Plasma Autoantibodies Research Articles

Deficiency of CFHR plasma proteins and autoantibody positive hemolytic uremic syndrome: treatment rationale, outcomes, and monitoring.

Deficiency of Complement Factor H Related (CFHR) plasma proteins and Autoantibody Positive Hemolytic Uremic Syndrome (DEAP-HUS) is a subtype of atypical hemolytic uremic syndrome, known to be associated with significant morbidity. Its pathogenesis is linked to the production of IgG autoantibodies against complement factor H, a regulator of the alternative complement pathway. The binding of the autoantibodies to the C terminal of complement factor H interferes with its regulatory function, leading to increased activation of the alternative complement pathway and consequent endothelial cellular damage. Early diagnosis and initiation of appropriate therapy is reported to lead to favorable outcomes. Institution of plasma exchange therapy within 24h of diagnosis has been shown to rapidly lower antibody levels, leading to clinical improvement. Adjunctive immunosuppression therapy suppresses antibody production and helps in maintaining long-term clinical remission of the disease. Available data advocates a treatment regimen that combines plasma therapy (preferably plasma exchange) and immunosuppression to halt disease process and sustain long-term disease remission.

Anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura do not hamper ELISA‐based quantification of ADAMTS13 antigen

BackgroundThe biological diagnosis of immune‐mediated thrombotic thrombocytopenic purpura (iTTP) is based on determination of ADAMTS13 activity (<10%) and anti‐ADAMTS13 autoantibodies. ADAMTS13 antigen levels are not routinely measured in iTTP patients, but studies have shown that antigen levels are a valuable prognostic factor. ObjectivesTo (a) report the validation of our in‐house developed ADAMTS13 antigen enzyme‐linked immunosorbent assay (ELISA) and determine ADAMTS13 antigen in a large cohort of healthy donor and iTTP patient plasma samples; and (b) to investigate whether ADAMTS13 antigen determination is not disturbed by the presence of anti‐ADAMTS13 autoantibodies. MethodsOur in‐house ADAMTS13 antigen ELISA was validated in terms of sensitivity, repeatability, and reproducibility. ADAMTS13 antigen levels were determined in plasma samples from 423 healthy donors and 112 acute iTTP patients. Purified IgGs from iTTP patients were added to normal human plasma to determine whether anti‐ADAMTS13 autoantibodies hampered ADAMTS13 antigen determination. ResultsOur in‐house ADAMTS13 antigen ELISA has a detection limit of 3% and low intra‐assay (coefficient of variation, %CV < 10%) and inter‐assay (%CV < 18%) variability. ADAMTS13 antigen levels were significantly reduced (P < .0001) in acute iTTP patients (15 ± 18%) compared to healthy donors (101 ± 18%). The anti‐ADAMTS13 autoantibodies in plasma of iTTP patients did not impede ADAMTS13 antigen determinations using our in‐house ELISA. ConclusionsOur in‐house ADAMT13 antigen ELISA is a powerful tool to correctly determine ADAMTS13 antigen levels in iTTP patients, which supports routine ADAMTS13 antigen measurements in these patients to have better insight into disease prognosis.

Adults with systemic lupus exhibit distinct molecular phenotypes in a cross-sectional study

BackgroundThe clinical and pathologic diversity of systemic lupus erythematosus (SLE) hinders diagnosis, management, and treatment development. This study addresses heterogeneity in SLE through comprehensive molecular phenotyping and machine learning clustering. MethodsAdult SLE patients (n = 198) provided plasma, serum, and RNA. Disease activity was scored by modified SELENA-SLEDAI. Twenty-nine co-expression module scores were calculated from microarray gene-expression data. Plasma soluble mediators (n = 23) and autoantibodies (n = 13) were assessed by multiplex bead-based assays and ELISAs. Patient clusters were identified by machine learning combining K-means clustering and random forest analysis of co-expression module scores and soluble mediators. FindingsSLEDAI scores correlated with interferon, plasma cell, and select cell cycle modules, and with circulating IFN-α, IP10, and IL-1α levels. Co-expression modules and soluble mediators differentiated seven clusters of SLE patients with unique molecular phenotypes. Inflammation and interferon modules were elevated in Clusters 1 (moderately) and 4 (strongly), with decreased T cell modules in Cluster 4. Monocyte, neutrophil, plasmablast, B cell, and T cell modules distinguished the remaining clusters. Active clinical features were similar across clusters. Clinical SLEDAI trended highest in Clusters 3 and 4, though Cluster 3 lacked strong interferon and inflammation signatures. Renal activity was more frequent in Cluster 4, and rare in Clusters 2, 5, and 7. Serology findings were lowest in Clusters 2 and 5. Musculoskeletal and mucocutaneous activity were common in all clusters. InterpretationMolecular profiles distinguish SLE subsets that are not apparent from clinical information. Prospective longitudinal studies of these profiles may help improve prognostic evaluation, clinical trial design, and precision medicine approaches. FundingUS National Institutes of Health

