Multiplex Autoantibody Detection in Patients with Autoimmune Polyglandular Syndromes

The diagnosis of autoimmune polyglandular syndrome (APS) types 1/2 is difficult due to their rarity and nonspecific clinical manifestations. APS-1 development can be identified with assays for autoantibodies against cytokines, and APS-2 development with organ-specific antibodies. In this study, a microarray-based multiplex assay was proposed for simultaneous detection of both organ-specific (anti-21-OH, anti-GAD-65, anti-IA2, anti-ICA, anti-TG, and anti-TPO) and APS-1-specific (anti-IFN-ω, anti-IFN-α-2a, and anti-IL-22) autoantibodies. Herein, 206 serum samples from adult patients with APS-1, APS-2, isolated autoimmune endocrine pathologies or non-autoimmune endocrine pathologies and from healthy donors were analyzed. The prevalence of autoantibodies differed among the groups of healthy donors and patients with non-, mono- and multi-endocrine diseases. APS-1 patients were characterized by the presence of at least two specific autoantibodies (specificity 99.5%, sensitivity 100%). Furthermore, in 16 of the 18 patients, the APS-1 assay revealed triple positivity for autoantibodies against IFN-ω, IFN-α-2a and IL-22 (specificity 100%, sensitivity 88.9%). No anti-cytokine autoantibodies were found in the group of patients with non-APS-1 polyendocrine autoimmunity. The accuracy of the microarray-based assay compared to ELISA for organ-specific autoantibodies was 88.8–97.6%. This multiplex assay can be part of the strategy for diagnosing and predicting the development of APS.

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Defective Suppressor Function of Human CD4+ CD25+ Regulatory T Cells in Autoimmune Polyglandular Syndrome Type II

Journal of Experimental Medicine ◽

10.1084/jem.20032158 ◽

2004 ◽

Vol 199 (9) ◽

pp. 1285-1291 ◽

Cited By ~ 260

Author(s):

Martin A. Kriegel ◽

Tobias Lohmann ◽

Christoph Gabler ◽

Norbert Blank ◽

Joachim R. Kalden ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Autoimmune Diseases ◽

Type I ◽

Type Ii ◽

Central Tolerance ◽

Autoimmune Polyglandular Syndrome ◽

Healthy Donors ◽

Organ Specific ◽

Autoimmune Polyglandular Syndrome Type

In autoimmune polyglandular syndromes (APS), several organ-specific autoimmune diseases are clustered. Although APS type I is caused by loss of central tolerance, the etiology of APS type II (APS-II) is currently unknown. However, in several murine models, depletion of CD4+ CD25+ regulatory T cells (Tregs) causes a syndrome resembling human APS-II with multiple endocrinopathies. Therefore, we hypothesized that loss of active suppression in the periphery could be a hallmark of this syndrome. Tregs from peripheral blood of APS-II, control patients with single autoimmune endocrinopathies, and normal healthy donors showed no differences in quantity (except for patients with isolated autoimmune diseases), in functionally important surface markers, or in apoptosis induced by growth factor withdrawal. Strikingly, APS-II Tregs were defective in their suppressive capacity. The defect was persistent and not due to responder cell resistance. These data provide novel insights into the pathogenesis of APS-II and possibly human autoimmunity in general.

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A Rapid Immunochromatography Test Based on Hcp1 Is a Potential Point-of-Care Test for Serological Diagnosis of Melioidosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00346-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 8

Author(s):

Phornpun Phokrai ◽

Wisansanee Karoonboonyanan ◽

Nida Thanapattarapairoj ◽

Chidchanok Promkong ◽

Adul Dulsuk ◽

...

Keyword(s):

Point Of Care ◽

Clinical Manifestations ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Target Antigen ◽

Northeast Thailand ◽

Serum Samples ◽

Serological Method ◽

Healthy Donors ◽

Wide Range

ABSTRACTMelioidosis is a fatal infectious disease caused by the environmental bacteriumBurkholderia pseudomallei. It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations, and definitive diagnosis requires bacterial culture, which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by enzyme-linked immunosorbent assay (ELISA) that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects, including the following: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors; and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively, and the specificity for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA results with a kappa value of 0.829, indicating that the method is much improved compared with the current serological method, the indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as in resource-limited areas.

