Autoantibody Activity Research Articles

'That Obscure Object of Desire': in systemic lupus erythematosus B-cell activating factor/B-lymphocyte stimulator is targeted both by the immune system and by physicians.

Systemic lupus erythematosus (SLE) is characterized by autoantibodies that mediate tissue injury. However, the pathogenesis of SLE remains poorly understood and available therapeutic approaches are not fully satisfactory. Belimumab, a monoclonal antibody that neutralizes B-cell activating factor (BAFF), was the first drug approved to treat SLE in more than 50 years. However, it is not labelled for use in severe lupus nephritis. Recently, a novel high-throughput multiplex protein microarray platform to profile circulating immunoglobulin G (IgG) autoantibodies in SLE patients identified IgG autoantibodies against several cytokines and growth factors at higher titres in SLE patients than in controls. The presence of autoantibodies to BAFF was validated in a subset of SLE patients by enzyme-linked immunosorbent assay. Low levels of anti-BAFF autoantibodies were also present in healthy controls. The association of anti-BAFF reactivity to clinical features and response to therapy was not addressed. However, preliminary data suggested an association to an interferon-α-responsive mRNA signature, itself associated with severity. Functional studies disclosed a neutralizing activity of autoantibodies against BAFF. These findings raise new questions regarding the role of BAFF in SLE and the functional and therapeutic significance of anti-BAFF and anti-cytokine autoantibodies.

Atrial tachyarrhythmias induced by the combined effects of β1/2-adrenergic autoantibodies and thyroid hormone in the rabbit.

Activating autoantibodies (AAb) to β-adrenergic receptors (βAR) are associated with atrial fibrillation in patients with Graves' disease. In the present study, we examined the interaction of thyroid hormone with β1/2AR-AAb in inducing atrial tachyarrhythmias in the rabbit. Immunization of rabbits with a β1AR or β2AR second extracellular loop peptide produced high titers of β1AR-AAb or β2AR-AAb. Thyroid hormone in combination with β1AR-AAb or β2AR-AAb induced a significant number of sustained sinus tachycardia and atrial tachycardia, respectively. Both combinations resulted in significantly increased inductions of sustained arrhythmias compared to AAb alone. Thyroid hormone alone induced sustained sinus and junctional tachycardia. Sera from immunized rabbits specifically bound to and activated β1AR or β2AR in transfected cells in vitro. This study demonstrates thyroid hormone qualitatively accentuates the specific arrhythmogenic action of these AAb and quantitatively enhances their rate. Our data support a dual role of AAb and thyroid hormone in Graves'-associated tachyarrhythmias.

AB0018 Confirmation of Genetic Association between Rs6822844 at the Il2–Il21 Region and Rheumatoid Arthritis in an Algerian Population

BackgroundIncreasing evidence suggest that single nucleotide polymorphism (SNP) rs6822844, within KIAA1109-TENR-IL2-IL21 gene cluster, is strongly associated with autoimmune diseases in Caucasian population, including rheumatoid arthritis (RA) [1, 2, 3].ObjectivesThe aim...

Evidence for Follicle-stimulating Hormone Receptor as a Functional Trimer

Follicle-stimulating hormone receptor (FSHR), a G-protein coupled receptor, is an important drug target in the development of novel therapeutics for reproductive indications. The FSHR extracellular domains were observed in the crystal structure as a trimer, which enabled us to propose a novel model for the receptor activation mechanism. The model predicts that FSHR binds Asnα(52)-deglycosylated FSH at a 3-fold higher capacity than fully glycosylated FSH. It also predicts that, upon dissociation of the FSHR trimer into monomers, the binding of glycosylated FSH, but not deglycosylated FSH, would increase 3-fold, and that the dissociated monomers would in turn enhance FSHR binding and signaling activities by 3-fold. This study presents evidence confirming these predictions and provides crystallographic and mutagenesis data supporting the proposed model. The model also provides a mechanistic explanation to the agonist and antagonist activities of thyroid-stimulating hormone receptor autoantibodies. We conclude that FSHR exists as a functional trimer.