Analysis of plasma autoantibodies for inflammatory cytokines in patients with first-episode schizophrenia among a Chinese population

Neuroinflammation has been considered to be involved in the development of schizophrenia. This study aimed to study circulating autoantibodies for inflammatory cytokines in first-episode schizophrenia. A total of 181 patients and 197 controls were recruited for detection of plasma IgG antibodies against peptide antigens derived from interleukin 1α (IL1α), IL1ß, IL6, IL8 and tumour necrosis factor alpha (TNFα). The major finding was that patients with schizophrenia had significantly higher levels of anti-IL1ß IgG, anti-IL6 IgG and anti-IL8 IgG, and a significantly lower level of anti-IL1α IgG. This study suggests that inflammatory response may contribute to the development of schizophrenia.

Tissue-Resident PSGL1loCD4+ T Cells Interact with B Cells Via PD-1 and PD-L2 in Gvhd Target Tissues to Augment Autoimmune-like Chronic Gvhd

Chronic graft versus host disease (cGVHD) is a systemic autoimmune-like syndrome, and donor-type CD4+ T interaction with B cells plays an important role in its pathogenesis. Our recent publication has demonstrated that cGVHD pathogenesis does not require germinal center formation and is associated with expansion of extrafollicular CD44hiCD62LloPSGL1loCD4+ (PSGL1loCD4+) T cells in the spleen and GVHD target tissues. The causal role of PSGL1loCD4+ T cells in cGVHD pathogenesis remains unclear. With murine and humanized murine models, we show that both murine and human PSGL1loCD4+ T cells reside in tissues and do not circulate in the blood, and their high expression of PD-1 and ICOS and production of IL-21 suggest that they provide B cell help. In murine models, PSGL1loCD4+ T cells augment plasma cell expansion and autoantibody production via their PD-1 interaction with PD-L2 on B. In humanized mouse models, human PSGL1loCD4+ T cells from GVHD target tissues augment memory B cell differentiation into plasma cells and antibody production via their production of IL-21.These results indicate that tissue-resident PSGL1loCD4+ T cells augment autoantibody production and cGVHD pathogenesis. Targeting tissue-resident T cells may represent a novel approach for treating cGVHD. Disclosures Nakamura: Alexion: Other: support to a lecture at a Japan Society of Transfusion/Cellular Therapy meeting ; Kirin Kyowa: Other: support for an academic seminar in a university in Japan; Celgene: Other: support for an academic seminar in a university in Japan; Merck: Membership on an entity's Board of Directors or advisory committees. Martin:Abgenomics: Research Funding; Enlivex Therapeutics: Consultancy; Genentech: Consultancy, Other: One-time advisory board; Neovii: Other: One-time advisory board; Pfizer: Other: Data and Safety Monitoring Committee; Pharmacyclics LLC: Other: One-time advisory board; Procter and Gamble: Equity Ownership; Xenikos: Research Funding.

SLE Plasma Profiling Identifies Unique Signatures of Lupus Nephritis and Discoid Lupus

Systemic lupus erythematosus (SLE) impacts multiple organ systems, although the causes of many individual SLE pathologies are poorly understood. This study was designed to elucidate organ-specific inflammation by identifying proteins that correlate with SLE organ involvement and to evaluate established biomarkers of disease activity across a diverse patient cohort. Plasma proteins and autoantibodies were measured across seven SLE manifestations. Comparative analyses between pathologies and correlation with the SLE Disease Activity Index (SLEDAI) were used to identify proteins associated with organ-specific and composite disease activity. Established biomarkers of composite disease activity, SLE-associated antibodies, type I interferon (IFN), and complement C3, correlated with composite SLEDAI, but did not significantly associate with many individual SLE pathologies. Two clusters of proteins were associated with renal disease in lupus nephritis samples. One cluster included markers of infiltrating leukocytes and the second cluster included markers of tissue remodelling. In patients with discoid lupus, a distinct signature consisting of elevated immunoglobulin A autoantibodies and interleukin-23 was observed. Our findings indicate that proteins from blood samples can be used to identify protein signatures that are distinct from established SLE biomarkers and SLEDAI and could be used to conveniently monitor multiple inflammatory pathways present in different organ systems.

A Link Between Plasma Microbial Translocation, Microbiome, and Autoantibody Development in First-Degree Relatives of Systemic Lupus Erythematosus Patients.

