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.

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.

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.

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.

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.

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.

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.

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.

Autoimmune Hepatitis: Factors Involved in Initiation and Methods of Diagnosis and Treatment.

Autoimmune hepatitis is an acute or mostly chronic liver disease that can affect both adults and children and has a clear prevalence for the female sex. A definite etiology has not been established, but it is known that genetic predisposing profiles and exogenous trigger factors are involved. The main diagnostic criteria include typical histological features, the occurrence of serum auto-antibodies, and increased levels of transaminases and gamma-globulins. Instances of autoimmune hepatitis sharing features with other autoimmune liver diseases have also been observed. An imbalance of the immune system with persistent activation of effector T cells has been emphasized to account for the sustained liver injury. Clinical manifestations are variable both at presentation and throughout the course of the disease, ranging from an asymptomatic state or the occurrence of non-specific symptoms to the features of end-stage liver disease such as jaundice, ascites, and gastrointestinal bleeding. A clinical and biochemical remission is achieved in at least 80% of patients receiving corticosteroids with or without the addition of azathioprine. Alternative therapeutic schedules have been proposed for unresponsive and intolerant patients. Given that relapse often occurs after therapy withdrawal, maintenance treatment is usually required.

Changes in anti-cyclic citrullinated peptide antibodies and rheumatoid factor isotypes serum levels in patients with rheumatoid arthritis following treatment with different biological drugs.

OBJECTIVES: Anti-cyclic citrullinated peptide antibodies (anti-CCP) are a serological marker of rheumatoid arthritis (RA), and also have a prognostic value for more aggressive disease. Whether anti-CCP levels may change during treatment according to clinical response is matter of debate. Likewise, it is unknown whether different biological drugs have peculiar effects on anti-CCP levels. This study aimed to investigate changes in anti-CCP serum levels in RA patients on biological drugs with different mechanism of action. METHODS: We studied 71 patients with active RA tested positive for anti-CCP who started a first biological drug (54 anti-TNF-α drug, 9 rituximab, 8 tocilizumab). In 14 patients stopping anti-TNF-α treatment for ineffectiveness, rituximab was started. Anti-CCP and rheumatoid factor (RF) isotypes (IgM, IgA, IgG) levels were measured at entry, 12 months and again at 12 months after swapping to rituximab. RESULTS: After 1 year of therapy of the first biological drug, patients taking anti-TNF-α drugs showed a significant reduction of the anti-CCP levels (p=0.002), and all RF isotypes (p=0.003). Also patients treated with rituximab or tolicizumab had a significant decrease in anti-CCP (p=0.01) and RF isotype levels (p=0.01). Anti-CCP levels did not correlated with DAS28 over time. In patients switching to rituximab after failure of TNF-α blockers, anti-CCP levels did not change at 12 months (p=0.06), despite of the reduction of DAS28 (p=0.02) and RFs levels (p=0.02). CONCLUSIONS: Our study showed that anti-CCP levels may change during RA course, regardless of the biological drug used and the clinical response.

A New Immunodot Assay for Multiplex Detection of Autoantibodies in a Cohort of Italian Patients With Idiopathic Inflammatory Myopathies.

BACKGROUND: Autoantibody detection has been assessed as tool for the diagnosis and the definition of idiopathic inflammatory myopathies (IIM). The aim of the study was to characterize the autoantibody profiling of a cohort of Italian patients with IIM. METHODS: Sera of 53 adult patients with definite IIM, according to Bohan-Peter criteria, were tested for anti-nuclear autoantibodies (ANA), using indirect immunofluorescence (IIF) method, and for myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), using two new commercial immunodot assays. RESULTS: MSAs and/or MAAs were detected in 29 of 53 (54.7%) patients with IIM. Twenty-three patients (43.4%) were positive for at least one MSAs: 13 (24.5%) had anti-histidyl-tRNA synthetase autoantibodies (Jo1), 4 (7.5%) had other anti-aminoacyl-tRNA synthetases autoantibodies (anti-ARS), 1 (1.8%) had anti-transcription intermediary factor 1 gamma autoantibodies (anti-TIF1γ), 2 (3.7%) had anti-nuclear helicase protein Mi-2 autoantibodies (anti-Mi-2), 4 (7.5%) had anti-small ubiquitin like modifier activating enzyme heterodimer autoantibodies (anti-SAE). Moreover, 17 patients (32%) were positive for at least one MAAs. Coexisting MSAs and MAAs were observed in 9 of 53 (16.9%) patients, anti-Jo1/SS-A autoantibodies in most cases. Overall sensitivity of immunodot assays was 54.7%, the specificity was almost absolute. At cut-off value of 1:160, the sensitivity of ANA-IIF was 52.8%, increasing to 66% if cytoplasmatic fluorescence reaction was reported. Notably, two (5.7%) ANA-IIF negative patients had MSAs, detected only by immunodot assays. CONCLUSION: It was possible to identify MSAs otherwise undetectable because of the use of new assays. Immunodot can reveal MSAs even when IIF results are inconclusive or, in some cases, ANA negative.