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Multiple Autoantibodies Display Association with Lymphopenia, Proteinuria, and Cellular Casts in a Large, Ethnically Diverse SLE Patient Cohort

Autoimmune Diseases ◽

10.1155/2012/819634 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Rufei Lu ◽

Julie M. Robertson ◽

Benjamin F. Bruner ◽

Joel M. Guthridge ◽

Barbara R. Neas ◽

...

Keyword(s):

High Throughput ◽

Lupus Erythematosus ◽

Conditional Logistic Regression ◽

Clinical Manifestations ◽

Study Participant ◽

Ethnically Diverse ◽

Racial Groups ◽

Serum Samples ◽

Autoantibody Detection ◽

Ribosomal P

Purpose. This study evaluates high-throughput autoantibody screening and determines associated systemic lupus erythematosus (SLE) clinical features in a large lupus cohort.Methods. Clinical and demographic information, along with serum samples, were obtained from each SLE study participant after appropriate informed consent. Serum samples were screened for 10 distinct SLE autoantibody specificities and examined for association with SLE ACR criteria and subcriteria using conditional logistic regression analysis.Results. In European-American SLE patients, autoantibodies against 52 kD Ro and RNP 68 are independently enriched in patients with lymphopenia, anti-La, and anti-ribosomal P are increased in patients with malar rash, and anti-dsDNA and anti-Sm are enriched in patients with proteinuria. In African-American SLE patients, cellular casts associate with autoantibodies against dsDNA, Sm, and Sm/nRNP.Conclusion. Using a high-throughput, bead-based method of autoantibody detection, anti-dsDNA is significantly enriched in patienets with SLE ACR renal criteria as has been previously described. However, lymphopenia is associated with several distinct autoantibody specificities. These findings offer meaningful information to allow clinicians and clinical investigators to understand which autoantibodies correlate with select SLE clinical manifestations across common racial groups using this novel methodology which is expanding in clinical use.

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POS0755 PREVALENCE AND RELEVANCE OF ANTIBODIES AGAINST CITRULLINATED ALPHA ENOLASE (ANTI-CEP1) IN CONNECTIVE TISSUE DISEASES

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2825 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 630.1-630

Author(s):

A. Alunno ◽

O. Bistoni ◽

F. Carubbi ◽

M. Antonucci ◽

S. Calvacchi ◽

...

Keyword(s):

Connective Tissue ◽

Lupus Erythematosus ◽

Bone Erosion ◽

Disease Onset ◽

Connective Tissue Diseases ◽

Clinical Manifestations ◽

Igg Antibodies ◽

Serum Samples ◽

Healthy Donors

Background:Anti-citrullinated alpha enolase antibodies have been investigated in rheumatoid arthritis and associated with bone erosion and interstitial lung disease but little is known about their prevalence and role in connective tissue diseases (CTDs).Objectives:The aim of this study was to investigate the prevalence and relevance of anti-CEP1 antibodies in CTDs.Methods:Serum samples from five independent patient cohorts were assessed: 1) established (est) primary Sjogren’s syndrome (pSS) N=78, 2) est-systemic lupus erythematosus (SLE) N=52, 3) est-systemic sclerosis (SSc) N=71, 4) pSS at disease onset N=30, 5) SLE at disease onset N=46 (cohorts 4 and 5 had at least 3 years of follow-up). Samples from ninety sex and age matched healthy donors (HD) and 200 patients with est-RA (disease controls) were also tested. Anti-CEP1 IgG antibodies were measured with a commercially available ELISA kit (Euroimmun, Luebeck, Germany).Results:Anti-CEP1 titer was significantly higher in est-pSS, est-SLE and est-SSc compared to HD, significantly lower in est-pSS and est-SSc compared to est-RA and comparable in est-SLE versus est-RA. We divided patients in every CTD group based on whether their anti-CEP1 titer was below or above the 25th, 50th and 75th percentile. In est-SLE anti-CEP1 values over the 25th percentile were associated with articular involvement (odds ratio, OR (95% confidence interval, CI)=11.5; 1.9-70.6, p=0.008). In est-pSS, no relationship between anti-CEP1>25th percentile and articular involvement was found but rather an association with rheumatoid factor positivity (OR (95% CI)=4.8, 1.6-14.1, p=0.004) and salivary gland swelling (OR (95% CI)=6.2, 1.3-29.1, p=0.021). In est-SSc no difference could be detected across the 3 groups. Anti-CEP-1 titers in pSS and SLE at onset did not differ from each other, were comparable also to those of HD and significantly lower than those of est-pSS, est-SLE and est-RA patients (all p<0.0001).). Of interest, we could retrieve a serum sample collected at the time of diagnosis for 5 patients from the cohort of established pSS and we observed that anti-CEP1 titers were significantly lower at pSS onset than during follow up (at least 12 months after the diagnosis, p=0.0024). No difference was observed in the clinical presentation at disease onset according to different anti-CEP1 titer and they did not predict the development of new clinical manifestations during follow-up.Conclusion:Anti-CEP-1 antibodies can be detected in CTDs at different title during the disease course and may increase overtime, at least in pSS. Although anti-CEP1 antibodies are associated with specific clinical manifestation in est-CTDs, such as articular involvement in est-SLE, they seem to lack a predictive value for future manifestations when measured at disease onset.References:[1]Alunno A, Bistoni O, Pratesi F et al Rheumatology (Oxford) 2018.[2]Manca ML, Alunno A, D’Amato C et al. Joint Bone Spine 2018.Disclosure of Interests:None declared