Systemic lupus erythematosus (SLE) is characterized by the production of antibodies against self antigens. However, the events underlying autoantibody formation in SLE remain unclear. This study was undertaken to investigate the role of plasma autoantibody levels, microbial translocation, and the microbiome in SLE. Plasma samples from 2 cohorts, one with 18 unrelated healthy controls and 18 first-degree relatives and the other with 19 healthy controls and 21 SLE patients, were assessed for autoantibody levels by autoantigen microarray analysis, measurement of lipopolysaccharide (LPS) levels by Limulus amebocyte assay, and determination of microbiome composition by microbial 16S ribosomal DNA sequencing. First-degree relatives and SLE patients exhibited increased plasma autoantibody levels compared to their control groups. Parents and children of lupus patients exhibited elevated plasma LPS levels compared to controls (P = 0.02). Plasma LPS levels positively correlated with plasma anti-double-stranded DNA IgG levels in first-degree relatives (r = 0.51, P = 0.03), but not in SLE patients. Circulating microbiome analysis revealed that first-degree relatives had significantly reduced microbiome diversity compared to their controls (observed species, P = 0.004; Chao1 index, P = 0.005), but this reduction was not observed in SLE patients. The majority of bacteria that were differentially abundant between unrelated healthy controls and first-degree relatives were in the Firmicutes phylum, while differences in bacteria from several phyla were identified between healthy controls and SLE patients. Bacteria in the Paenibacillus genus were the only overlapping differentially abundant bacteria in both cohorts, and were reduced in first-degree relatives (adjusted P [Padj ] = 2.13 × 10-12 ) and SLE patients (Padj = 0.008) but elevated in controls. These results indicate a possible role of plasma microbial translocation and microbiome composition in influencing autoantibody development in SLE.

Longitudinal Changes of Plasma ADAMTS13 Activity, Antigen, and Autoantibodies in Patients With Immune-Mediated Thrombotic Thrombocytopenic Purpura

Abstract Background Severe deficiency of plasma ADAMTS13 activity resulting from anti-ADAMTS13 IgG is the primary cause of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Anti-ADAMTS13 IgG may bind and inhibit plasma ADAMTS13 activity and/or accelerate clearance of ADAMTS13 from the circulation. The present study aims to determine the initial and longitudinal changes of plasma ADAMTS13 activity, antigen, and anti-ADAMTS13 IgG and their relationships with clinical responses and outcomes of patients with iTTP after standard treatment. Methods Thirty-eight patients with a confirmed diagnosis of iTTP who underwent therapeutic plasma exchange (TPE) at UAB Medical Center were enrolled into the study. The study spanned from May 2015 to December 2018. An informed consent was obtained from each participant. Clinical and laboratory information was extracted from the electronic medical record and stored in the Alabama TTP Registry database. Plasma samples were collected prior to the initiation of and every 3 to 5 days after TPE until discharge. Plasma ADAMTS13 activity, antigen, and anti-ADAMTS13 IgG were determined using commercially available reagents. Mann-Whitney test, Fisher’s exact test, Spearman rank correlation, Cox proportional hazard regression, and Kaplan-Meier survival analysis were used to determine statistical significances. Results The median age of this cohort was 46.5 years old; 26 (68%) patients were female and 12 (32%) were male. Twenty-three patients (60%) were experiencing their initial episode while 15 (39%) had an exacerbation/relapse at the time of enrollment. All patients were diagnosed based on the findings of thrombocytopenia, microangiopathic hemolytic anemia, plasma ADAMTS13 activity (&lt;10 U/dL), and inhibitor titer ≥0.4 BU or elevated anti-ADAMTS13 IgG. Following treatment with standard therapy (TPE, corticosteroids, and/or other immunosuppressives), plasma levels of ADAMTS13 activity and antigen increased with a concurrent reduction of anti-ADAMTS13 IgG. However, there were at least three distinct patterns of dynamic changes of these markers over time: (1) rapid increase, (2) slow increase or fluctuation, and (3) persistently low. More interestingly, those with the highest quartile of anti-ADAMTS13 IgG (HR = 4.2) and inhibitor titer ≥1.2 BU (HR = 3.2) at presentation, ADAMTS13 activity &lt;20 U/dL 3 to 7 days during therapy (HR = 2.5), and ADAMTS13 activity &lt;20 U/dL (HR = 3.2) or the lowest quartile of ADAMTS13 antigen at clinical response (HR = 2.8) were all associated with a higher risk of TTP exacerbation (the disease recurred within 30 days following a sustained normalization of platelet counts). Conclusion Initial and longitudinal assessment of plasma ADAMTS13 activity, antigen, inhibitor titer, and anti-ADAMTS13 IgG may be useful not only for diagnosis but also for predicting the risk of exacerbation. This may influence how we select a therapeutic modality for a better outcome. Long-term follow-up is necessary to determine whether these ADAMTS13 biomarkers at patient discharge are predictive of relapse and mortality.

