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.

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.

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.

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.

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.

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.

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.

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.

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.

IL-6/IL-10 Ratio as A Prognostic and Predictive Marker of the Severity of Inherited Epidermolysis Bullosa.

BACKGROUND: Recent studies have shown that cytokines have an important role in the pathogenesis of inflammatory diseases and can be used as prognostic markers. OBJECTIVE: To evaluate the IL-6/IL-10 ratio in patients with Inherited Epidermolysis Bullosa (EB) as a prognostic marker. METHODS: Serum levels of IL-6 and IL-10 were measured in 13 patients with recessive dystrophic EB (RDEB) as well as 10 with EB Simplex (EBS), and in 18 healthy subjects. Receiver Operating Characteristics (ROC) analyses were used to assess the diagnostic accuracy of the IL-6/IL-10 ratio for detecting severe form of EB. RESULTS: The IL-6/IL-10 ratio was statistically higher in RDEB patients than in EBS patients and healthy subjects. The IL-6/IL-10 ratio significantly correlated with BEBS score. CONCLUSION: Our findings suggest that IL-6/IL-10 ratio >5.6 has a good diagnostic accuracy to identify patients with the highest severity of disease.

The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist's intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test.

Anti-DFS70 antibodies detected by immunoblot methods: A reliable tool to confirm the dense fine speckles ANA pattern.

BACKGROUND: Autoantibodies to the DFS70 (dense fine speckles 70) protein have been identified among the antinuclear antibodies (ANA) in patients with various disorders. However, the ANA test in indirect immunofluorescence (IIF) is not a reliable method to identify anti-DFS70 antibodies. We undertook this study to evaluate the diagnostic performance of two new immunoblot methods for the detection of anti-DFS70 antibodies and to investigate whether their different DFS70 antigen composition could affect diagnostic accuracy in detecting anti-DFS70 antibodies. METHODS: 62 samples showing a DFS70 staining pattern by IIF were tested by dot blot (Alphadia) and line blot (Euroimmun) methods. The dot blot method employs a truncated sequence of the DFS70 antigen (residues 349-435), while the line blot uses the full-length protein (aa 1-530). The 62 samples were previously assayed by a chemoluminescent (CLIA) method also using a truncated antigen (aa 349-435): 27 were CLIA positive and 35 were CLIA negative. 120 sera from subjects with infectious diseases were used as controls. RESULT: Both immunoblot methods were positive in the 27 IIF/CLIA positive samples; in addition, the Alphadia dot blot identified another seven DFS70 samples and the Euroimmun line blot was positive in five samples that were negative by CLIA. Among the 120 control samples, two false positives were recorded for the CLIA method, six for the Alphadia method and four for the Euroimmun method. Therefore, in this selected series of samples, sensitivity and specificity were 43.5% and 98.3% for the CLIA method, 54.8% and 95% for the dot blot and 51.6% and 96.6% for the line blot, respectively. CONCLUSIONS: Because of great inconsistency in assessing the DFS70 pattern using the ANA-IIF test, specific assays should be used to confirm anti-DFS70 antibodies. The results of this study show that there is no difference in the overall diagnostic accuracy among methods that use the truncated or the full-length DFS70 antigenic sequence and that it is likely that antibodies directed against antigens other than DFS70 may be responsible for producing a DFS70-like ANA-IIF pattern.

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.

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.

Serological epitope profile of anti-Ro52-positive patients with systemic autoimmune rheumatic diseases.

BACKGROUND: Ro52 is an interferon-inducible protein of the tripartite motif family. Antibodies against Ro52 have been described in patients with different autoimmune diseases, such as systemic lupus erythematosus and Sjögren's syndrome, that are often associated with anti-Ro60 antibodies. The Ro52 autoantigen is extraordinarily immunogenic, and its autoantibodies are directed against both linear and conformational epitopes. The aim of this study was to evaluate the prevalence of antibodies to the five Ro52 domains, as well as to Ro52 176- to 196-amino acid (aa) and 200-239-aa peptides, in different systemic autoimmune rheumatic diseases (SARDs). We also aimed to verify whether antibodies to a single domain or domain association could increase their diagnostic specificity for any SARD. METHODS: Serum samples were obtained from 100 anti-Ro52 antibody-positive patients with SARDs and from 68 controls (50 healthy donors and 18 patients with other autoimmune or allergic diseases). A special line immunoassay was created containing a full-length Ro52 antigen expressed in insect cells using the baculovirus system, five recombinant Ro52 antigen fragments [Ro52-1, Ro52-2, Ro52-3, Ro52-4 (partly overlapping Ro52-1 and Ro52-2), and Ro52-5 (partly overlapping Ro52-2 and Ro52-3)], and two Ro52 peptides (176-196 aa and 200-239 aa), all expressed in Escherichia coli. RESULTS: In patients with SARDs, fragment prevalence rates were as follows: Ro52-1 = 3 %, Ro52-2 = 97 %, Ro52-3 = 0 %, Ro52-4 = 9 %, Ro52-5 = 28 %, Ro52 175-196-aa peptide = 6 %, and Ro52 200-239-aa peptide = 74 %. All control samples were negative for the full-length Ro52 and for the five fragments tested. CONCLUSIONS: The main epitope of the Ro52 antigen was localized on fragment 2 (aa 125-267), and the majority (97 %) of SARD sera had antibodies that target this fragment. As most of the samples were positive for fragment 2 and only some for fragments 4 or 5, which partially overlap fragment 2, it seems that the target epitope is localized in the middle of fragment 2 or in the area between fragments 4 and 5. No antibody against a single epitope or a combination of epitopes was linked to any of the single SARDs.

