Autoantibodies In Sera Of Patients Research Articles

Case Report: Longitudinal Evaluation and Treatment of a Melanoma-Associated Retinopathy Patient.

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous metastatic melanoma in which patients develop vision deficits that include reduced night vision, poor contrast sensitivity, and photopsia. MAR is caused by autoantibodies targeting TRPM1, an ion channel found in melanocytes and retinal ON-bipolar cells (ON-BCs). The visual symptoms arise when TRPM1 autoantibodies enter ON-BCs and block the function of TRPM1, thus detection of TRPM1 autoantibodies in patient serum is a key criterion in diagnosing MAR. Electroretinograms are used to measure the impact of TRPM1 autoantibodies on ON-BC function and represent another important diagnostic tool for MAR. To date, MAR case reports have included one or both diagnostic components, but only for a single time point in the course of a patient's disease. Here, we report a case of MAR supported by longitudinal analysis of serum autoantibody detection, visual function, ocular inflammation, vascular integrity, and response to slow-release intraocular corticosteroids. Integrating these data with the patient's oncological and ophthalmological records reveals novel insights regarding MAR pathogenesis, progression, and treatment, which may inform new research and expand our collective understanding of the disease. In brief, we find TRPM1 autoantibodies can disrupt vision even when serum levels are barely detectable by western blot and immunohistochemistry; intraocular dexamethasone treatment alleviates MAR visual symptoms despite high levels of circulating TRPM1 autoantibodies, implicating antibody access to the retina as a key factor in MAR pathogenesis. Elevated inflammatory cytokine levels in the patient's eyes may be responsible for the observed damage to the blood-retinal barrier and subsequent entry of autoantibodies into the retina.

Cell-based vs enzyme-linked immunosorbent assay for detection of anti-Tribbles homolog 2 autoantibodies in Chinese patients with narcolepsy.

Narcolepsy type 1 is attributed to a deficiency in cerebrospinal fluid orexin and is considered linked to autoimmunity. The levels of anti-Tribbles homolog 2 (TRIB2) autoantibodies are elevated in the sera of some patients with narcolepsy with cataplexy. Additionally, injecting mice with serum immunoglobulin from patients with narcolepsy with positive anti-TRIB2 antibodies can induce hypothalamic neuron loss and alterations in sleep patterns. Consequently, we hypothesized the existence of a potential association between anti-TRIB2 antibodies and narcolepsy. To test this possibility, we used cell-based assays (CBAs) and enzyme-linked immunosorbent assays (ELISAs) to detect the presence of anti-TRIB2 antibodies in Chinese patients with narcolepsy. We included 68 patients with narcolepsy type 1, 39 patients with other central disorders of hypersomnolence, and 43 healthy controls. A CBA and a conventional ELISA were used to detect anti-TRIB2 antibody levels in patients' sera. CBA was used to detect serum anti-TRIB2 antibodies in Chinese patients with narcolepsy, and the results were negative. However, when the ELISA was used, only 2 patients with narcolepsy type 1 had TRIB2 antibody titers higher than the mean titer plus 2 standard deviations of the healthy controls. In our study, ELISA identified TRIB2 autoantibodies in sera of patients with narcolepsy where CBA failed to demonstrate them. Contrary to our hypothesis, this intriguing finding deserves further research to elucidate the potential association between TRIB2 and narcolepsy type 1. Exploring the implications of TRIB2 autoantibodies in narcolepsy and disparate outcomes between ELISA and CBA could provide crucial insights. Zhong X, Yuan Y, Zhan Q, etal. Cell-based vs enzyme-linked immunosorbent assay for detection of anti-Tribbles homolog 2 autoantibodies in Chinese patients with narcolepsy. J Clin Sleep Med. 2024;20(6):941-946.