Schizophrenia Plasma Autoantibodies Promote ‘Biased Agonism’ at the 5-Hydroxytryptamine 2A Receptor: Neurotoxicity is Positively Modulated by Metabotropic Glutamate 2/3 Receptor Agonism

To test whether neurite-inhibitory plasma autoantibodies in chronic schizophrenia activate Gq/11- and Gi- coupled signaling pathways downstream of 5-hydroxytryptamine 2A receptor activation; and for modulation of serotonergic signaling by the metabotropic 2/3 receptor agonist LY379268. Plasma from five older adults with chronic schizophrenia and eight age-matched patients having another neuropsychiatric, immune or metabolic disorder was subjected to Protein-A affinity chromatography to obtain IgG autoantibodies. Mean neurite retraction (5 minutes) or cell survival (24 hours) was determined in mouse N2A neuroblastoma cells incubated with autoantibodies in the presence or absence of specific antagonists of the Gq/11/PLC/IP3R signaling pathway, Gi-coupled, beta-arrestin2-directed pathways, or LY379268. Chronic schizophrenia plasma autoantibodies- mediated dose- and time-dependent acute N2A neurite retraction was completely prevented by M100907, a selective 5-hydroxytryptamine 2A receptor antagonist. LY379268 promoted autoantibody-induced neurite retraction causing a shift-to-the-left in the dose-response curve. Antagonists of the RhoA/Rho kinase and Gq/11/PLC/IP3R signaling pathways blocked autoantibody-mediated neurite retraction. Chronic schizophrenia plasma autoantibodies mediated increased N2A cell survival which was blocked by LY379268, pertussis toxin, and antagonists of PI3-kinase- mediated survival signaling. Schizophrenia plasma autoantibodies activate the 5-hydroxytryptamine 2A receptor positively coupled to Gq/11/PLC/IP3R pathway and RhoA/Rho kinase signaling activation in promoting acute N2A cell neurite retraction. Autoantibodies in a subset of patients experiencing hallucinations promoted increased N2A cell survival mediated (in part) via a pertussis-toxin sensitive, Gi-coupled, PI3-kinase-dependent mechanism. Positive modulation of 5-HT2AR-mediated neurite retraction by LY379268 suggests the autoantibodies may target (in part) the 5-HT2AR/mGlu2R heteromer.

Docosahexaenoic Acid (DHA) Suppresses Broad Spectrum of Pathogenic Autoantibodies Elicited in Murine Model of Lupus Flaring (OR12-03-19)

ObjectivesIntranasal instillation of lupus-prone mice with crystalline silica (cSiO2), a respirable particle linked to human lupus, mimics human lupus flaring by accelerating systemic autoimmunity and glomerulonephritis. Notably, these effects were prevented when mouse diets were supplemented with DHA. A broad-spectrum pathogenic autoantibodies (AAs) can be elicited relative to specificity and isotype during human lupus, each of which could differentially contribute to tissue injury. Here, we assessed how DHA influences the autoantibody spectrum over time in this novel preclinical model. MethodsFemale NZBWF1 mice at 6 weeks(wks) of age were fed isocaloric diets AIN-93 G supplemented with DHA a 0, 4, or 10 g/kg diet, beginning either 2 wk before cSiO2 instillation (prevention) or 1 wk after cSiO2 instillation (treatment). For both groups, the mice were intranasally instilled with 1 mg cSiO2 or saline vehicle alone once per wk for 4 wks, starting at 8 wks of age. Cohorts were sacrificed 1, 5, 9, or 13 wks post-instillation (PI) of the last cSiO2 dose. Plasma was subjected to high-throughput AA profiling of different isotypes (IgG, IgM, IgA, and IgE) using a microarray containing 120 autoantigens. ResultscSiO2 triggered production of IgG AAs against components from the cell nucleus (nucleic acids and associated proteins), complement family, cytoplasm, membrane, and matrix proteins within 9 and 13 wk PI A supplementation before and after cSiO2 installation significantly inhibited the IgG AAs production. Importantly, known lupus markers such as anti-DNA, anti-RNP, anti-SM, anti-histone, anti-ds DNA, anti-Ro/SSA, and anti-La/SSB and anti-complement AAs, were inhibited with DHA. Spontaneous elevation of some IgG AAs in plasma, likely genome-driven, was evident at 13 wk in control groups compared to earlier time points. The prevention strategy with DHA showed a more robust effect compared to therapeutic strategy. The preventive effects of DHA treatment on IgM and IgA AAs also mimicked the effect on IgG at 13 wks. ConclusionsOur results highlight a newly identified function of DHA in suppressing production of pathogenic AAs. The data provide preclinical proof of concept for the use of DHA supplementation as an adjunct to established therapeutics for preventing and treating the flaring of lupus and other autoimmune diseases. Funding SourcesNIEHS grant, Lupus Foundation, and Dr. Robert and Carol Diebel Family Endowment.

