The Binding Mechanisms of Antibodies to DNA from Healthy Subjects and Patients with Systemic Lupus Erythematosus: The Role of Monogamous Bivalency and Fc Dependence.

Abs to DNA (anti-DNA) are a unique population of Abs that bind structural determinants on the DNA molecule. In systemic lupus erythematosus (SLE), anti-DNA Abs bind to conserved antigenic determinants, with the phosphodiester backbone being the most likely. In contrast, otherwise healthy subjects (HS) express anti-DNA that bind selectively to nonconserved sites on certain bacterial and viral DNA. As shown previously, SLE anti-DNA bind by a mechanism termed Fc-dependent monogamous bivalency. In this mechanism, both Fab sites interact with determinants on the same extended DNA molecule, reflecting the low affinity of each Fab site; the requirement for the Fc region suggests some contribution of the C region to increase avidity. In this study, we investigated whether anti-DNA from HS also bind to bacterial DNA by Fc-dependent monogamous bivalency. For this purpose, we compared the activity of intact IgG with Fab and F(ab')2 fragments prepared from the plasmas of SLE patients and HS using ELISAs with DNA from calf thymus or Micrococcus luteus These studies showed that Fab fragments from all plasmas tested, both SLE and HS, failed to bind significantly to DNA compared with intact IgG. By contrast, some, but not all, F(ab')2 preparations from both SLE patients and HS showed binding to M. luteus DNA; F(ab')2 fragments from SLE plasmas, however, did not bind significantly to calf thymus DNA. Together, these findings suggest that although anti-DNA Abs, whether from SLE or HS, bind by monogamous bivalency, binding to bacterial DNA does not require the Fc region.

Findings and feasibility of major salivary gland ultrasound in childhood-onset systemic lupus erythematosus: a pilot study.

BACKGROUND: Childhood-onset systemic lupus erythematosus (cSLE) is a complex autoimmune disorder with multi-organ manifestations and can be associated with other rheumatic diseases including Sjögren's syndrome (SS). Salivary gland ultrasound (SGUS) represents a noninvasive tool to screen for salivary gland disease in rheumatic disease patients. The aims of this cross-sectional study were to determine feasibility of major SGUS in a clinic setting and to identify characteristics in a cohort of cSLE patients (without confirmed SS) that may be associated with salivary gland abnormalities consistent with secondary SS. METHODS: Patients with SLE onset prior to age 18 were recruited. Patients completed questionnaires rating symptoms and underwent major SGUS examination. Disease and demographic differences were compared between cSLE patients with abnormal SGUS vs. cSLE patients with normal SGUS using t-tests and Fisher's exact tests. RESULTS: Thirty-one cSLE patients were recruited, 84% were female, 55% were Caucasian. The average disease duration among all patients was 5 years. Average time to complete the SGUS examination and scoring protocol was 7 min. 35% of SGUS scores were abnormal and significantly associated with IgG level at diagnosis, and anti-Ro and anti-La antibodies. CONCLUSIONS: This is one of the first studies to our knowledge that assesses major SGUS in a cohort of patients with cSLE without prior diagnoses of SS. The SGUS protocol was feasible to perform by rheumatologists in a clinic setting. Although the sample size was small, SGUS abnormalities were identified in one-third of patients. IgG level at diagnosis and anti-Ro and anti-La antibodies may be associated with SGUS abnormalities.

Evaluation of a novel particle-based multi-analyte technology for the detection of anti-fibrillarin antibodies.

Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren's syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1-96.6%) sensitivity with 100.0% (95% CI: 96.4-100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman's rho = 0.89, 95% CI: 0.77.9-0.95%, p < 0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ). In conclusion, this first study on anti-fibrillarin antibodies measured using a novel PMAT assay shows promising results where the new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. The availability of novel AFA assays such as PMAT might facilitate the clinical deployment, additional studies, standardization efforts, and potentially consideration of AFA for next generations of the classification criteria.

Update of the simplified criteria for autoimmune hepatitis: Evaluation of the methodology for immunoserological testing.