High-throughput autoantibody profiling of different stages of Schistosomiasis japonica

Infection by the Schistosoma japonicum can result in acute, chronic and late-stage manifestations. The latter often presents with severe organ failures and premature death. Importantly, infection can also produce autoimmune phenomena reflected by the development of autoantibodies. We wished to explore and profile the presence of autoantibodies in sera of patients with different stages of S. japonicum infection with the added aim of providing a reference assisting diagnosis. Blood samples from 55 patients with chronic and 20 patients with late-stage schistosomiasis japonica together, with a control group of 50 healthy people were randomly investigated against a microarray of 121 different autoantigens. In addition, the frequency of antibodies against Schistosoma egg antigen (SEA) was examined. In the sera from patients with chronic schistosomiasis japonica, 14 different highly expressed autoantibodies were detected, while patients with late-stage schistosomiasis were found to express as many as 43 autoantibody specificities together with a significantly higher frequency of antibodies against SEA compared to the control group. The findings presented suggest that autoantibody-based classification of schistosomiasis japonica represents a promising approach for the elucidation of subtypes of the disease. This approach may reflect differential disease mechanisms, which could ultimately lead to better treatment.

Serum Autoantibodies in Patients with Dry and Wet Age-Related Macular Degeneration

To assess the serum autoantibody profile in patients with dry and exudative age-related macular degeneration compared with healthy volunteers to detect potential biomarkers, e.g., markers for progression of the disease. IgG Immunoreactivities were compared in patients suffering from dry age-related macular degeneration (AMD) (n = 20), patients with treatment-naive exudative AMD (n = 29) and healthy volunteers (n = 21). Serum was analysed by customized antigen microarrays containing 61 antigens. The statistical analysis was performed by univariate and multivariate analysis of variance, predictive data-mining methods and artificial neuronal networks were used to detect specific autoantibody patterns. The immunoreactivities of dry and wet AMD patients were significantly different from each other and from controls. One of the most prominently changed reactivity was against alpha-synuclein (p ≤ 0.0034), which is known from other neurodegenerative diseases. Furthermore, reactivities against glyceraldehyde-3-phosphat-dehydrogenase (p ≤ 0.031) and Annexin V (p ≤ 0.034), which performs a major role in apoptotic processes, were significantly changed. Some immunoreacitvities were antithetic regulated in wet and dry-AMD, such as Vesicle transport-related protein (VTI-B). Comparison of autoantibody profiles in patients with dry and wet AMD revealed significantly altered immunoreactivities against proteins particularly found in immunological diseases, further neurodegenerative, apoptotic and autoimmune markers could be observed. A validation study has to explore if these antibody pattern can help to understand the underlying differences in pathogenesis, evaluate their prognostic value and if those could be possibly useful as additional therapeutic targets.

Clinical significance of serum autoantibodies in patients with sarcoidosis

Background The association between sarcoidosis and autoimmunity has been reported for years. However, the significance of autoantibodies in the pathophysiology and clinical management of sarcoidosis is not well understood. No autoantibodies that can be used as serologic biomarkers to diagnose the disease, monitor the state of the disease, and predict the prognosis of patients are established. Methods We performed a comprehensive analysis of serum autoantibodies and analyzed their associations with clinical features of sarcoidosis. Results Patients with systemic autoimmune rheumatic diseases-associated autoantibodies had a higher prevalence of advanced radiographic stage and consolidations in high-resolution computed tomography than patients without autoantibodies (p < .05). Age, sex, clinical history, pulmonary function tests, serum angiotensin-converting enzyme levels, rheumatoid factor, and the number of involved organs were not significantly different between the two groups. Conclusions There may be an association between autoantibodies and more advanced pulmonary lesions in patients with sarcoidosis. Further investigations are needed to establish the significance of autoantibodies.

Transient positive antimitochondrial M2 in sera of patients with connective tissue diseases after intravenous immunoglobulin infusions