Human MOSPD2: A bacterial Lmb mimicked auto-antigen is involved in immune infertility

Autoantibody production is one of the leading factors of immune infertility, an autoimmune disease of the male reproductive system. The potential involvement of MHC-class II derived self-peptides against bacterial proteins in the antisperm antibody (ASA) production has been reported previously. Apparently, Streptococcus agalactiae has been considered as an important pathogen to impart infection-induced infertility in a bacteriospermia associated leukocytospermia (LCS/BS) state. Hence, the present study attempts to confirm S. agalactiae specific Laminin binding protein (Lmb) derived self-peptide (‘KDSYTKKAKAFKKEA’) namely human Motile Sperm domain-containing protein 2 (MOSPD2) as an auto-antigen in LCS/BS condition. Semen samples were collected from infertile men with LCS/BS (n ​= ​17) and their fertile counterparts (n ​= ​10). Gram-positive bacteria were predominantly identified in the entire 17 LCS samples using culture method followed by 16S rDNA sequencing technique. TLRs 2 and 4 expression used as markers of immune response in spermatozoa and sperm dysfunction were elevated in the LCS/BS spermatozoa as compared to their fertile counterparts. A significant increase in oxidative stress indices i.e., protein carbonylation, lipid peroxidation and acridine orange test (AOT), was also observed in the LCS/BS spermatozoa. Spermatozoa lysate (both auto and heterologous), bacterial lysate (control) and synthesized MOSPD2 self-peptide were used to test their antigenicity against the autoantibodies by rocket immunoelectrophoresis (RIEP) assay. Seminal plasma from LCS/BS patients with S. agalactiae was used as the source of autoantibodies. Spermatozoa and bacteria lysate; and MOSPD2 self-peptide were able to bind autoantibodies in the seminal plasma. Besides, the self-peptide showed a dose dependent increase in the precipitation of antibody. T-cell epitope mapping of 48 Enterococcus faecalis and 91Staphylococcus aureus surface proteins confirmed MOSPD2 as a global auto-antigen. Thus, augmentation of TLR expression in LCS/BS spermatozoa inferred MOSPD2 to be a putative immunogen. Altogether, these findings will delineate the significance of MOSPD2 auto-antigen in a bacteria derived immune infertility condition.

Inhibition of IL-17 ameliorates systemic lupus erythematosus in Roquinsan/san mice through regulating the balance of TFH cells, GC B cells, Treg and Breg

Systemic lupus erythematosus (SLE) is mediated by a chronic and dysregulated inflammatory response. Interleukin (IL)-17, a proinflammatory cytokine, and T helper (Th)17 cells are associated with chronic autoimmune diseases. We hypothesized that inhibition of IL-17 would decrease the numbers of T cell subsets that function as B-cell helpers, as well as B-cell differentiation into plasma cells and autoantibody expression. The IL-17 level was increased markedly in Roquinsan/san mice. Loss of IL-17 in Roquinsan/san mice improved nephritis by downregulating immunoglobulin (Ig)G, IgG1, and IgG2a production. Formation of germinal centers (GCs), and follicular B- and T-cell differentiation was reduced, whereas the number of regulatory T (Treg) cells and immature B cells was increased, by IL-17 deficiency in Roquinsan/san mice. These results suggest that IL-17 inhibition can ameliorate SLE by inhibiting B-cell differentiation into GCs. Therefore, IL-17–producing Th17 cells show promise as a target for development of novel therapeutics for SLE.

Endothelial Cell Growth Promoting Activity in Graves’ Disease Sera is Neutralized by Anti-Basic Fibroblast Growth Factor Antibodies in Patients with Fat Expansive but Not Infiltrative Orbitopathy