BACKGROUND & AIMS: The simplified criteria for the diagnosis of autoimmune hepatitis (AIH) include immunofluorescence testing (IFT) of antinuclear and smooth muscle autoantibodies (ANA and SMA) on rodent tissue sections. We aimed to establish scoring criteria for the implementation of ANA IFT on human epithelioma-2 (HEp-2) cells and ELISA-based testing. METHODS: ANA and SMA reactivity of 61 AIH sera and 72 non-alcoholic fatty liver disease controls were separately assessed on tissue sections and HEp-2 cells to compare the diagnostic value at increasing titers. A total of 113 patients with AIH at diagnosis and 202 controls from 3 European centers were assessed by IFT as well as 3 different commercially available ANA ELISA and 1 anti-F-actin ELISA. RESULTS: ANA assessment by IFT on liver sections had 83.6% sensitivity and 69.4% specificity for AIH at a titer of 1:40. On HEp-2 cells, sensitivity and specificity were 75.4% and 73.6%, respectively, at an adjusted titer of 1:160. Area under the curve (AUC) values of ANA ELISA ranged from 0.70-0.87, with ELISA coated with HEp-2 extracts in addition to selected antigens performing significantly better. SMA assessment by IFT had the highest specificity for the SMA-VG/T pattern and anti-microfilament reactivity on HEp-2 cells. ELISA-based anti-F-actin evaluation was a strong predictor of AIH (AUC 0.88) and performed better than SMA assessment by IFT (AUC 0.77-0.87). CONCLUSION: At adjusted cut-offs, both ANA IFT using HEp-2 cells and ELISA-based autoantibody evaluation for ANA and SMA are potential alternatives to tissue-based IFT for the diagnosis of AIH. LAY SUMMARY: Autoantibodies are a hallmark of autoimmune hepatitis and are traditionally tested for by immunofluorescence assays on rodent tissue sections. Herein, we demonstrate that human epithelioma cells can be used as a reliable substrate for immunofluorescence testing. ELISA-based testing is also a potentially reliable alternative for autoantibody assessment in autoimmune hepatitis. We propose the implementation of these testing methods into the simplified criteria for the diagnosis of autoimmune hepatitis.

Frecuencia de ANCA positivos en una población con síntomas clínicos sugestivos de enfermedad autoinmune y la interferencia de ANA en su interpretación / Frequency of positive ANCA test in a population with clinical symptoms suggestive of autoimmune disease and the interference of ANA in its interpretation

ANTECEDENTES: Los anticuerpos anticitoplasma del neutrófilo (ANCA) se asocian con vasculitis. Existen diferentes métodos para determinar su presencia. Se ha descrito la interferencia de anticuerpos antinucleares (ANA) en la diferenciación de los patrones P-ANCA y C-ANCA. OBJETIVO: Determinar la frecuencia de ANCA en una población con manifestaciones de enfermedad autoinmune; y evaluar la interferencia de los ANA en su interpretación. MATERIALES Y MÉTODOS: Estudio de corte transversal retrospectivo, descriptivo no experimental incluyendo 3.330 datos con diagnóstico presuntivo de enfermedad autoinmune y solicitud de ANCA. Las determinaciones de ANCA y de ANA se realizaron mediante inmunofluorescencia indirecta, L-ANCA® y CytoBead® ANCA. Antiproteinasa 3 y antimieloperoxidasa fueron determinados mediante ELISA y CytoBead® ANCA. RESULTADOS: Se encontraron ANCA positivos en el 10,21% y el 12,64% con ANCA positivos presentaban ANA positivos. La concordancia kappa para antiproteinasa 3 entre CytoBead® ANCA y ELISA fue del 100% (K=1; p < 0,05), La concordancia entre antimieloperoxidasa por ELISA y CytoBead®ANCA fue alta (K=0,94; p < 0,05). El 30% de aquellos con ANCA positivos tenía diagnóstico de algún tipo de vasculitis, el 20% cursaba con alguna enfermedad autoinmune. CONCLUSIONES: Los resultados indican una solicitud sobreestimada de este marcador como ayuda diagnóstica en consulta de atención primaria no direccionada. Para una adecuada evaluación de ANCA se debe implementar la técnica de inmunofluorescencia indirecta para tamizaje y confirmar con la determinación de antígenos específicos para antiproteinasa 3 y antimieloperoxidasa por cualquiera de los ensayos confirmatorios. La alta concordancia mostrada por CytoBeads® ANCA hace que planteemos el empleo de dicha alternativa para la determinación de ANCA y su confirmación. Dada la interferencia de los ANA, se recomienda solicitar la prueba ANA por inmunofluorescencia indirecta ante la presencia de resultados P-ANCA positivos, con el fin de minimizar «falsos positivos»