Autoantibody Profiling in a Cohort of Pediatric and Adult Patients With Autoimmune Hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a rare condition characterized by the presence of autoantibodies distinctive of type 1 AIH (AIH-1) and type 2 AIH (AIH-2). The aim of this study was to evaluate the autoantibody profile in a cohort of pediatric and adult AIH patients, using both indirect immunofluorescence (IIF) and a new multiplexed line-blot assay. METHODS: Sera from 63 pediatric and 53 adult AIH patients were tested for antinuclear (ANA), antismooth muscle (SMA), anti-liver kidney microsome 1 (anti-LKM1), anti-liver cytosol 1 (anti-LC1) autoantibodies using IIF methods; for anti-LKM1, anti-LC1, and soluble liver antigen/liver-pancreas (anti-SLA/LP) autoantibodies using the line-blot; for anti-F-actin autoantibodies using IIF both on VSM47 cell-line and on rat intestinal epithelial cells. RESULTS: AIH-1 was the most common type of AIH in the adult cohort (73.6%), while AIH-2 was the most common AIH in the pediatric cohort (61.9%). Both in adult and pediatric AIH-2 anti-LKM1 were the prevalent autoantibodies. In pediatric AIH-2 anti-LC1 autoantibodies were more frequent than in adult AIH-2 (59 vs. 28.6%), and in 35.9% of cases they were present alone. In 17 patients anti-LC1 autoantibodies were detected only with the line-blot assay. The levels of anti-LKM1 and of anti-LC1 were not different between adult and pediatric AIH, and the overall agreement between the results obtained with the two IIF methods for F-actin detection was 98.8% (CI 95%: 94.4-99.7%). CONCLUSIONS: The line-blot assay showed a higher sensitivity than IIF for anti-LC1 detection. Anti-LKM1 and anti-LC1 autoantibody levels are not different in adults and children. An almost perfect agreement between the two IIF methods for anti-F-actin detection has been observed.

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.

Clinical usefulness of the serological gastric biopsy for the diagnosis of chronic autoimmune gastritis.

AIM: To assess the predictive value for chronic autoimmune gastritis (AIG) of the combined assay of anti-parietal-cell antibodies (PCA), anti-intrinsic-factor antibodies (IFA), anti-Helicobacter pylori (Hp) antibodies, and measurement of blood gastrin. METHODS: We studied 181 consecutive patients with anemia, due to iron deficiency resistant to oral replacement therapy or to vitamin B12 deficiency. RESULTS: 83 patients (45.8%) tested positive for PCA and underwent gastroscopy with multiple gastric biopsies. On the basis of the histological diagnosis, PCA-positive patients were divided into 4 groups: (1) 30 patients with chronic atrophic gastritis; they had high concentrations of PCA and gastrin and no detectable IFA; (2) 14 subjects with metaplastic gastric atrophy; they had high PCA, IFA, and gastrin; (3) 18 patients with nonspecific lymphocytic inflammation with increased PCA, normal gastrin levels, and absence of IFA; (4) 21 patients with multifocal atrophic gastritis with "borderline" PCA, normal gastrin, absence of IFA and presence of anti-Hp in 100% of the cases. CONCLUSIONS: The assay of four serological markers proved particularly effective in the diagnostic classification of gastritis and highly correlated with the histological profile. As such, this laboratory diagnostic profile may be considered an authentic "serological biopsy."

Anti-skin specific autoantibodies detected by a new immunofluorescence multiplex biochip method in patients with autoimmune bullous diseases.

BACKGROUND: Autoimmune blistering skin diseases are a heterogeneous group of diseases characterized by autoantibodies against structural components of the skin. In pemphigus vulgaris (PV) autoantibodies react mainly with desmoglein 3 (Dsg3) alone and/or in combination with desmoglein 1 (Dsg1). In bullous pemphigoid (BP) autoantibodies target two hemidesmosomal proteins, BP180 and BP230. OBJECTIVE: To evaluate the diagnostic accuracy of a new indirect immunofluorescence (IIF) multiplex biochip method for the detection of anti-skin specific autoantibodies. METHODS: Sera from 36 patients with PV and from 40 patients with BP were collected. The control group included 54 patients with other skin diseases and 40 healthy subjects. The detection of circulating autoantibodies to Dsg1, Dsg3, BP230 and BP180 was performed with a new IIF multiplex biochip method and with two currently commercially available ELISA methods. RESULTS: The multiplex IIF method showed a high diagnostic sensitivity (100%) for PV on cells transfected with Dsg3. In patients with BP, the positivity to the BP180 antigen was higher (90%) than that on monkey esophagus (50%) and on cells transfected with BP230 (40%). A good rate of agreement was observed among methods (IIF vs. ELISA) and among ELISA systems. CONCLUSIONS: The new multiplex biochip IIF method has a high diagnostic accuracy for the diagnosis of PV and BP, comparable to ELISA methods, and is able to screen autoimmune bullous diseases.

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 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.

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.

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.