To report a case of orbital fat expansion leading to globe prolapse in a Graves' disease patient undergoing high-dose glucocorticoid therapy. To evaluate the growth factor receptor specificities of plasma autoantibodies in Graves' disease patients who exhibited contrasting subtypes of thyroid-associated ophthalmopathy, i.e. orbital fat expansion-type vs. infiltrative. Sera from Graves' orbitopathy and control patients with or without Graves' disease were subjected to protein-A affinity chromatography to obtain immunoglobulin G. A (1/50th to 1/1600th) range in dilutions of the protein-A eluate fraction was incubated for four days at 37 degrees C with bovine pulmonary artery endothelial cells to test for endothelial cell inhibition or stimulation. Growth stimulatory autoantibodies were co-incubated with specific neutralizing anti-insulin like growth factor 1 receptor antibodies or anti-basic fibroblast growth factor antibodies to assess autoantibody specificity in contrasting Graves' orbitopathy subtypes. We observed increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from eighteen patients with active Graves' disease (117 ± 28%, n = 18) compared to mean endothelial cell activity (89 ± 10%, n = 13, P = 0.003) in thirteen adults without Graves' disease. The protein-A eluate fraction in acute infiltrative-type Graves' orbitopathy contained a high titer (> 1:1000) of endothelial cell stimulatory activity which was significantly neutralized by specific monoclonal anti-human insulin-like growth factor 1 receptor antibodies. The protein-A eluate fraction in fat expansion-type Graves' orbitopathy contained endothelial cell inhibitory activity (at low titers) and stimulatory activity (at high titers), and the latter stimulatory activity was completely neutralized by specific anti-basic fibroblast growth factor antibodies. Graves' disease suffering globe prolapse secondary to marked orbital fat-expansion had coexisting plasma fibroblast growth factor-inhibitory and -stimulatory autoantibodies. The latter was completely neutralized by anti-basic fibroblast growth factor antibodies.

An ELISA for quantifying GPIHBP1 autoantibodies and making a diagnosis of the GPIHBP1 autoantibody syndrome

BackgroundAutoantibodies against GPIHBP1, the endothelial cell transporter for lipoprotein lipase (LPL), cause severe hypertriglyceridemia (“GPIHBP1 autoantibody syndrome”). Affected patients have low serum GPIHBP1 and LPL levels. We report the development of a sensitive and specific ELISA, suitable for routine clinical use, to detect GPIHBP1 autoantibodies in serum and plasma. MethodsSerum and plasma samples were added to wells of an ELISA plate that had been coated with recombinant human GPIHBP1. GPIHBP1 autoantibodies bound to GPIHBP1 were detected with an HRP-labeled antibody against human immunoglobulin. Sensitivity, specificity, and reproducibility of the ELISA was evaluated with plasma or serum samples from patients with the GPIHBP1 autoantibody syndrome. ResultsA solid-phase ELISA to detect and quantify GPIHBP1 autoantibodies in human plasma and serum was developed. Spiking recombinant human GPIHBP1 into the samples reduced the ability of the ELISA to detect GPIHBP1 autoantibodies. The ELISA is reproducible and sensitive; it can detect GPIHBP1 autoantibodies in samples diluted by >1000-fold. ConclusionWe have developed a sensitive and specific ELISA for detecting GPIHBP1 autoantibodies in human serum and plasma; this assay will make it possible to rapidly diagnose the GPIHBP1 autoantibody syndrome.

NF-κB-94ins/del ATTG Genotype Contributes to the Susceptibility and Imbalanced Th17 Cells in Patients with Immune Thrombocytopenia.

Background The NLRP3 inflammasome plays important roles in the pathogenesis of autoimmune diseases. However, the role of the NLRP3 inflammasome in the pathophysiology of immune thrombocytopenia (ITP) remains unclear. Methods RT-PCR was used to examine the polymorphism and expression of genes involved in the NLRP3 inflammasome in ITP patients. T helper cells and apoptosis of PBMC from ITP patients were analyzed by flow cytometry. The antiplatelet autoantibodies in plasma were determined by modified monoclonal antibody-specific immobilization of platelet antigens (MAIPA). Results We found that the NF-κB-94ins/del ATTG genotype contributed to the susceptibility of ITP. Furthermore, the platelet counts of ITP patients with the WW genotype or WD genotype were lower than those with the DD genotype of NF-κB-94ins/del ATTG. Compared with controls, NF-κB gene expression was significantly decreased and WW or WD genotype ITP patients displayed higher mRNA expression than DD individuals. Similarly, the mRNA expression of NLRP3 was also increased in the WW genotype. There was a significant gene dose effect of the percentage of Th17 cells for the WW, WD, and DD genotypes (WW < WD < DD) in the unstimulated group and no significant difference was found after being stimulated. The activation of the NLRP3 inflammasome could upregulate Th17 in ITP patients. Conclusion The NF-κB-94ins/del ATTG genotype might serve as a novel biomarker and potential target for ITP.

Circulating Neurotoxic 5-HT2A Receptor Agonist Autoantibodies in Adult Type 2 Diabetes with Parkinson's Disease.