BACKGROUND: Antibodies against neutrophil cytoplasm (ANCA) are associated with vasculitis. There are different methods to determine their presence. The interference of antinuclear antibodies (ANA) in the differentiation between P-ANCA and C-ANCA patterns has been described. OBJECTIVE: To determine the frequency of ANCA in a population with manifestations of autoimmune disease, and evaluate the interference of ANA in its interpretation. MATERIALS AND METHODS: Retrospective, descriptive nonexperimental cross-sectional study, including 3,330 data. The presumptive diagnosis was autoimmune disease and a test for ANCA was requested. The ANCA and ANA determinations were made by indirect immunofluorescence, L-ANCA® and CytoBead® ANCA. Anti-proteinase 3 and anti-myeloperoxidase were detected by ELISA and CytoBead® ANCA. RESULTS: ANCAs were positive in 10.21% and 12.64% of those positive for ANCA were positive for ANA. The inter-rater agreement statistic (Kappa) for anti-PR3 between CytoBead ANCA and ELISA was 100% (K=1.00; P<.05) and the agreement between anti- myeloperoxidase by ELISA and CytoBead® ANCA was high (K=0.94; P<.05). 30% of those with ANCAs had a diagnosis of a type of vasculitis; 20% of them had an autoimmune disease. CONCLUSIONS: The results suggest an overestimated request for ANCAs as a diagnostic aid in primary care which was not addressed. For an adequate evaluation of ANCAs, the indirect immunofluorescence technique should be implemented for the control and confirmation with the determination of specific antigens for anti- proteinase 3 and anti- myeloperoxidase in any of the confirmatory assays. The high concordance shown by ANCA CytoBeads makes us consider the use of this alternative for the determination of ANCAs and the confirmation. Given the interference of ANAs, the ANA test by IFI in the presence of positive P-ANCA results is recommended in order to minimize "false positives"

Sobre la capacidad discriminativa de diferentes autoanticuerpos en el diagnóstico del Lupus Eritematoso Sistémico / On the discriminative capacity of several autoantibodies for the diagnosis of Systemic Lupus Erythematosus

INTRODUCCIÓN: Se han propuesto varios autoanticuerpos en el diagnóstico diferencial del Lupus Eritematoso Sistémico (LES). El uso de estos autoanticuerpos puede resultar en diferentes características operacionales. OBJETIVO: Evaluar las características operacionales de autoanticuerpos selectos en el diagnóstico diferencial del LES. DISEÑO DEL ESTUDIO: Retrospectivo, analítico. SERIE DE ESTUDIO: Ciento sesenta y nueve sujetos (LES: 35.5 %; Enfermedades del tejido conectivo diferentes del LES: 38.5 %; Sujetos aparentemente sanos: 26.0 %; Mujeres: 84.6 %; Edad promedio: 30.9 ± 17.9 años; Tiempo promedio de evolución de la enfermedad lúpica: 3.9 ± 4.7 años). MÉTODOS: Los anticuerpos anti-ADN de doble cadena (anti-ADNdc), anti-nucleosomas (anti-Nuc), anti-proteína Smith (anti-Sm) y anti-ribosomas P (anti-Rib P) se determinaron mediante técnicas inmunoenzimáticas (Orgentec Diagnostika, Mainz, Alemania). Los anticuerpos antinucleares (ANA) se determinaron indistintamente mediante técnicas inmunoenzimáticas (ANA-EIA) e inmunofluorescentes con células Hep2 (ANA-Hep2). Se calcularon las características operacionales de los autoanticuerpos en el diagnóstico diferencial del LES. Asimismo, se obtuvieron las correspondientes curvas ROC en cada instancia de análisis, y se estimó la exactitud diagnóstica del anticuerpo del área bajo la curva ROC (AROC). RESULTADOS: La exactitud de los anticuerpos empleados en el diagnóstico diferencial del LES fue como sigue (en orden descendente): anti-ADNdc: 0.9528 ± 0.0288; anti-Nuc: 0.7851 ± 0.0675; ANA-EIA: 0.7755 ± 0.2633; anti-Sm: 0.5926 ± 0.0790; anti-Rib P: 0.5770 ± 0.2641; y ANA-Hep2: 0.5341 ± 0.2635; respectivamente. Excepción hecha de los anti-ADNdc, las características operacionales de los otros anticuerpos fueron dependientes del punto de corte, la subpoblación muestreada, y el método analítico. CONCLUSIONES: Los anticuerpos anti-ADNdc son los más exactos en el diagnóstico diferencial del LES