Abstract 256: Activation of Cell Surface GRP78 by Anti-GRP78 Autoantibodies Accelerates Lesion Development by Promoting Endothelial Cell Activation

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is a molecular chaperone localized to the endoplasmic reticulum (ER) in healthy cells that functions to assist in the correct folding of newly synthesized proteins and to prevent aggregation of folding intermediates. In addition, GRP78 is also present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a membrane signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies isolated from the serum of cancer patients. We have demonstrated previously that high levels of anti-GRP78 autoantibodies accelerate lesion development in apoE -/- mice. However, the role of anti-GRP78 autoantibody activation of cell surface GRP78 on endothelial cells and how this contributes to atherogenesis is unknown. The objective of this study is to identify factors that mediate surface GRP78 expression on endothelial cells and to determine the mechanism by which surface GRP78 activation on endothelial cells contributes to atherogenesis. Here we demonstrate that induction of ER stress by tunicamycin increased surface GRP78 expression in cultured human aortic endothelial cells. We also show that activation of surface GRP78 on endothelial cells by anti-GRP78 autoantibodies significantly increases gene expression of the adhesion molecules ICAM-1 and VCAM-1. Pretreatment of these cells with a calcium chelator or an inhibitor of NF-B activation attenuated the anti-GRP78 autoantibody-induced promotion of ICAM-1 and VCAM-1. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation, an important initiating event in atherogenesis. These findings further our understanding of the role of anti-GRP78 autoantibodies and the activation of surface GRP78 in endothelial cell function and lesion development. Furthermore, inhibiting the interaction of anti-GRP78 autoantibodies with surface GRP78 could present a novel therapeutic strategy to modulate lesion growth and thereby reduce the risk for atherosclerosis and cardiovascular disease.

Classifying Patients for Breast Cancer by Detection of Autoantibodies against a Panel of Conformation-Carrying Antigens

Patients with breast cancer elicit an autoantibody response against cancer proteins, which reflects and amplifies the cellular changes associated with tumorigenesis. Detection of autoantibodies in plasma may provide a minimally invasive mechanism for early detection of breast cancer. To identify cancer proteins that elicit a humoral response, we generated a cDNA library enriched for breast cancer genes that encode membrane and secreted proteins, which are more likely to induce an antibody response compared with intracellular proteins. To generate conformation-carrying antigens that are efficiently recognized by patients' antibodies, a eukaryotic expression strategy was established. Plasma from 200 patients with breast cancer and 200 age-matched healthy controls were measured for autoantibody activity against 20 different antigens designed to have conformational epitopes using ELISA. A conditional logistic regression model was used to select a combination of autoantibody responses against the 20 different antigens to classify patients with breast cancer from healthy controls. The best combination included ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN, and LRRC15; however, autoantibody responses against GFRA1, GRN, and LRRC15 were inversely correlated with breast cancer. When the autoantibody responses against the 7 antigens were added to the base model, including age, BMI, race and current smoking status, the assay had the following diagnostic capabilities: c-stat (95% CI), 0.82 (0.78-0.86); sensitivity, 73%; specificity, 76%; and positive likelihood ratio (95% CI), 3.04 (2.34-3.94). The model was calibrated across risk deciles (Hosmer-Lemeshow, P = 0.13) and performed well in specific subtypes of breast cancer including estrogen receptor positive, HER-2 positive, invasive, in situ and tumor sizes >1 cm.

Hydrolytical activity of autoantibodies to double-stranded DNA in autoimmune thyroiditis

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

0149: Agonistic anti β3-adrenoceptor antibodies do not affect the β1-adrenoceptor-mediated inotropy and the β3 adrenoceptor-mediated vasorelaxation in Lewis rat