AbstractBackgroundAlthough antinuclear antibodies (ANAs), anti‐SSA and anti‐Ro52, are present in immunoglobulin preparations, it is unknown whether intravenous immunoglobulin (IVIG) therapy influences the testing of serum autoantibodies in patients with connective tissue diseases (CTDs). The present study aimed to investigate the dynamic change over time of serum ANA‐related autoantibodies in patients with CTDs receiving IVIG therapy.MethodsSerum ANA‐related autoantibodies were monitored in two patients with CTD before IVIG therapy and at different times after therapy. These autoantibodies were tested in different batches of immunoglobulin preparations from seven pharmaceutical companies.ResultsOne patient developed a new ANA pattern (cytoplasmic dense fine speckled pattern, AC‐19) just after IVIG therapy. Both patients developed de novo positivity for AMA‐M2 and anti‐SSA, but returned negative 1 month after IVIG therapy. The residual liquid in patients' immunoglobulin preparations showed positive ANAs with a high titer of AC‐19 (1:640), a low titer of the nuclear fine speckled pattern (AC‐4, 1:80), positive AMA‐M2, and positive anti‐SSA. ANA‐related autoantibodies were tested in 16 batches of immunoglobulin preparations and all had positive ANAs with two patterns: AC‐19 (1:640 or 1:320) and AC‐4 (1:80). AMA‐M2 and anti‐SSA were positive in 100% of the batches.ConclusionOur study highlights high‐titer AMA‐M2 autoantibodies in immunoglobulin preparations and suggests their transient transfer into a patient's circulation via IVIG therapy. To avoid incorrect clinical decisions based on postinfusion antibody titers, our data recommend retesting 1–2 months after high‐dose IVIG immunomodulatory treatment.

Evaluation of Diagnostic Accuracy of Alpha-Fodrin Antibody in Iranian Patients with Sjogren's Disease.

Background:Sjogren's syndrome, as a chronic autoimmune disease, involves in lymphocytic infiltration in the exocrine glands. As the result of exocrine glands disruption, the clinical hallmark of this disease including dryness of mouth and eyes along with fatigue and joint pain occur. However, heterogeneity of clinical presentations among newly diagnosed adult patients with Sjogren's syndrome leads to difficulty in its diagnosis. One of the diagnostic criteria for Sjogren's syndrome is the presence of autoantibodies in patient serum. One of the novel biomarkers suggested for diagnosis of Sjogren is alpha-fodrin antibody. In this study, we aimed to evaluate the diagnostic power of anti-α-fodrin antibody among the Iranian population for the first time.Materials and Methods:We recruited 82 individuals in this study. Alpha-fodrin were measured in case and control with Elisa kit as 16.71 (9.84) and 18.44 (11.54).Results:There was no any significant difference between two groups regarding alpha-fodrin level (P = 0.35). Then we applied the receiver operating characteristic (ROC) curve analysis to determine the predictive value of alpha-fodrin for diagnosing Sjogren's disease. The area under curve of the ROC curve was calculated as 0.5453. Also, there were significant association between age and alpha-fodrin antibody.Conclusions:Alpha-fodrin test did not have acceptable predictive power for predicting Sjogren's disease; however, it could be associated with disease progression.

Evidence of Classical Complement Pathway Involvement in a Subset of Patients with Warm Autoimmune Hemolytic Anemia

Introduction: Autoimmune Hemolytic Anemia (AIHA) is caused by autoantibodies that react with red blood cells (RBCs) resulting in predominantly extravascular hemolysis in an FcR and/or complement-dependent manner. In warm AIHA (wAIHA), autoantibodies are generally of the IgG isotype, while in cold agglutinin disease (CAD) they are predominantly of the IgM isotype. It is well established that the classical complement cascade is critical for the pathogenesis of CAD based on therapeutic clinical studies. Published data also suggest that complement activation plays a role in wAIHA, although it is not clear which patients would most benefit from complement-based therapy. To help address this question, we utilized an assay that measures the ability of autoantibodies in patient sera to induce complement deposition on the surface of donor RBCs (based on Meulenbroek, et al., 2015).Methods: Sera were collected retrospectively from 12 wAIHA patients whose direct antiglobulin tests (DAT) were either IgG+/C3+ or IgG+/C3-. Sera retrospectively collected from two CAD patients were used as positive controls. Individual patient sera were examined in the in vitro complement deposition assay using RBCs from type O+ healthy donors. RBCs and sera were incubated at 37 oC in the presence of either EDTA or an inhibitory antibody against C1q as inhibitors of the classical pathway. RBCs were then stained and processed by flow cytometry to determine the level of C4 deposition.Results: Sera from both CAD patients deposited C4 on the surface of ~70% of healthy human RBCs in vitro. Four out of twelve (33%) sera from wAIHA patients displayed this activity, and all four of these patients were identified as IgG+/C3+ on DAT. Complement deposition ranged from ~10-60% of the RBCs in wAIHA, suggesting heterogeneity in antibody activity for complement deposition in sera from wAIHA patients. Addition of EDTA or an inhibitory antibody against C1q fully blocked deposition of C4 on RBCs by wAIHA sera, indicating dependence of the classical complement pathway. These results indicate differences in the frequency of classical pathway involvement in CAD versus wAIHA and may help identify a subset of wAIHA patients most likely to respond to anti-C1q therapy.Conclusions: The hypothesis of classical complement cascade involvement in wAIHA disease in a subset of patients is supported by our results. Critically, complement deposition on the surface of cells by anti-C1q prevented the deposition of a downstream complement marker, C4. Inhibition of C1q has been shown to block activation of all downstream classical complement components, including C3b and C4b involved in extravascular hemolysis and C5b involved in direct cell lysis. The therapeutic potential of blocking classical complement pathway activity in wAIHA is currently being evaluated in an ongoing Phase 2 interventional trial (NCT04691570) assessing efficacy of an anti-C1q drug candidate in wAIHA patients, focusing on those with evidence of classical complement pathway activity. DisclosuresTeigler: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Low: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Rose: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Cahir-Mcfarland: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Yednock: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Kroon: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Keswani: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Barcellini: Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees.