Proinflammatory Cytokines and Antiskin Autoantibodies in Patients With Inherited Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a rare disorder characterized by inherited skin adhesion defects with abnormal disruption of the epidermal-dermal junction in response to mechanical trauma. Our aim was to investigate a set of cytokine levels in serum samples from patients suffering from epidermolysis bullosa simplex (EBS), dystrophic epidermolysis bullosa (DEB), and healthy controls (HCs), exploring their potential correlations with antiskin autoantibody titers and disease activity. Forty patients afferent to the Dermatological Ward of Bari City Hospital and 9 HCs were enrolled and subdivided according to the dystrophic (DEB) and simplex forms (EBS). We found a significant increase in interleukin (IL)-1ß plasmatic levels of DEB (P = 0.0224) and EBS (P = 0.0465) patients compared to HCs; IL-6 levels were significantly higher in DEB than in EBS patients (P = 0.0004) or HCs (P = 0.0474); IL-2 levels were significantly increased in DEB compared with EBS (P = 0.0428). Plasmatic tumor necrosis factor-ß and interferon-γ were higher in DEB patients than in HCs (P = 0.0448 and 0.0229). Conversely, tumor necrosis factor-α was significantly decreased in DEB (P = 0.0034). IL-5 correlated with anti-BP180 (r = -0.5018, P = 0.0338), anti-BP230 (r = -0.6097, P = 0.0122), and anticollagen VII (r = -0.5166, P = 0.0405) autoantibodies; interferon-γ correlated with anti-BP180 (r = 0.9633, P < 0.0001), anti-BP230 (r = 0.9071, P < 0.0001), and anticollagen VII (r = 0.8619, P = 0.0045) autoantibodies. Score of disease severity was significantly correlated with IL-6 (r = 0.6941, P = 0.029) and IL-12 (r = 0.5503, P = 0.0272). The present study supports that EB might be considered a systemic inflammatory disease rather than a skin-limited disorder; clinical disease activity scores could be also integrated by laboratory data such as IL-6 and IL-12 dosage; biotherapies targeting specific cytokine networks probably represent a way to go in the future.

Specific chemoluminescence and immunoasdorption tests for anti-DFS70 antibodies avoid false positive results by indirect immunofluorescence.

OBJECTIVE: To evaluate two new diagnostic methods for the identification of anti-DFS70 antibodies in samples showing a DFS70-staining pattern by indirect immunofluorescence (IIF). METHODS: We studied 731 patients: 576 were collected consecutively among those that in the ANA test on HEp-2 cells had produced a DFS70 fluorescence pattern and 155 were a consecutive series of patients sent by referring physicians for routine ANA testing. As controls we studied 50 patients with autoimmune diseases and 120 patients with active infectious disease. All 731 sera were assayed for anti-DFS70 antibodies by a specific chemoluminescence assay (CLIA); 70 randomly selected IIF-positive sera and 35 samples from patients with autoimmune diseases were studied by inhibition tests using the HEp-2 Select method. RESULTS: Assays performed with the CLIA-DFS70 method were positive in 30.4% of the samples presenting a DFS70 pattern by IIF, in 1.3% of the routine ANA sera, in 1.6% of the infectious sera and in none of the 50 autoimmune controls. However, as the IIF-DFS70 positive group included 106 patients with systemic autoimmune rheumatic diseases (SARD), 11 of which were DFS70 positive by CLIA, the prevalence of DFS70 antibodies in SARD was 7.5%. The ANA test performed after the use of HEp-2 Select showed an inhibition in 95.7% of the sera. No change in fluorescence intensity and pattern morphology between the native sera and the same sera tested with the solution containing the DFS70 antigen was observed in the 35 samples from patients with autoimmune diseases. CONCLUSIONS: To avoid misinterpretation of ANA pattern and consequent diagnostic errors, confirmation of the DFS70-IIF pattern by CLIA or other specific methods is mandatory before reporting the presence of anti-DFS70 antibodies. The HEp-2 Select test in most cases eliminates the interference by anti-DFS70 antibodies and avoids the possible reporting of false positive results.

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."