Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1.

Background/Objective: Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. Methods/Results: 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). Conclusions: The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥.

Birth weight and its relationship with endothelial function and pattern of endothelium-derived microparticles during childhood: New insight about early vascular damage.

AIMS: To investigate whether a specific endothelium-derived microparticles (EMPs) phenotype could be associated with birth weight and microvascular endothelial function in children. MATERIALS AND METHODS: A total of 95 children aged 6-14 years were recruited. Anthropometric measurements, blood pressure measurement, microvascular endothelial function testing, and biochemical profile analyses were performed. Standardized flow cytometry methods were used to identify and quantify the circulating CD144+, CD31+/annexin V+, and CD62E+ EMPs. KEY FINDINGS: The circulating number of CD31+/annexin V+ EMPs and CD144+ EMP levels were correlated with birth weight, systolic blood pressure, microvascular endothelial function, total cholesterol, and low-density lipoprotein cholesterol (LDL-C) level. In the multivariable logistic regression models, we identified strong evidence of a higher risk of microvascular endothelial dysfunction among children with low birth weight (LBW) and increased levels of both CD31+/annexin V+ EMPs and LDL-C; LBW and elevated LDL-C levels were independent predictors of high circulating numbers of CD31+/annexin V+ and CD144+ > 75th percentile EMPs. SIGNIFICANCE: Our data provide evidence that children with LBW values showed greater numbers of circulating CD31+/annexin V+ and CD144+ EMPs. In addition, LBW and high levels of CD31+/annexin V+ and LDL-C were significant risk factors for the presence of microvascular endothelial dysfunction.

High circulating levels of CD62E+ and CD31+/Annexin V+ endothelium-derived microparticles in children with overweight/obesity: Evidence of early vascular damage.

BACKGROUND: Obesity perturbs endothelium integrity, leading to endothelial activation, which predisposes the release of endothelium-derived microparticles (EMP). We measured the CD31+/annexin V+ and CD62E+ EMP levels to improve our understanding of their contribution to endothelial damage in children with overweight/obesity. SUBJECT AND METHODS: In this cross-sectional study, 107 children with normal weight and 35 children with overweight/obesity were evaluated. Anthropometric measurement, blood pressure, biochemical profile was performed. Standardized flow cytometry methods were used to identify and quantify circulating CD31+/annexin V+ and CD62E+ EMP. RESULTS: Children with overweight/obesity had significantly higher circulating levels of CD31+/annexin V+ (750 [600]) and CD62E+ (1400 [700]) EMP than those with normal weight (P < 0.001 for both). We found that EMP levels were positively correlated with body mass index (BMI), waist circumference, blood pressure, total cholesterol, low-density lipoprotein cholesterol (LDLc), and triglycerides. The multivariable logistic regression model revealed that the risks of having high EMP levels (> 75th percentile) were high in children with both large waist circumference and elevated LDLc level. Receiver operating characteristic (ROC) curves demonstrated that the LDLc levels showed significantly greater discrimination than waist circumference for both CD31+/annexin V+ (P = 0.031) as CD62E+ EMPs (P = 0.041). CONCLUSIONS: Children with overweight/obesity have high circulating CD31+/annexin V+ and CD62E+ EMP levels, which may be an early sign of endothelial apoptosis and inflammatory activation in response to injury. These EMP levels were positively associated with several cardiometabolic risk factors. Our data underscore the negative influence of high-risk metabolic profiles on endothelial integrity in the early stages of childhood obesity.

Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern.