Differential expression of sputum and serum autoantibodies in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a complex and progressive respiratory disease. Autoimmune processes have been hypothesized to contribute to disease progression; however, the presence of autoantibodies in the serum has been variable. Given that COPD is a lung disease, we sought to investigate whether autoantibodies in sputum supernatant would better define pulmonary autoimmune processes. Matched sputum and serum samples were obtained from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study and at the Guangzhou Institute of Respiratory Health (GIRH). Samples were collected from patients with varying severity of COPD, asymptomatic smokers, and healthy control subjects. IgG and IgM autoantibodies were detected in sputum and serum of all subjects in both cohorts using a broad-spectrum autoantigen array. No differences were observed in sputum autoantibodies between COPD and asymptomatic smokers in either cohort. In contrast, 16% of detectable sputum IgG autoantibodies were decreased in subjects with COPD compared to healthy controls in the ADEPT cohort. Compared to asymptomatic smokers, approximately 13% of detectable serum IgG and 40% of detectable serum IgM autoantibodies were differentially expressed in GIRH COPD subjects. Of the differentially expressed specificities, anti-nuclear autoantibodies were predominately decreased. A weak correlation between increased serum IgM anti-tissue autoantibodies and a measure of airspace enlargement was observed. The differential expression of specificities varied between the cohorts. In closing, using a comprehensive autoantibody array, we demonstrate that autoantibodies are present in subjects with COPD, asymptomatic smokers, and healthy controls. Cohorts displayed high levels of heterogeneity, precluding the utilization of autoantibodies for diagnostic purposes.

LRPAP1 autoantibodies in mantle cell lymphoma are associated with superior outcome

Low-density lipoprotein (LDL) receptor-related protein-associated protein 1 (LRPAP1) had been identified by B-cell receptor (BCR) expression cloning and subsequent protein array screening as a frequent and proliferation-inducing autoantigen of mantle cell lymphoma (MCL). Of interest, high-titered and light chain–restricted LRPAP1 autoantibodies were detected in 8 of 28 patients with MCL. In the present study, LRPAP1 autoantibodies in sera of patients treated within the Younger and Elderly trials of the European MCL Network were analyzed regarding frequency, association with disease characteristics, and prognostic impact. LRPAP1 autoantibodies were detected in 41 (13%) of 312 evaluable patients with MCL. These LRPAP1 autoantibodies belonged predominantly to the immunoglobulin G (IgG) class and were clonally light chain restricted (27 with κ light chains, 14 patients with λ light chains). Titers ranged between 1:400 and 1:3200. The presence of LRPAP1 autoantibodies was not significantly associated with any baseline clinical characteristic, however, it was associated with a superior 5-year probability for failure-free survival (FFS) of 70% (95% confidence interval [CI], 57% to 87%) vs 51% (95% CI, 44% to 58%), P = .0052; and for overall survival (OS) of 93% (95% CI, 85% to 100%) vs 68% (95% CI, 62% to 74%), P = .0142. LRPAP1-seropositive patients had a Mantle Cell Lymphoma International Prognostic Index–adjusted hazard ratio for FFS of 0.48 (95% CI 0.27-0.83, P = .0083) and for OS of 0.47 (95% CI 0.24-0.94, P = .032). LRPAP1 autoantibodies were frequently detected in a large cohort of MCL patients treated within prospective multicenter clinical trials. Our results suggest better outcomes for LRPAP1-autoantibody seropositive patients.

