common finding while testing for anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells. However, despite many efforts and numerous studies, the clinical significance of anti-DFS70 antibodies is still unknown as they can be found in patients with various disorders and even in healthy subjects. In this study we aimed at verifying whether these antibodies are associated with thrombotic events or with unexplained recurrent pregnancy loss (RPL). We studied 443 patients with venous or arterial thrombosis or RPL and 244 controls by IIF on HEp-2 cells and by a DFS70-specific chemiluminescent immunoassay (CIA). The DFS pattern was observed in IIF in 31/443 (7.0%) patients and in 6/244 (2.5%) controls (p = 0.01) while anti-DFS70 specific antibodies were detected by CIA in 11 (2.5%) patients and in one (0.4%) control (p = 0.06). Positive samples, either by IIF or by CIA, were then assayed by a second DFS70-specific line-immunoassay (LIA) method: 83.3% of the CIA positive samples were confirmed DFS70 positive versus only 29.7% of the IIF positive samples. These findings show that IIF overestimates anti-DFS70 antibody frequency and that results obtained by specific CIA and LIA assays do not indicate that venous or arterial thrombosis or RPL are linked to a higher prevalence of anti-DFS70 antibodies.

Autophagy markers LC3 and p62 accumulate in immune-mediated necrotizing myopathy.

INTRODUCTION: The molecular mechanism of immune-mediated necrotizing myopathy (IMNM) remains unknown. Autophagy impairment, described in autoimmune diseases, is a key process in myofiber protein degradation flux and muscle integrity and has not been studied in IMNM. METHODS: Muscle biopsies from patients with IMNM (n = 40), dermatomyositis (DM; 24), polymyositis (PM; 8), polymyositis with mitochondrial pathology (4), sporadic inclusion body myositis (8), and controls (6) were compared by immunohistochemistry. RESULTS: The proportions of myofibers containing autophagy markers LC3b and p62 were higher in IMNM than in DM or PM and correlated with creatine kinase levels. In IMNM, compartmentalized LC3b puncta were located in regenerating and degenerating myofibers surrounded by major histocompatibility complex type II+ inflammatory cells. Several IMNM myofibers accumulated ubiquitin and misfolded protein. DISCUSSION: The detection of LC3b+ or p62+ myofibers could be used in differentiating IMNM from PM. The identification of autophagy-modifying molecules potentially could improve patients' outcomes. Muscle Nerve, 2019.

A new M23-based ELISA assay for anti-aquaporin 4 autoantibodies: diagnostic accuracy and clinical correlation.

PURPOSE: Although many assays have been developed to detect anti-aquaporin-4 (AQP4) antibodies, most of these assays require sophisticated techniques and are thus only available at specialized laboratories. The aim of this study was to evaluate the analytical and clinical performance of a new commercial enzyme-linked immunosorbent assay (ELISA RSR, AQP4 Ab Version 2) to detect anti-AQP4 antibodies performed on a fully automated system (SkyLAB 752). METHODS: Serum samples from 64 patients with neuromyelitis optica spectrum disorders (NMOSD) (including NMO, longitudinally extensive myelitis-LETM, optical neuritis and myelitis) and 27 controls were tested for anti-AQP4 antibodies. All sera were previously tested using an indirect immunofluorescence (IIF) method on primate tissue, as the reference method. Commercial control sera were used to determine within-run, between-day and within-laboratory precision (CLSI guidelines). RESULTS: At a cut-off value of 2.1 U/mL as determined by ROC curves, sensitivity and specificity for NMO were 83.3% and 100%, respectively. The ELISA assay provided 100% concordant results with the reference IIF method. The median concentration of anti-AQP4 antibodies was statistically higher in patients with NMO than in patients with LETM (p = 0.0006) or with other NMOSD and in controls (p < 0.0001). At the concentration of 12.4 and 28.1 U/mL, the within-run, between-day and within-laboratory coefficients of variation (CV) were 3.2% and 3%, 7.6% and 7.4%, and 8.2% and 8.0%, respectively. CONCLUSIONS: This new ELISA method performed on a fully automated system, showed high sensitivity and absolute specificity, good CV in precision tests, and provided observer-independent quantitative results.

Definition of a new cut-off for the anti-phospholipase A2 receptor (PLA2R) autoantibody immunoassay in patients affected by idiopathic membranous nephropathy.

Autoantibodies against phospholipase A2 receptor (PLA2R) are a sensitive and specific marker for idiopathic membranous nephropathy (IMN). The aim of our study was to redefine the cut-off value for the measurement of anti-PLA2R autoantibody levels by an automated enzyme immunoassay, in a large single-center cohort of Italian IMN patients at the time of diagnosis. Sixty-seven consecutive incident patients, with biopsy-proven IMN, were recruited. All patients were naïve to preceding immunosuppressive therapeutic regimens. The patient population had a mean age of 57 years and included 48 males and 19 females. Also, 200 patients with other renal diseases and 36 healthy subjects were studied as controls. The anti-PLA2R autoantibody levels were measured using the commercial enzyme-linked immunosorbent assay kit at the time of renal biopsy. At a cut-off value of 2.7 RU/ml (significantly lower than the manufacturer's recommended value of 14 RU/ml), calculated by receiver operating characteristic curves, the sensitivity and specificity of anti-PLA2R autoantibodies in the diagnosis of IMN was 88.1 and 96% respectively. The adapted cut-off value of 2.7 UI/ml increased sensitivity without affecting the specificity and it should be the recommended value for this method. Additionally our study confirmed the correlation, at baseline, between anti-PLA2R autoantibody levels and other biomarkers of disease activity.