Introduction: The indirect immunofluorescence assay on HEp-2 cells (HEp-2/IFA) is used worldwide for screening for autoantibodies to cellular antigens. Cell culture and fixation methods influence the cell distribution of autoantigens and the preservation of epitopes. Therefore, discrepancy of results obtained using different HEp-2/IFA kits (interkit nonreproducibility) is a common phenomenon in the clinical laboratory routine. Objective: This study evaluated the interkit nonreproducibility of HEp-2/IFA results using samples from patients with systemic autoimmune disease (SAD), nonautoimmune diseases (NAD), and healthy blood donors (HBD). Methods: Serum from 275 SAD patients, 293 NAD patients, and 300 HBD were processed at 1:80 dilution using four HEp-2 kits according to the manufacturers' instructions. Interkit reproducibility was determined for positive/negative results and patterns. The agreement of positive/negative results among kits for each sample was determined as the reactivity agreement score (RAS). The pattern reproducibility score (PRS) in each sample was calculated as a function of the number of kits showing equivalent patterns. Qualitative variables and ordinal variables were analyzed by the Chi-square and Mann-Whitney U tests, respectively. Results: A total of 402 samples were nonreactive in all kits and were considered devoid of autoantibodies. Further analysis included the 466 reactive samples (238 SAD, 119 NAD, 109 HBD). Reactivity to the nucleus had the highest interkit reproducibility (RAS = 83.6), followed by the metaphase plate (RAS = 78.9), cytoplasm (RAS = 77.4), and nucleolus (RAS = 72.4). Interkit reproducibility was higher in SAD (RAS = 78.0) than in NAD (RAS = 70.6) and HBD (RAS = 71.3) groups. Samples with strong reactivity (++++/4 and +++/4) had higher interkit reproducibility than those with weak reactivity (+/4). In the SAD group, RAS for nuclear reactivity was 87.5% for strongly reactive samples as opposed to 4.4% for weakly reactive samples, and the same was observed for NAD and HBD samples. The most robust patterns were the centromere AC-3 (PRS = 78.4), multiple nuclear dots AC-6 (PRS = 73.6), nuclear coarse speckled AC-5 (PRS = 71.3), nuclear homogeneous AC-1 (PRS = 67.9), and the reticular cytoplasmic AC-21 (PRS = 68.6). Conclusion: Interkit nonreproducibility in HEp-2/IFA is prevalent and occurs with the highest frequency with weakly reactive samples. International initiatives with the engagement of in vitro diagnostic industry are encouraged to promote the harmonization of the properties and performance of HEp-2/IFA commercial kits.

Changes in the result of antinuclear antibody immunofluorescence assay on HEp-2 cells reflect disease activity status in systemic lupus erythematosus.

Background The objective of the study was to determine whether the staining pattern and titer of indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) are associated with systemic lupus erythematosus (SLE) disease activity. Methods A total of 269 consecutive patients meeting the ACR and SLICC criteria for SLE were classified into three groups according to the SLE Disease Activity Index 2000 (SLEDAI2K): Remission (SLEDAI2K = 0; n = 47); Intermediate (SLEDAI2K = 1-5; n = 111); Active (SLEDAI2K ≥ 6; n = 111). All subjects were assessed for HEp-2 IFA titer and staining pattern and nine traditional parameters of SLE disease activity. After a 6 to 12-month interval, 101 of the 269 patients were reassessed. Results HEp-2 IFA homogeneous nuclear pattern (AC-1) occurred more frequently in the Active Group compared to the Remission Group (p < 0.001). Fine speckled nuclear pattern (AC-4) tended to occur more frequently in the Remission Group compared to the Active Group (p = 0.054). Subjects with AC-1 pattern had higher SLEDAI (8.8 ± 7.6) than those with AC-4 (4.8 ± 5.2) (p < 0.001). HEp-2 IFA titer and anti-nuclear antibody by enzyme-linked immunosorbent assay (ANA-ELISA) values were lower in the Remission Group compared to the other two groups (p < 0.001). Multivariate analyses identified only ELISA anti-dsDNA as an independent variable associated with disease activity. In follow-up analysis, HEp-2 IFA titer decreased significantly in the 33 subjects with decreased disease activity (p = 0.002). Receiver operator characteristic (ROC) curve analysis for determination of disease activity showed equivalent areas under the curve (AUC) for HEp-2 IFA titer and traditional disease activity parameters. Conclusions HEp-2 IFA pattern and titer can reflect SLE disease activity and may be considered in conjunction with other laboratory and clinical parameters in the assessment of SLE disease activity.

Biological anti-TNF drugs: immunogenicity underlying treatment failure and adverse events.