Association between positivity of serum autoantibodies and liver disease severity in patients with biopsy-proven NAFLD

Background and aimsSome previous studies reported serum autoantibody positivity in patients with nonalcoholic fatty liver disease (NAFLD). The clinical significance of these findings remains uncertain. We aimed to investigate the association between the presence of serum autoantibodies and liver disease severity in NAFLD. Methods and resultsA total of 388 consecutive patients with biopsy-proven NAFLD were included in the study. Various serum autoantibodies (including also anti-nuclear antibodies [ANA]) were detected by indirect immunofluorescent or immunoblotting assays. Overall, 84 (21.6%) patients with biopsy-confirmed NAFLD had positivity for at least one of the measured serum autoantibodies. ANA positivity was present in 50 (12.9%) patients, whereas anti-U1RNP or pANCA antibodies were detectable in 9 (2.3%) and 6 (1.5%) patients, respectively. Multivariate logistic regression analysis showed that ANA positivity (adjusted-odds ratio: 4.51, 95%CI: 1.77–11.5; P = 0.002) or positivity of any serum autoantibodies (adjusted-odds ratio: 3.14, 95%CI: 1.30–7.62; P = 0.01) were independently associated with advanced liver fibrosis (stages F3–F4). In serum autoantibody/ANA-positive patients, the proportion of those with advanced fibrosis was also greater among carriers of PNPLA3 rs738409 GG or CG than among those carrying PNPLA3 rs738409 CC genotype. ConclusionsSerum autoantibody positivity was independently associated with advanced liver fibrosis in patients with biopsy-proven NAFLD. The presence of serum autoantibodies in patients with advanced fibrosis occurred more frequently amongst those carrying PNPLA3 rs738409 GG or CG genotypes.

Dissociation between airway and systemic autoantibody responses in chronic obstructive pulmonary disease

BackgroundAutoimmune processes have been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the relationship between airway and systemic autoantibody responses remains unclear. The aim of this study was to elucidate this relationship in patients with stable COPD by investigating the correlation patterns between sputum and serum autoantibodies.MethodsIn this cross-sectional study, sputum supernatant and serum obtained from 47 patients with stable COPD were assayed for the presence of IgG antibodies against ten autoantigens: Smith antigen (Sm), ribosomal phosphoprotein P0 (P0), Ro/Sjögren syndrome type A antigen (Ro/SSA), La/Sjögren syndrome type B antigen (La/SSB), DNA topoisomerase I (Scl-70), histidyl-tRNA synthetase (Jo-1), U1 small nuclear ribonucleoprotein (U1-SnRNP), thyroid peroxidase (TPO), proteinase-3 (PR3), and myeloperoxidase (MPO). A second cohort of 55 stable COPD patients was recruited for validation, and a group of 59 non-COPD controls and a group of 20 connective-tissue disease-associated interstitial lung disease (CTD-ILD) patients were also recruited for comparison. Hierarchical clustering and network analysis were used to evaluate the correlation patterns between sputum and serum autoantibody profiles.ResultsBoth hierarchical clustering and network analysis showed that sputum and serum autoantibody profiles were distinct in either analytic COPD cohort or validation cohort. In contrast, the autoantibodies of the two compartments in non-COPD controls and CTD-ILD patients were inadequately distinguished using either hierarchical clustering or network analysis. Many autoantibodies in the sputum were found to have significant correlations with lung function, symptom score and frequency of prior exacerbations in COPD patients, but the antibodies in the serum were not.ConclusionsWe observed a dissociation between sputum autoantibodies and serum autoantibodies in patients with stable COPD, suggesting that airway and systemic immune status may play very different roles in the disease. Sputum autoantibodies are more clinically relevant than serum autoantibodies. Focusing on airway autoimmunity may help improve understanding of the immunopathological mechanism of COPD.