RATIONALE: Several autoantibodies have been proposed for the differential diagnosis of Systemic Lupus Erythematosus (SLE). Use of these autoantibodies might result in different operational characteristics. OBJECTIVE: To assess the operational characteristics of selected autoantibodies in the differential diagnosis of SLE. STUDY DISIG: Retrospective analytical. STUDY SERIE: One-hundred and sixty-nine subjects (SLE: 35.5 %; Non-SLE diseases of connective tissue: 38.5 %; Apparently healthy subjects: 26.0 %; Women: 84.6 %; Average age: 30.9 ± 17.9 years; Average evolution time of lupic disease: 3.9 ± 4.7 years). METHODS: Anti-double stranded DNA (anti-dsDNAdc), anti-nucleosomes (anti-Nuc), anti-Smith protein (anti-Sm) and anti-ribosomes (anti-Rib) autoantibodies were determined by immunoenzymatic techniques (Orgentec Diagnostika, Mainz, Alemania). Anti-nuclear antibodies (ANA) were indistinctely determined either by immunoenzimatic techniques (ANA-EIA) or immunofluorescent methods with Hep2 cells (ANA-Hep2). Operational characteristics of the autoantibodies in the differential diagnosis of SLE were calculated. In addition, corresponding ROC curves for each instance of analysis were obtained, and diagnostic accuracy was estimated from the area under the ROC curve (AROC). RESULTS: Accuracy of the autoantibodies used in the differential diagnosis of SLE was as follows (in descending order): anti-dsDNA: 0.9528 ± 0.0288; anti-Nuc: 0.7851 ± 0.0675; ANA-EIA: 0.7755 ± 0.2633; anti-Sm: 0.5926 ± 0.0790; anti-Rib: 0.5770 ± 0.2641; and ANA-Hep2: 0.5341 ± 0.2635; respectively. With the exception of anti-dcDNA, operational characteristics of the remaining antibodies were dependent upon cut-off value, sampled subpopulation, and analytical method. CONCLUSIONS: anti-dsDNA antibodies were the most accurate in the differential diagnosis of SLE

Trombopenia aislada no siempre es trombopenia inmune primaria / Isolated thrombopenia is not always primary immune thrombopenia

El lupus eritematoso sistémico (LES) es una enfermedad autoinmune crónica, de etiología desconocida, que afecta a múltiples órganos y sistemas. El LES pediátrico es más frecuente en las niñas y presenta un pico de incidencia entre los 12-16 años. Las manifestaciones clínicas más frecuentes son los síntomas constitucionales, la artritis, el eritema malar y las alteraciones hematológicas. La trombopenia aparece en el 50% de los casos y es la manifestación inicial en el 15% de los casos de LES de inicio pediátrico, aunque puede preceder varios años a la aparición del resto de manifestaciones de LES. Entre el 20 y el 30% de los niños con trombopenia inmune primaria y ANA positivos en suero desarrollarán posteriormente LES