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Reassessing the Role of the Active TGF-β1 as a Biomarker in Systemic Sclerosis: Association of Serum Levels with Clinical Manifestations

Disease Markers ◽

10.1155/2016/6064830 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 13

Author(s):

Andréa Tavares Dantas ◽

Sayonara Maria Calado Gonçalves ◽

Anderson Rodrigues de Almeida ◽

Rafaela Silva Guimarães Gonçalves ◽

Maria Clara Pinheiro Duarte Sampaio ◽

...

Keyword(s):

Systemic Sclerosis ◽

Clinical Manifestations ◽

Serum Levels ◽

Vascular Involvement ◽

Digital Ulcers ◽

Serum Samples ◽

Peripheral Blood Mononuclear ◽

Pbmc Culture ◽

Significant Difference ◽

Skin Biopsies

Objective. To determine active TGF-β1 (aTGF-β1) levels in serum, skin, and peripheral blood mononuclear cell (PBMC) culture supernatants and to understand their associations with clinical parameters in systemic sclerosis (SSc) patients.Methods. We evaluated serum samples from 56 SSc patients and 24 healthy controls (HC). In 20 SSc patients, we quantified spontaneous or anti-CD3/CD28 stimulated production of aTGF-β1 by PBMC. The aTGF-β1 levels were measured by ELISA. Skin biopsies were obtained from 13 SSc patients and six HC, and TGFB1 expression was analyzed by RT-PCR.Results. TGF-β1 serum levels were significantly higher in SSc patients than in HC (p< 0.0001). Patients with increased TGF-β1 serum levels were more likely to have diffuse subset (p= 0.02), digital ulcers (p= 0.02), lung fibrosis (p< 0.0001), positive antitopoisomerase I (p= 0.03), and higher modified Rodnan score (p= 0.046). Most of our culture supernatant samples had undetectable levels of TGF-β1. No significant difference in TGFB1 expression was observed in the SSc skin compared with HC skin.Conclusion. Raised active TGF-β1 serum levels and their association with clinical manifestations in scleroderma patients suggest that this cytokine could be a marker of fibrotic and vascular involvement in SSc.

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Transcriptomic Data Analysis Reveals a Down-Expression of Galectin-8 in Schizophrenia Hippocampus

Brain Sciences ◽

10.3390/brainsci11080973 ◽

2021 ◽

Vol 11 (8) ◽

pp. 973

Author(s):

Maria Cristina Petralia ◽

Rosella Ciurleo ◽

Alessia Bramanti ◽

Placido Bramanti ◽

Andrea Saraceno ◽

...