Abstract PR03: Immuno-mass spectrometric identification of serum biomarkers of response and toxicity to pembrolizumab

Abstract Immune checkpoint blockade (ICB) is a breakthrough form of cancer immunotherapy that employs antibody targeting of specific inhibitory receptors and ligands, such as cytotoxic T-lymphocyte associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed cell death ligand 1 (PD-L1). The major limitations of ICB are high cost, limited success rate (10-40%), and potential severe toxicity due to immune-related adverse effects (IRAEs), which resemble autoimmune disease. Predictive biomarkers of ICB are not currently widespread in clinical use, despite the growing need for a personalized approach to cancer treatment. Effective immunotherapy causes tumor cell death, which releases tumor-associated antigens (TAAs) into circulation. This results in abnormal presentation of these antigens to immune cells, which leads to B-cell autoantibody production against them. Autoantibodies are effective biomarkers of some autoimmune diseases and may be present before disease onset. We hypothesized that patients who develop immune-related toxicity from immunotherapy will produce specific autoantibodies that are indicative of an autoimmune-like response. Furthermore, we hypothesized that responders to pembrolizumab will develop high titers of serum autoantibodies against TAAs, indicative of a strong humoral immune response to TAAs released during immunotherapy. Likewise, nonresponders will have low levels of these autoantibodies, due to a weaker or nonexistent antitumor and humoral immune response. We used a novel immuno-mass spectrometry method to screen for autoantibodies in the sera of patients with various tumors treated with PD-1 inhibition in the clinical trial called INSPIRE (INvestigator-initiated Phase II Study of Pembrolizumab Immunological Response Evaluation; NCT02644369) at Princess Margaret Cancer Centre. Our methodology involves immunoprecipitation of proteome-wide target antigens of autoantibodies in patient sera with the use of protein G magnetic beads, followed by shotgun mass spectrometry analysis. We analyzed autoantibody responses in the sera before and after immunotherapy initiation in a total of 24 patients, subdivided into 4 patient groups based on their objective response and toxicity status. Candidate autoantibody target antigens, including thyroglobulin, thyroid peroxidase, and ficolin-2, were identified by our pilot study. Validation with additional datasets is planned. Furthermore, we identified PD-1 as an antibody target exclusively in the post-immunotherapy patient sera of all 4 patient groups. This finding confirms the efficacy of our method since pembrolizumab is a humanized antibody targeting PD-1. Predictive biomarkers of cancer immunotherapy will save significant resources, ensure proper patient selection for cancer treatment, and spare certain patients from the toxic effects of immunotherapy. This abstract is also being presented as Poster B05. Citation Format: Milena Music, Marco Iafolla, Antoninus Soosaipillai, Ihor Batruch, Ioannis Prassas, Lillian L. Siu, Eleftherios P. Diamandis. Immuno-mass spectrometric identification of serum biomarkers of response and toxicity to pembrolizumab [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR03.

Autoantibodies as diagnostic markers and potential drivers of inflammation in ulcerative colitis.