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown etiology that affects multiple organs and systems. Pediatric SLE is more frequent in girls and has a peak incidence between 12-16 years. The most frequent clinical manifestations are constitutional symptoms, arthritis, malar erythema and hematological abnormalities. Thrombopenia appears in 50% of cases and is the initial manifestation in 15% of cases of SLE of pediatric onset, although it may take several years before the appearance of other manifestations of SLE. Between 20 and 30% of children with primary immune thrombopenia and serum positive ANA will subsequently develop SLE

A multi-centre study for standardization of antinuclear antibody indirect immunofluorescence screening with automated system.

INTRODUCTION: ndirect immunofluorescence assay (IFA) using HEp-2 as substrate plays a consolidate role for the detection and measurement of ANA, which is currently considered as the reference method for detection. Manual operation is still very common in China, therefore, the need of standardization and automation for ANA-IFA detecting has been highlighted. OBJECTIVE: The current multi-center study is aimed to evaluate if HELIOS (AESKU Diagnostics, Wendelsheim, Germany) contributes to comparability of ANA screening results among different labs，and establish application specification of HELIOS for standardization of ANA detection. METHODS: ANA detection by manual IFA method and HELIOS on 230 clinical serum samples in eight laboratories. The performance to discriminate positive/negative screening results, endpoint titer estimation and pattern recognition were evaluated in HELIOS and manual visual. RESULTS: The positive coincident rate for ANA detection by manual IFA ranges from 87.7% to 97.8%, the negative coincidence rate ranges from 68.8% to 100%, the correctly estimated titer evaluation were 80 to 171 cases, the correct pattern in 146 to 161 cases, respectively. The positive coincident rate of HELIOS for ANA detection ranges from 91.2% to 97.7%, the negative coincidence rate ranges from 96.5% to 100%, the correctly estimated titer evaluation were 145 to 157 cases, the correct pattern in 123 to 140 cases, respectively. CONCLUSION: HELIOS could provide accurate diagnostic results, this include not only positive/negative results, but also endpoint titer, common patterns. The application of this system can help to promote standardization of ANA detection.

Successful evolution of morphea after hepatitis C virus eradication with direct-acting antiviral agent treatment

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Enfermedad indiferenciada del tejido conectivo y enfermedad pulmonar intersticial: intentando definir patrones / Undifferentiated connective tissue disease and interstitial lung disease: trying to define patterns

Objetivos. Agrupar a los pacientes con enfermedad pulmonar intersticial (EPI) asociada a enfermedad indiferenciada del tejido conectivo (EITC) según la presencia o no de ciertas manifestaciones clínicas o inmunológicas, esperando encontrar diferentes expresiones tomográficas o funcionales. Métodos. Estudio de cohortes retrospectivas. Se incluyeron pacientes que cumplían criterios de Kinder para EITC. Se consideraron variables predictoras: manifestaciones «altamente específicas de enfermedad del tejido conectivo (ETC)» (Raynaud, xeroftalmia o artritis), títulos altos de anticuerpos antinucleares (ANA) (mayores a 1:320) y patrones específicos de ANA (centromérico, citoplásmico y nucleolar). El cambio en la capacidad vital forzada% (CVF%) en el tiempo y el patrón en TCAR fueron las variables de resultado estudiadas. Resultados. Se incluyeron 66 pacientes. Veintinueve presentaron al menos una manifestación «altamente específica de ETC» (43,94%), 16 ANA específico (28,57%) y 29 ANA alto título (43,94%). Aquellos con manifestaciones «altamente específicas de ETC» presentaron menor frecuencia de sexo masculino (10,34% vs 48,65%, p<0,001), menor edad en años (media 52 [DE14,58] vs 62,08 [9,46], p<0,001) y menor mediana de declinación de CVF% (1[RIC −1 a 10] vs −6 [RIC −16 a −4], p<0,006). En el análisis de regresión lineal múltiple la presencia de manifestaciones «altamente específicas de ETC» se asoció con mejoría en CVF% (coeficiente B de 13,25 [IC95% 2,41 a 24,09]). No encontramos asociaciones en cuanto al patrón en TACAR. Conclusiones. La presencia de manifestaciones «altamente específicas de ETC» se asoció con sexo femenino, menor edad al inicio y una evolución más favorable en cuanto a la CVF%, lo cual evidencia el impacto de las manifestaciones clínicas en la evolución de estos pacientes (AU)