To test whether circulating neurotoxic autoantibodies increase in adult type 2 diabetes mellitus with Parkinson's disease (PD) or dementia. To identify the G-protein coupled receptor on neuroblastoma cells mediating neural inhibitory effects in diabetic Parkinson's disease plasma autoantibodies. To determine the mechanism of accelerated neuroblastoma cell death and acute neurite retraction induced by diabetic Parkinson's disease and dementia autoantibodies. Protein-A eluates from plasma of twelve older adult male diabetic patients having Parkinson's disease (n=10) or dementia (n=2), and eight age-matched control diabetic patients were tested for ability to cause accelerated N2A neuroblastoma cell death and acute neurite retraction. Specific antagonists of G protein coupled receptors belonging to the G alpha q subfamily of heterotrimetric G-proteins, the phospholipase C/inositol triphosphate/Ca2+ pathway, or the RhoA/Rho kinase pathway were tested for ability to block diabetic Parkinson's disease/dementia autoantibody-induced neurite retraction or N2A accelerated cell loss. Sequential Liposorber LA-15 dextran sulfate cellulose/protein-A affinity chromatography was used to obtain highly-purified fractions of diabetic Parkinson's disease autoantibodies. Mean accelerated neuroblastoma cell loss induced by diabetic Parkinson's disease or dementia autoantibodies significantly exceeded (P = 0.001) the level of N2A cell loss induced by an identical concentration of the diabetic autoantibodies in control patients without these two co-morbid neurodegenerative disorders. Co-incubation of diabetic Parkinson's disease and dementia autoantibodies with two-hundred nanomolar concentrations of M100907, a highly selective 5-HT2AR antagonist, completely prevented autoantibody-induced accelerated N2A cell loss and neurite retraction. A higher concentration (500 nM-10μM) of alpha-1 adrenergic, angiotensin II type 1, or endothelin A receptor antagonists did not substantially inhibit autoantibody-induced neuroblastoma cell death or prevent neurite retraction. Antagonists of the inositol triphosphate receptor (2-APB, 50μM), the intracellular calcium chelator (BAPTA-AM, 30 μM) and Y27632 (10 μM), a selective RhoA/Rho kinase inhibitor, each completely blocked acute neurite retraction induced by sixty nanomolar concentrations of diabetic Parkinson's disease autoantibodies. Co-incubation with 2-APB (1-2 μM) for 8 hours' prevented autoantibody-induced N2A cell loss. The highly-purified fraction obtained after Liposorber LA/protein-A affinity chromatography in hypertriglyceridemic diabetic dementia and Parkinson's disease plasmas had apparent MWs > 30 kD, and displayed enhanced N2A toxicity requiring substantially higher concentrations of 5-HT2AR antagonists (M100907, ketanserin, spiperone) to effectively neutralize. These data suggest increased autoantibodies in older adult diabetes with Parkinson's disease or dementia cause accelerated neuron loss via the 5-hydroxytryptamine 2 receptor coupled to inositol triphosphate receptor-mediated cytosolic Ca2+ release.

Multiplex immunoassay detection of autoimmune disease autoantibodies in serum and plasma

Abstract Autoantibody formation is principal to the pathogenesis of a variety of autoimmune diseases. Dysregulated apoptosis and the subsequent defective clearance of cellular debris leads to the exposure of autoantigens and the generation of autoantibodies. The presence of autoantibodies may indicate disease activity, prognosis, and clinical associations related to a variety of autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren’s Syndrome, Scleroderma, Polymyositis/Dermatomyositis, and various overlap syndromes of these diseases. Here we report the development of a multiplex immunoassay to monitor 20 autoantibodies present in blood that are involved in a variety of autoimmune diseases: SSA/Ro60, SSA/Ro52, SSB/La, RNP, RNP/Sm, Sm, Ribosomal P, Proteinase 3, Myeloperoxidase, PCNA, β 2-Glycoprotein, CENP-A, CENP-B, Scl-70, Jo-1, C1q, PM/Scl-100, Ku, Mi-2, and PL-12. Using the 20-plex immunoassay, we measured expression of autoantibodies in serum and plasma from SLE patients with various disease manifestations and overlap syndromes, healthy controls, and CDC reference samples available for 11 autoantibodies. Additionally, control beads were included in the immunoassay. Levels of SSA/Ro60, SSA/Ro52, SSB/La, RNP, RNP/Sm, and Sm matched clinical laboratory features noted at the time of SLE sample collection. Additionally, anti-C1q antibody levels were associated with lupus nephritis. The CDC controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly. This study demonstrates the value of using multiplex technology to evaluate multiple autoantibodies, allowing for an extensive autoantibody profile to evaluate patients with various autoimmune diseases.