Keyword(s):

Inflammatory Responses ◽

Clinical Manifestations ◽

Antipsychotic Agents ◽

Standards Of Care ◽

Specific Gene ◽

Contributing Factors ◽

Healthy Donors ◽

Dorsolateral Prefrontal ◽

Inflammatory Processes ◽

Disease Specific

Schizophrenia (SCZ) is a severe psychiatric disorder with several clinical manifestations that include cognitive dysfunction, decline in motivation, and psychosis. Current standards of care treatment with antipsychotic agents are often ineffective in controlling the disease, as only one-third of SCZ patients respond to medications. The mechanisms underlying the pathogenesis of SCZ remain elusive. It is believed that inflammatory processes may play a role as contributing factors to the etiology of SCZ. Galectins are a family of β-galactoside-binding lectins that contribute to the regulation of immune and inflammatory responses, and previous reports have shown their role in the maintenance of central nervous system (CNS) homeostasis and neuroinflammation. In the current study, we evaluated the expression levels of the galectin gene family in post-mortem samples of the hippocampus, associative striatum, and dorsolateral prefrontal cortex from SCZ patients. We found a significant downregulation of LGALS8 (Galectin-8) in the hippocampus of SCZ patients as compared to otherwise healthy donors. Interestingly, the reduction of LGALS8 was disease-specific, as no modulation was observed in the hippocampus from bipolar nor major depressive disorder (MDD) patients. Prediction analysis identified TBL1XR1, BRF2, and TAF7 as potential transcription factors controlling LGALS8 expression. In addition, MIR3681HG and MIR4296 were negatively correlated with LGALS8 expression, suggesting a role for epigenetics in the regulation of LGALS8 levels. On the other hand, no differences in the methylation levels of LGALS8 were observed between SCZ and matched control hippocampus. Finally, ontology analysis of the genes negatively correlated with LGALS8 expression identified an enrichment of the NGF-stimulated transcription pathway and of the oligodendrocyte differentiation pathway. Our study identified LGALS8 as a disease-specific gene, characterizing SCZ patients, that may in the future be exploited as a potential therapeutic target.

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Atypical anti-glomerular basement membrane disease with anti-GBM antibody negativity and ANCA positivity: a case report

BMC Nephrology ◽

10.1186/s12882-021-02232-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Na Guo ◽

Qinghua Yin ◽

Song Lei ◽

Yanjun He ◽

Ping Fu

Keyword(s):

Basement Membrane ◽

Glomerular Basement Membrane ◽

Rare Case ◽

Test Results ◽

Serum Samples ◽

Tissue Samples ◽

Case Presentation ◽

Severe Renal Insufficiency ◽

Laboratory Test Results ◽

Organ Specific

Abstract Background Anti-glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disease that involves the lung and kidneys and leads to rapid glomerulonephritis progression, with or without diffuse alveolar hemorrhage, and even respiratory failure. Classic cases of anti-GBM disease are diagnosed based on the presence of the anti-GBM antibody in serum samples and kidney or lung biopsy tissue samples. However, atypical cases of anti-GBM disease are also seen in clinical practice. Case presentation We herein report the rare case of a patient with atypical anti-GBM disease whose serum was negative for the anti-GBM antibody but positive for the myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (p-ANCA) and another atypical ANCA. Laboratory test results showed severe renal insufficiency with a creatinine level of 385 μmol/L. Renal biopsy specimen analysis revealed 100% glomeruli with crescents; immunofluorescence showed immunoglobulin G (IgG) linearly deposited alongside the GBM. Finally, the patient was discharged successfully after treatment with plasmapheresis, methylprednisolone and prednisone. Conclusion This patient, whose serum was negative for the anti-GBM antibody but positive for p-ANCA and another atypical ANCA, had a rare case of anti-GBM disease. Insights from this unusual case might help physicians diagnose rare forms of glomerulonephritis and treat affected patients in a timely manner.

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Expanding Assay Dynamics: A Combined Competitive and Direct Assay System for the Quantification of Proteins in Multiplexed Immunoassays

Clinical Chemistry ◽

10.1373/clinchem.2007.099812 ◽

2008 ◽

Vol 54 (6) ◽

pp. 956-963 ◽

Cited By ~ 17

Author(s):

Michael Hartmann ◽

Monika Schrenk ◽

Anette Döttinger ◽

Sarah Nagel ◽

Johan Roeraade ◽

...