Cardiac activity of autoantibodies (AABs) directed against β1-adrenoceptor (β1-AR) has been proposed to play an important role in the pathogenesis of dilated cardiomyopathy. Our previous work has shown that the immunization of rats with the second extracellular loop (ECII) of β1-AR induced endothelial dysfunction in aorta and mesenteric arteries. However, until now, no study has explored the cardiovascular effects of β3-AABs alone or combined with β1-AABs. To evaluate whether β3-AABs possess β3-AR agonistic effect and whether active immunization producing β3-AABs and/or β1-AABs has deleterious effects on cardiac and vascular reactivity in Lewis rats. Lewis rats were immunized for 3 months with peptidic sequences corresponding to the ECII of β3-AR and/or β1-AR.? The agonistic effect of β3-AABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Inotropy studies and isolated aorta and mesenteric artery studies were also conducted on immunized rats. SR58611A (10 nM), a preferential β3-AR agonist and purified β3-AABs (25 μg/mL) induced a decrease of cell shortening (–39.6±4.4% (n=11) and –18.5±3.9% (n=10) respectively). This decrease was significantly inhibited when the cardiomyocytes were preincubated with the L-748337 (1 μM), a selective β3-AR antagonist (p<0.05). The cell shortening of cardiomyocytes from rats immunized against the β1-AR, in response to isoprenaline (10 nM), was significantly decreased (p<0.05). In contrast, this effect was conserved in rats immunized against β3-AR or β3/β1-AR. Vasorelaxations induced by acetylcholine and SR58611A in both aorta and mesenteric arteries were unaltered by immunization. These results show that β3-AABs induced a β3-AR agonist-like activity. They would not have a cardiovascular pathogenic action but would offset the cardiac and endothelial dysfunctions caused by β1-AABs.

The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders: recommendations from the European Myeloma Network.

Monoclonal gammopathy of undetermined significance is one of the most common pre-malignant disorders. IgG and IgA monoclonal gammopathy of undetermined significance are precursor conditions of multiple myeloma; light-chain monoclonal gammopathy of undetermined significance of light-chain multiple myeloma; and IgM monoclonal gammopathy of undetermined significance of Waldenström's macroglobulinemia and other lymphoproliferative disorders. Clonal burden, as determined by bone marrow plasma cell percentage or M-protein level, as well as biological characteristics, including heavy chain isotype and light chain production, are helpful in predicting risk of progression of monoclonal gammopathy of undetermined significance to symptomatic disease. Furthermore, alterations in the bone marrow microenvironment of monoclonal gammopathy of undetermined significance patients result in an increased risk of venous and arterial thrombosis, infections, osteoporosis, and bone fractures. In addition, the small clone may occasionally be responsible for severe organ damage through the production of a monoclonal protein that has autoantibody activity or deposits in tissues. These disorders are rare and often require therapy directed at eradication of the underlying plasma cell or lymphoplasmacytic clone. In this review, we provide an overview of the clinical relevance of monoclonal gammopathy of undetermined significance. We also give general recommendations of how to diagnose and manage patients with monoclonal gammopathy of undetermined significance.

Molecular and Cellular Pathways of Immunoglobulin G Activity In Vivo

In retrospect, the therapeutic potential of immunoglobulins was first demonstrated by von Behring and Kitasato in the late nineteenth century by protecting mice from the lethal effects caused by tetanus and diphtheria toxin via injection of a hyperimmune serum generated in rabbits. Even today, hyperimmune sera generated from human donors with high serum titers against a certain pathogen are still in use as a means of providing passive protection. More importantly, therapeutic antibodies specific for malignant or autoreactive cells have become included in the standard of care in diseases such as breast cancer and malignant lymphoma. Despite this clinical success, we are only at the beginning of understanding the precise molecular and cellular pathways responsible for immunoglobulin G (IgG) activity in vivo. Since then, an enormous amount of information about the mechanism of IgG activity has been obtained in various model systems. The aim of this review is to provide a comprehensive overview of our current understanding of how IgG antibodies mediate their activity in vivo and how we can use this knowledge to enhance the activity of therapeutic antibodies or block the proinflammatory and tissue pathology inducing activity of autoantibodies.