Direct correlation between plasma microbial translocation and autoantibodies in first-degree relatives of patients with systemic lupus erythematosus

Abstract Background The process of events underlying autoantibody production in systemic lupus erythematosus (SLE) is unknown. Methods Plasma from 36 female African-Americans (18 unrelated healthy controls (UHCs) and 18 first-degree relatives of SLE patients (FDRs)) were assessed for autoantibodies by autoantibody array, lipopolysaccharide (LPS) levels by the limulus amebocyte assay and microbiome by 16S rDNA analysis. Results Compared to UHCs, FDRs exhibited elevated plasma autoantibody levels but not similar total IgGs. The median plasma IgGs were 346 (interquartile range, 236–465) and 480 (286–891) for anti-double strand DNA (anti-dsDNA, P = 0.04, Mann-Whitney U-test), 3422 (2863–4076) and 4815 (3447–8464) for anti-single strand DNA (anti-ssDNA, P = 0.03), and 4593 (4050–5521) and 4714 (4004–5278) for total IgGs (P = 0.95), in UHCs and FDRs respectively. Furthermore, parent/child of patients exhibited elevated plasma LPS compared to UHCs. Plasma LPS levels were 59.1 (53.3–68.6) and 71.2 (62.2–80.4) for UHCs and parent/child of patients respectively (P = 0.03). Notably, plasma LPS was positively correlated with IgG anti-dsDNA (r = 0.53, P = 0.02, Spearman correlation) and anti-ssDNA (r = 0.53, P = 0.02) in FDRs but not in UHCs (P &amp;gt; 0.70). Plasma microbiome revealed a reduced diversity (P &amp;lt; 0.02) and lower abundance of Streptococcaceae (unadjusted P = 0.01, adjusted for multiple comparisons P = 0.10) in FDRs compared to UHCs. The ratios of Firmicutes to Bacteroidetes were 6.75 (0.66–42.5) in UHCs and 3.37 (0.63–22.5) in FDRs but did not achieve significance. Conclusion Taken together, these results indicate a possible role for plasma microbial translocation and microbiome composition in autoantibody development in SLE.

Peli1 negatively regulates noncanonical NF-\u03baB signaling to restrain systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by uncontrolled secretion of autoantibodies by plasma cells. Although the functional importance of plasma cells and autoantibodies in SLE has been well established, the underlying molecular mechanisms of controlling autoantibody production remain poorly understood. Here we show that Peli1 has a B cell-intrinsic function to protect against lupus-like autoimmunity in mice. Peli1 deficiency in B cells induces autoantibody production via noncanonical NF-κB signaling. Mechanically, Peli1 functions as an E3 ligase to associate with NF-κB inducing kinase (NIK) and mediates NIK Lys48 ubiquitination and degradation. Overexpression of Peli1 inhibits noncanonical NF-κB activation and alleviates lupus-like disease. In humans, PELI1 levels negatively correlate with disease severity in SLE patients. Our findings establish Peli1 as a negative regulator of the noncanonical NF-κB pathway in the context of restraining the pathogenesis of lupus-like disease.

Identification of the antigenic epitopes of maternal autoantibodies in autism spectrum disorders

Several groups have described the presence of fetal brain-reactive maternal autoantibodies in the plasma of some mothers whose children have autism spectrum disorder (ASD). We previously identified seven autoantigens targeted by these maternal autoantibodies, each of which is expressed at significant levels in the developing brain and has demonstrated roles in typical neurodevelopment. To further understand the binding repertoire of the maternal autoantibodies, as well as the presence of any meaningful differences with respect to the recognition and binding of these ASD-specific autoantibodies to each of these neuronal autoantigens, we utilized overlapping peptide microarrays incubated with maternal plasma samples obtained from the Childhood Autism Risk from Genetics and Environment (CHARGE) Study. In an effort to identify the most commonly recognized (immunodominant) epitope sequences targeted by maternal autoantibodies for each of the seven ASD-specific autoantigens, arrays were screened with plasma from mothers with children across diagnostic groups (ASD and typically developing (TD)) that were positive for at least one antigen by western blot (N = 67) or negative control mothers unreactive to any of the autoantigens (N = 18). Of the 63 peptides identified with the discovery microarrays, at least one immunodominant peptide was successfully identified for each of the seven antigenic proteins using subsequent selective screening microarrays. Furthermore, while limited by our relatively small sample size, there were peptides that were distinctly recognized by autoantibodies relative to diagnosis For example, reactivity was observed exclusively in mothers of children of ASD towards several peptides, including the LDH-B peptides DCIIIVVSNPVDILT (9.1% ASD vs. 0% TD; odds ratio (95% CI) = 6.644 (0.355–124.384)) and PVAEEEATVPNNKIT (5.5% ASD vs. 0% TD; odds ratio (95% CI) = 4.067 (0.203–81.403)).These results suggest that there are differences in the binding repertoire between the antigen positive ASD and TD maternal samples. Further, the autoantibodies in plasma from mothers of children with ASD bound to a more diverse set of peptides, and there were specific peptide binding combinations observed only in this group. Future studies are underway to determine the critical amino acids necessary for autoantibody binding, which will be essential in developing a potential therapeutic strategy for maternal autoantibody related (MAR) ASD.