Objectives. To identify clinical or immunological features in patients with undifferentiated connective tissue disease (UCTD) associated interstitial lung disease (ILD), in order to group them and recognize different functional and high resolution computed tomography (HRCT) behavior. Methods. Retrospective cohort study. Patients meeting Kinder criteria for UCTD were included. We defined the following predictive variables: ‘highly specific’ connective tissue disease (CTD) manifestations (Raynaud's phenomenon, dry eyes or arthritis), high antinuclear antibody (ANA) titer (above 1: 320), and ‘specific’ ANA staining patterns (centromere, cytoplasmic and nucleolar patterns). We evaluated the following outcomes: change in the percentage of the predicted forced vital capacity (FVC%) during the follow-up period, and HRCT pattern. Results. Sixty-six patients were included. Twenty-nine (43.94%) showed at least one ‘highly specific’ CTD manifestation, 16 (28.57%) had a ‘specific’ ANA staining pattern and 29 (43.94%) high ANA titer. Patients with ‘highly specific’ CTD manifestations were younger (mean [SD] 52 years [14.58] vs 62.08 years [9.46], P<.001), were more likely men (10.34% vs 48.65%, P<.001) and showed a smaller decline of the FVC% (median [interquartile range] 1% [−1 to 10] vs -6% [−16 to −4], P<.006). In the multivariate analysis, the presence of highly specific manifestations was associated with improvement in the FVC% (B coefficient of 13.25 [95% confidence interval, 2.41 to 24.09]). No association was observed in relation to the HRCT pattern. Conclusion. The presence of ‘highly specific’ CTD manifestations was associated with female sex, younger age and better functional behavior. These findings highlight the impact of the clinical features in the outcome of patients with UCTD ILD (AU)

[THE CLINICAL INFORMATIVENESS OF AUTOMATED METHODS OF SCREENING DETECTION OF ANTI-NUCLEAR ANTIBODIES USING INDIRECT REACTION OF IMMUNE FLUORESCENCE, ENZYME-LINKED IMMUNOSORBENT ASSAY AND MULTIPLEX XMAP TECHNOLOGY UNDER SYSTEMIC LUPUS ERYTHEMATOSUS].

Thew antinuclear antibodies (ANA) consist heterogeneous group of auto antibodies reacting with various components of nucleus and cytoplasm. The ANA is a main serological marker of systemic lupus erythematosus (SLE). The implementation in clinical practice of new highly productive techniques of immune analysis using automated systems sets up prerequisites for standardization and amelioration of reproducibility of detection of ANA. The study was carried out to compare diagnostic significance of automated techniques of screening detection of ANA (indirect immunofluorescence test on cells HEp-2 (IIFT-HEp-2)), enzyme-linked immunosorbent assay (ELISA) and multi-complex immune analysis (MIA, using suspension technology xMAP) in serum of patients with SLE. The serums from 94 patients with SLE were analyzed. The comparison group included 70 patients with other rheumatic diseases. The control group consisted of 30 healthy donors. The screening detection of ANA using technique IIFT-HEp-2 was implemented on automated platform AKLIDES, ELISA - on automated analyzer ALEGRIA and MIA on automated analyzer BioPlex 2200. The technique IIFT-HEp-2 demonstrated the most high diagnostic sensitivity as compared with ELISA and MIA- BioPlex 2200 (96.8%; 79.8% and 82.9% correspondingly). The general diagnostic specificity of detection of ANA using technique IIFT-HEp-2 was lower than in case of ELISA and MIO-BioPlex 2200 (40%, 70% and 57% correspondingly). In the group of healthy donors the lowest diagnostic specificity was observed in ANA screening analysis using MIA-BioPlex 2200 (80%) while in case of applying IIFT-HEp-2 and ELISA indices of diagnostic specificity made up 93.3% and 96.7% correspondingly. The ANA analysis of mix of 26 nuclear antigens using ELISA technique was a reliable laboratory test for diagnostic of SLE (likelihood ratio of positive result - 2.66). By the level of likelihood ratio of negative result of the IIFT-HEp-2 technique was more informative test for exclusion of diagnosis of SLE than techniques of ELISA and MIA-BioPlex 2200 (0.08; 0.29 and 0.3 correspondingly). The detection of ANA using technique of is the most preferable primary screening test for diagnostic of SLE. The ELISA of antibodies to mix of nuclear antigens and MIA on the basis of xMAP technology are less preferable screening tests for diagnostic of SLE as compared with IIFT-HEp-2 because of false-negative results in 20% and 17% of cases correspondingly. ELISA and MIA are to applied as confirmatory screening tests permitting to detect antigen-specific ANA in patients with SLE with positive results of IIFT-HEp-2.