Keyword(s):

Dynamic Range ◽

Protein Microarray ◽

Measurement Range ◽

Multiplex Assay ◽

Competitive System ◽

Direct Immunoassay ◽

Sandwich Configuration ◽

Readout Channel ◽

Autoantibody Detection ◽

Detection And Quantification

Abstract Background: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. Methods: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system’s dynamic range is possible. Results: We implemented the method in a planar protein microarray–based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray–based system reduced spot-to-spot variation and intraassay variation. Conclusions: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

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Enoxaparin-induced Wunderlich syndrome in a young patient with anti-GAD 65-associated opsoclonus and limbic encephalitis: a rare complication in a rare disease

BMJ Case Reports ◽

10.1136/bcr-2021-244916 ◽

2021 ◽

Vol 14 (10) ◽

pp. e244916

Author(s):

Saranya B Gomathy ◽

Animesh Das ◽

Awadh Kishor Pandit ◽

Achal Kumar Srivastava

Keyword(s):

Rare Disease ◽

Young Patient ◽

Clinical Condition ◽

Limbic Encephalitis ◽

Rare Complication ◽

Clinical Manifestations ◽

Autoimmune Encephalitis ◽

Rare Condition ◽

Gad 65 ◽

Good Improvement

Wunderlich syndrome is a rare condition characterised by acute spontaneous non-traumatic renal haemorrhage into the subcapsular and perirenal spaces. Our case of anti-GAD65-associated autoimmune encephalitis (AE), aged 30 years, developed this complication following use of enoxaparin and was managed by selective glue embolisation of subsegmental branches of right renal cortical arteries. Our case had opsoclonus as one of the clinical manifestations, which has till now been described in only two patients of this AE. This patient received all forms of induction therapies (steroids, plasmapheresis, intravenous immunoglobulin and rituximab) following which she had good improvement in her clinical condition. The good response to immunotherapy is also a point of discussion as this has been rarely associated with anti-GAD65 AE.

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New Serology Assays Can Detect Gluten Sensitivity among Enteropathy Patients Seronegative for Anti–Tissue Transglutaminase

Clinical Chemistry ◽

10.1373/clinchem.2009.129668 ◽

2010 ◽

Vol 56 (4) ◽

pp. 661-665 ◽

Cited By ~ 29

Author(s):

Emilia Sugai ◽

Hui Jer Hwang ◽

Horacio Vázquez ◽

Edgardo Smecuol ◽

Sonia Niveloni ◽

...

Keyword(s):

Villous Atrophy ◽

Tissue Transglutaminase ◽

Simultaneous Detection ◽

Mucosal Damage ◽

Intestinal Biopsy ◽

Gluten Sensitivity ◽

Serum Samples ◽

Antibody Isotypes ◽

Igg Isotypes ◽

Inova Diagnostics

Abstract Background: Some patients with celiac disease (CD) may be seronegative with the commonly used test for IgA anti–tissue transglutaminase (anti-tTG) antibodies. Our aim was to explore whether newer assays incorporating synthetic deamidated gliadin-related peptides (DGPs) or other TG isoenzymes as antigen are useful for detecting gluten sensitivity in IgA anti-tTG–seronegative patients. Methods: We assayed serum samples obtained at diagnosis from (a) anti-tTG–seronegative patients with a CD-like enteropathy (n = 12), (b) skin biopsy–proven dermatitis herpetiformis (DH) patients (n = 26), and (c) IgA anti-tTG–positive CD patients (n = 26). All patients had typical total IgA concentrations. All patients underwent intestinal biopsy and serum testing for (a) detection of IgA and IgG isotypes of both anti-DGP and anti-tTG in a single assay (tTG/DGP Screen; INOVA Diagnostics), (b) simultaneous detection of both IgA and IgG anti-DGP antibody isotypes (DGP Dual; INOVA Diagnostics), and (c) detection of antibodies to transglutaminase 3 (TG3) or transglutaminase 6 (TG6). Results: All anti-tTG–seropositive patients also tested positive in anti-DGP assays. Overall, tTG/DGP Screen detected 6 (31.6%) of the 19 anti-tTG seronegatives, and anti-DGP Dual produced positive results in 5 (26.3%) of these cases. Whereas both assays detected 2 anti-tTG–negative DH patients with partial villous atrophy, they were positive in only 2 of the 5 cases with no histologically discernible mucosal damage. Testing for antibodies to TG3 and TG6 identified 7 (36.8%) of the 19 anti-tTG–negative patients, 5 of which were also positive for anti-DGP. Conclusions: Detection of anti-DGP with tTG/DGP Screen or anti-DGP Dual, or detection of antibodies to other TG isoenzymes, enhances the sensitivity for detecting gluten sensitivity among non–IgA- deficient, anti-tTG–seronegative patients with CD-like enteropathy.

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