INTRODUCTION: Genetically engineered monoclonal antibodies and fusion proteins directed against cytokines or their receptors represent a breakthrough in the treatment of various chronic immune-inflammatory diseases. Areas covered: Studies show high remission rates in several diseases, but clinical practice shows a significant percentage of individuals who do not exhibit the desired response. Loss of therapeutic benefit after initial successful response is designated secondary failure. Immune-biological agents are not self-antigens and are therefore potentially immunogenic. Secondary failure is frequently caused by antibodies against immune-biologicals, known as anti-drug antibodies (ADA). ADA that neutralize circulating immune-biologicals and/or promote their clearance can reduce treatment efficacy. Furthermore, ADA can induce adverse events by diverse immunological mechanisms. This review provides a comprehensive overview of ADA in rheumatoid arthritis patients treated with anti-TNF immune-biologicals, and explores the concept of therapeutic drug monitoring (TDM) as an effective strategy to improve therapeutic management. Expert opinion: Monitoring circulating ADA and therapeutic immune-biological drugs is helpful when evaluating patients with secondary failure. However, immunological tests for ADA vary considerably regarding their ability to detect different types of ADA. Several assays are not designed to determine ADA-induced drug neutralizing capacity, and they may report clinically non-relevant data, especially if drug is present in test samples.

25-Hydroxivitamin D Serum Concentration, Not Free and Bioavailable Vitamin D, Is Associated with Disease Activity in Systemic Lupus Erythematosus Patients.

We aim to evaluate the prevalence of vitamin D deficiency in patients with systemic lupus erythematosus (SLE) and investigate the association between total, free and bioavailable vitamin D serum concentrations and disease activity. Patients with SLE (ACR 1997) consecutively seen at UNIFESP's outpatient's clinics had disease activity measured after clinical and laboratory evaluation using SLEDAI (Systemic Lupus Erythematosus Disease Activity Index). 25-hydroxyvitamin D (25(OH)D) serum concentrations measured by chemiluminescence and vitamin D binding protein (DBP) measured by ELISA were used to calculate free and bioavailable vitamin D. Healthy blood donors were used as controls. A total of 142 patients (71.4%) had 25(OH)D serum concentrations below 30 ng/mL. Total 25(OH)D serum concentration was associated with disease activity categorized in 5 continuous groups of SLEDAI. 25(OH)D serum concentrations were higher among patients with SLEDAI 1-5 and lower in those with severe activity (SLEDAI≥20) (p <0.05). On the other hand, no statistically significant difference was observed for DBP, free and bioavailable vitamin D measurements in the disease activity subgroups evaluated. Vitamin D deficiency is highly prevalent among patients with SLE and was associated with higher disease activity. DBP serum level and calculation of free and bioavailable vitamin D were not associated with SLE disease activity.

Differential capacity of therapeutic drugs to induce Rods/Rings structures in vitro and in vivo and generation of anti-Rods/Rings autoantibodies.

Some HCV patients using ribavirin and interferon alpha (IFN-α) develop anti-rods and rings (RR) autoantibodies, the main target of which is inosine monophosphate dehydrogenase (IMPDH), the rate-determining enzyme in de novo GTP biosynthesis. In vitro inhibition of IMPDH by ribavirin induces RR formation. Here we investigate whether other commonly used drugs that interfere with GTP biosynthesis can induce RR structures in vitro and vivo and elicit generation of autoantibodies. HEp-2 cells treated for 24h with ribavirin, mycophenolic acid (MPA), azathioprine, methotrexate or acyclovir were positive for RR structures. However, adefovir, entecavir, tenofovir and lamivudine did not induce RR structures in these cells. Structures induced by ribavirin in HEp-2 cells are stable after 24h drug-washout, while structures induced by other drugs are relatively labile, disappearing within 2h. Looking at patients treated with these drugs, HCV patients treated with ribavirin (n=17) showed higher average percentage of RR-positive peripheral mononuclear cells than autoimmune patients treated with RR-inducing immunosuppressant drugs (n=21). Serum from 173 autoimmune patients who had been treated with MPA, azathioprine or methotrexate was tested for presence of anti-RR autoantibodies, and only one sample was found to be positive. Conversely, of 48 anti-RR autoantibody positive samples identified at Fleury Laboratories over 30months, 94% were from HCV patients treated with ribavirin plus IFN-α. These data indicate that RR structures can be induced by a variety of drugs in vitro and in vivo, but anti-RR autoantibody production is mostly restricted to HCV patients under ribavirin+IFN-α treatment.