Computer-Assisted Classification Patterns in Autoimmune Diagnostics: The AIDA Project.

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%).

Feasibility of using DNA-immobilized nanocellulose-based immunoadsorbent for systemic lupus erythematosus plasmapheresis.

The goal of this project was to study the feasibility of using a DNA-immobilized nanocellulose-based immunoadsorbent for possible application in medical apheresis such as systemic lupus erythematosus (SLE) treatment. Calf thymus DNA was bound to high surface area nanocellulose membrane at varying concentrations using UV-irradiation. The DNA-immobilized samples were characterized with scanning electron microscopy, atomic force microscopy, and phosphorus elemental analysis. The anti-ds-DNA IgG binding was tested in vitro using ELISA. The produced sample showed high affinity in vitro to bind anti-ds-DNA-antibodies from mice, as much as 80% of added IgG was bound by the membrane. Furthermore, the binding efficiency was quantitatively dependent on the amount of immobilized DNA onto nanocellulose membrane. The described nanocellulose membranes are interesting immunoadsorbents for continued clinical studies.

ANA, lupus y hepatitis C. Comentario a «Infección por virus de la hepatitis C, interferón alfa y lupus, una curiosa asociación» / ANA, lupus and hepatitis C. Comment to «Infection with hepatitis C virus, interferon alfa and lupus: An odd association»

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Respuesta al comentario de «Infección por virus de la hepatitis C, interferón alfa y lupus, una curiosa asociación» / Reply to the comment «Infection with hepatitis C virus, interferon alfa and lupus: An odd association»

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Therapeutic plasma exchange in antisynthetase syndrome with severe interstitial lung disease.

Antisynthetase syndrome (ASS) is a rare condition characterized by interstitial lung disease (ILD), inflammatory myositis, fever, Raynaud phenomenon, mechanic's hand, and inflammatory polyarthritis in the setting of antibodies to amino acyl-transfer RNA synthetases, with anti-Jo-1 antibody being the most common. Prognosis is very poor especially when there is associated ILD. To date, there is no standardized treatment for ILD associated ASS. Therapy is based on the use of steroids alone or in combination with other immunosuppressive agents, especially in severe or refractory cases. The role of therapeutic plasma exchange (TPE) in the management of this rare condition has not been established. Here, we report a case of severe ILD associated ASS in a 41-year-old woman who did not show clinical or laboratory response after six doses of high dose steroids and a dose of IV cyclophosphamide. Because of the aggressive nature of her disease and poor prognostic indices present, a decision was made to add TPE to her treatment. She underwent five sessions of TPE. At the end of the 5th session, the anti-Jo-1 antibody levels dropped to 3.6 AI (antibody index) and her creatinine kinase (CK) level from 875 to 399 U L(-1) (Units per liter) with overall improvement in her respiratory status. This case suggests TPE may be a promising treatment option in patients with ILD associated ASS refractory to steroids and other immunosuppressive therapy, particularly those with severe disease.