To date, no comprehensive analysis of autoantibodies in sera of patients with ulcerative colitis has been conducted. To analyze the spectrum of autoantibodies and to elucidate their role serum-IgG from UC patients (n = 49) and non-UC donors (n = 23) were screened by using a human protein microarray. Screening yielded a remarkable number of 697 differentially-reactive at the nominal 0·01 significance level (FDR<0·1) of the univariate test between the UC and the non-UC group. CD99 emerged as a biomarker to discriminate between both groups (p = 1e-04, AUC = 0·8). In addition, cytokines, chemokines and growth factors were analyzed by Olink’s Proseek® Multiplex Inflammation-I 96×96 immuno-qPCR assay and 31 genes were significant at the nominal 0.05 level of the univariate test to discriminate between UC and non-UC donors. MCP-3, HGF and CXCL-9 were identified as the most significant markers to discriminate between UC patients with clinically active and inactive disease. Levels of CXCL10 (cor = 0.3; p = 0.02), CCL25 (cor = 0.25; p = 0.04) and CCL28 (cor = 0.3; p = 0.02) correlated positively with levels of anti CD99. To assess whether autoantibodies are detectable prior to diagnosis with UC, sera from nine donors at two different time points (T-early, median 21 months and T-late, median 6 months) were analyzed. 1201 features were identified with higher reactivity in samples at time points closer to clinical UC presentation. In vitro, additional challenge of peripheral mononuclear cells with CD99 did not activate CD4+ T cells but induced the secretion of IL-10 (-CD99: 20.21±20.25; +CD99: 130.20±89.55; mean ±sd; p = 0.015). To examine the effect of CD99 in vivo, inflammation and autoantibody levels were examined in NOD/ScidIL2Rγnull mice reconstituted with PBMC from UC donors (NSG-UC). Additional challenge with CD99 aggravated disease symptoms and pathological phenotype as indicated by the elevated clinical score (-CD99: 1·85 ± 1·94; +CD99: 4·25 ± 1·48) and histological score (-CD99: 2·16 ± 0·83; +CD99: 3·15 ± 1·16, p = 0·01). Furthermore, levels of anti-CD99 antibodies increased (Control: 398 ± 323; mean MFI ± sd; Ethanol + PBS: 358 ±316; Ethanol + CD99: 1363 ± 1336; Control versus Ethanol + CD99: p = 0.03). In a highly inflammatory environment, frequencies of pro-inflammatory M1 monocytes (CD14+ CD64+: unchallenged 8.09±4.72; challenged 14.2±8.62; p = 0.07; CD14+ CD1a+: unchallenged 16.29 ±6.97; challenged 43.81±14.4, p = 0.0003) increased and levels of autoantibodies in serum decreased in the NSG-UC mouse model. These results suggest that autoantibodies are potent biomarkers to discriminate between UC and non-UC and indicate risk to develop UC. In an inflammatory environment, auto-antibodies may promote the pathological phenotype by activating M1 monocytes in the NSG-UC animal model and also in patients with UC.

Keratinocyte footprint assay discriminates antilaminin-332 pemphigoid from all other forms of pemphigoid diseases.

SummaryBackgroundAntilaminin‐332 mucous membrane pemphigoid is a chronic severe pemphigoid disease characterized by autoantibodies to laminin‐332. At present no commercial assay is available to demonstrate antilaminin‐332 antibodies, and diagnosis relies on in‐house techniques with limited sensitivities.ObjectivesIn order to move, keratinocytes cultured in vitro secrete laminin‐332 to attach to the culture dish. In that way, they leave behind a unique footprint trail of laminin‐332. We aimed to develop a sensitive and specific laboratory assay to determine antilaminin‐332 autoantibodies in patient serum based on binding of patient IgG to these unique footprints.MethodsNormal human keratinocytes were grown on glass coverslips and incubated with patient or control serum for 1 h. The binding of IgG was then investigated by immunofluorescence. After validating the test for its ability to identify antilaminin‐332 autoantibodies it was converted into a daily available test based on binding of IgG to dried coverslips that can be stored frozen. The staining patterns of sera from patients with antilaminin‐332 pemphigoid were then compared with those of sera from patients with other autoimmune bullous diseases and normal human sera.ResultsIgG of all antilaminin‐332 pemphigoid sera (n = 16) bound to laminin‐332 footprints, while all normal human controls (n = 55) were negative. From the sera of patients with other diseases (n = 72) four sera tested positive. The footprint assay was also positive for sera that were negative by salt‐split skin analysis, demonstrating that it is a very sensitive technique.ConclusionsThe keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin‐332 autoantibodies. What's already known about this topic? Antilaminin‐332 mucous membrane pemphigoid is a severe form of pemphigoid, and patients may have an increased risk of malignancies.The diagnosis of antilaminin‐332 mucous membrane pemphigoid is complicated by the lack of specific commercial tests for antilaminin‐332 antibodies and can be confirmed only in specialized laboratories.Keratinocytes in culture need laminin‐332 for adhesion and migration and therefore deposit it on the bottom of the culture dish. What does this study add? The keratinocyte footprint assay detects antilaminin‐332 autoantibodies in patient serum using the native laminin‐332 produced by cultured keratinocytes. What is the translational message? The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin‐332 autoantibodies.