Hepatotoxicidad por simvastatina con elevación de anticuerpos antinucleares / Drug-induced liver injury caused by simvastatin associated with antinuclear antibodies

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Double positive CD4+CD8+ T cells: key suppressive role in the production of autoantibodies in systemic lupus erythematosus.

BACKGROUND & OBJECTIVES: The presence of CD4+CD8+ (double positive) T cells (DPT) in the target organs of several autoimmune diseases has been reported. The aim of this study was to investigate the pathogenic role of DPT in systemic lupus erythematosus (SLE). METHODS: A total of 175 SLE cases and 125 matched healthy controls were investigated for CD3+, CD4+, CD8+ lymphocytes and DPT by flow cytometry. Serum samples from SLE patients and controls were tested for antinuclear antibody (ANA), anti-double strain deoxyribonucleic acid (anti-dsDNA), anti-U1 ribonucleoprotein (anti-U1 RNP), anti-sjogren syndrome A (anti-SSA), anti-ribosomal P protein (anti-rib-P), anti-Smith (anti-Sm), anti-Sjogren syndrome B (anti-SSB), complement 3 (C3) and complement 4 (C4). RESULTS: The DPT median and 5-95 per cent range of SLE cases and healthy controls were 0.50 [0.10-2.60] and 0.80 [0.20-2.74] respectively (P<0.001). SLE patients were divided into a ≥1:1000 subgroup and a <1:1000 subgroup according to the ANA titre. The DPT of the former subgroup was significantly lower than that of the latter (P=0.032). The DPT medians of positive subgroups with anti-dsDNA (P<0.001), anti-U1RNP (P=0.018), anti-SSA (P=0.021) or anti-rib-P (P=0.039) were also significantly lower than the negative subgroups. Likewise, DPT was significantly lower in SLE subgroups with low concentration of C3 or C4 than those with high concentration (P<0.006). INTERPRETATION & CONCLUSIONS: Our findings show that the DPT cells may play a key suppressive role in the production of autoantibodies in SLE. Direct evidence that DPT regulates the pathogenesis of SLE needs to be investigated in future work.

Staining pattern classification of antinuclear autoantibodies based on block segmentation in indirect immunofluorescence images.

Indirect immunofluorescence based on HEp-2 cell substrate is the most commonly used staining method for antinuclear autoantibodies associated with different types of autoimmune pathologies. The aim of this paper is to design an automatic system to identify the staining patterns based on block segmentation compared to the cell segmentation most used in previous research. Various feature descriptors and classifiers are tested and compared in the classification of the staining pattern of blocks and it is found that the technique of the combination of the local binary pattern and the k-nearest neighbor algorithm achieve the best performance. Relying on the results of block pattern classification, experiments on the whole images show that classifier fusion rules are able to identify the staining patterns of the whole well (specimen image) with a total accuracy of about 94.62%.

A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells.

Cancer cells with defects in DNA repair are highly susceptible to DNA-damaging agents, but delivery of therapeutic agents into cell nuclei can be challenging. A subset of lupus autoantibodies is associated with nucleolytic activity, and some of these antibodies are capable of nuclear penetration. We hypothesized that such antibodies might have potential as therapeutic agents targeted towards DNA repair-deficient malignancies. We identified the lupus autoantibody 5C6 as a cell-penetrating nucleolytic antibody and found that 5C6 has a differential effect on a matched pair of BRCA2-proficient and deficient DLD1 colon cancer cells. 5C6 selectively induced γH2AX in, and suppressed the growth of, the BRCA2-deficient cells. These findings demonstrate the potential utility of 5C6 in targeted therapy for DNA repair-deficient malignancies and strengthen the rationale for studies of additional lupus autoantibodies in order to identify the best candidates for development as therapeutic agents. In addition, the toxic effect of 5C6 on BRCA2-deficient cells provides further support for the hypothesis that some lupus autoantibodies contribute to the lower risk of specific cancers associated with systemic lupus erythematosus.