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

BackgroundAutoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets.MethodsSerum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry.ResultsThe protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn’s disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control.ConclusionsThis study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.

Anti-kelch-like 12 autoantibodies in serum of patients with primary biliary cholangitis

Anti-kelch-like 12 autoantibodies in serum of patients with primary biliary cholangitis

AB043. P-11. Autoantibody screening in sera of patients with cholangiocarcinoma by serological proteomic approach

Background: Cholangiocarcinoma (CCA) is the second most common primary hepatic neoplasia. Antibodies are normally produced in response to a foreign protein or substance within the body. Autoantibodies can be used clinically for cancer detection which can be identified by proteomic analysis of tumor associated antigens (TAAs) that are potentially involved in malignant transformation. The purpose of the present study was to detect the autoantibodies in sera of patients with cholangiocarcinoma by serological proteomic approach for early cancer diagnosis.

Do elevated autoantibodies in patients with multiple sclerosis matter?

The incidence and clinical impact of serum autoantibodies in patients with multiple sclerosis (MS) are controversially discussed. The aim of the study was to reassess the value of elevated serum autoantibodies in our MS study cohort. In total, 176 MS patients were retrospectively analyzed for coexistence and clinical impact of increased serum autoantibody levels. The 18.8% of the MS cohort showed elevated serum autoantibody levels, but only 10.2% of all MS patients were diagnosed with a further autoimmune disease (AI). Patients with elevated serum autoantibodies (AABS) were not significantly more often diagnosed with a clinical manifest AI as compared to patients with negative autoantibodies (P=0.338). MS patients with disease duration of more than 10years showed no significant increase of positive autoantibodies as compared to patients with a more recent disease onset (P=1). MS patients with elevated serum autoantibodies did not exhibit a significantly worse disease course (P=0.428). According to our data, elevated serum autoantibodies do not have the potential to serve as a prognostic tool for disease severity in patients with MS Since MS patients with positive serum AABS did not significantly more often suffer from clinical manifest AIs than MS patients with negative serum AABS, the role of routine testing of serum AABS in MS patients should be critically called into question.

Evaluation of Auto-antibody Profiles in Patients with Tuberculosis

Background: Tuberculosis remains a major public health problem internationally, causing 9.6 million new cases and 1.5 million deaths worldwide, Tuberculosis (TB) has become one of the most important diseases in the past two decades. It leads to organ dysfunction, mortality and various clinical manifestations. Previous studies have shown that sera from patients with active TB may contain autoantibodies that are unique in autoimmune diseases. Objective: To detect the prevalence of wide array of autoantibodies in sera of patients with tuberculosis compared with healthy control subject. Patients and Methods: A consecutive patients with recently diagnosed pulmonary and extra pulmonary {axial & peripheral joints TB} , mean age 36.2 years . 41 males and 21 females . the autoantibodies are R.F lgM, ANA , anti cls DNA. P-ANCA , ACL, ENA,SSA, SSB . RNP SM SCL70}. Results: Mean duration of symptoms 15.69 & 15.70(SD) months . 82% had fever , 39.3% had cough and hemoptysis . 27% had arthralgia and myalgia , 31.1% are diabetic and 24% are smoker serum level above the upeer normal limits were found in 25.7% of patients with RF ,ANA was 8.8% , 20% , 6.3% in pulmonary , peripheral , axial TB respectively , P-ANCA 10% , 6.3% & for anti DNA was 5% , 10% ,7 6.3% for pulmonary , peripheral & axial :ACL was 33.3 , 12.5 pulmonary , axial TB,ENA was {SCL70 3.2% , SM 3.2% , RNP3.2%, SSA 8.1% SSB 6.5%}. Conclusion: R F: Rheumatoid Factor--Anti ds DNA: Anti-deoxyribonucleic acid (double strand).-ACL: Anti cardiolipin Antibody.-ANCA: Anti neutrophil cytoplasmic Antibody.-ANA: Anti nuclear Antibody.-ENA: Extractable Nuclear Antigen. S: Significant. N-S: Not Significant.Pul: Pulmonary.TB: Tuberculosis .AFB:Acidfastbacilli.