Laquinimod Delays and Suppresses Nephritis in Lupus‐Prone Mice and Affects Both Myeloid and Lymphoid Immune Cells

Lupus nephritis depends on autoantibody deposition and activation of multiple immune cell types that promote kidney inflammation, including lymphocytes and monocyte/macrophages. Laquinimod, currently in clinical trials for multiple sclerosis and lupus nephritis, reduces infiltration of inflammatory cells into the spinal cord in experimental autoimmune encephalomyelitis. Activated monocyte/macrophages infiltrate the kidneys during nephritis in systemic lupus erythematosus (SLE). We undertook this study to determine whether using laquinimod to reduce monocyte/macrophage-driven tissue damage as well as to alter lymphocytes in SLE nephritis could have greater therapeutic benefit than current treatments that primarily affect lymphocytes, such as mycophenolate mofetil (MMF). To test laquinimod efficacy, we used the (NZB × NZW)F1 mouse model of SLE, in which disease manifests as nephritis. Preventive and therapeutic studies were performed to determine whether laquinimod could prevent or delay nephritis, as measured by proteinuria, serum creatinine, survival, and renal pathology. Spleen and kidney leukocyte populations and suppression assays were analyzed by flow cytometry. Laquinimod prevented or delayed lupus manifestations at levels equal to or better than MMF. Laquinimod treatment was associated with reduced numbers of monocyte/macrophages, dendritic cells, and lymphocytes, as well as with induction of myeloid-derived suppressor cells in spleens and kidneys. Laquinimod suppressed macrophage-secreted tumor necrosis factor α and induced production of interleukin-10 (IL-10). In addition, laquinimod suppressed interferon-γ and IL-17 production by lymphocytes and down-regulated expression of activation/costimulatory markers on antigen-presenting cells. The effects of laquinimod on myeloid and lymphoid cells may contribute to improvements in (NZB × NZW)F1 mouse survival, proteinuria, and glomerulonephritis. Future development of laquinimod as a therapeutic agent for lupus nephritis is promising.

Autoimmune Mechanisms Activating the Angiotensin AT1 Receptor in 'Primary' Aldosteronism

The mechanisms causing excessive aldosterone production and hypertension in primary aldosteronism (PA) are complex and often incompletely recognized. Autoantibodies to the angiotensin AT1 receptor (AT1R) have been reported in some PA patients with an aldosterone-producing adenoma but not with idiopathic adrenal hyperplasia. We investigated whether these autoantibodies will activate AT1R and thereby potentially contribute to the pathophysiology of PA. AT1R autoantibody activity in sera and/or IgG purified from 13 biochemically confirmed PA patients was measured using AT1R-transfected cells, and their contractile effects were assayed using perfused rat cremaster arterioles. Aldosterone stimulation was measured in vitro using isolated human adrenal carcinoma (HAC15) adrenal cells. These data were compared with sera obtained from a group of normotensive control subjects who were expected to have negligible AT1R autoantibodies. Sera from each of the 13 PA patients significantly increased AT1R activation in AT1R-transfected cells compared with 20 control subjects, and this activity was inhibited by the selective AT1R blocker losartan. Sera and IgG purified from AT1R autoantibody-positive sera demonstrated significant vasoconstrictive effects in isolated rat cremaster arterioles and were blocked by losartan. Moreover, the AT1R autoantibody-positive IgG directly stimulated aldosterone production in the cultured adrenal cells and enhanced angiotensin-induced aldosterone production in these cells, and these effects were blocked by candesartan. These data support a probable pathophysiological role for AT1R autoantibodies in PA and thereby raise important etiological and therapeutic implications.

Inducible cardiac arrhythmias caused by enhanced β1-adrenergic autoantibody expression in the rabbit

Previous studies demonstrated burst pacing and intravenous infusion of ACh induced sustained atrial tachycardia when rabbits were immunized to produce β2-adrenergic receptor (β2AR)-activating autoantibodies. The objective of this study was to examine the arrhythmogenic effect of β1-adrenergic receptor (β1AR)-activating autoantibodies in the rabbit. Eight New Zealand white rabbits were immunized with a β1AR second extracellular loop peptide to raise β1AR antibody titers. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization. Arrhythmia occurrence was determined in response to burst pacing before and after ACh infusion in incremental concentrations of 10 μM, 100 μM, and 1 mM. The baseline sinus heart rate before and after immunization averaged 149 ± 17 per min and 169 ± 16 per min, respectively (P < 0.05). In the preimmune studies, there were five sustained (≥10 s) arrhythmias in 32 induction attempts, which occurred in only four of eight rabbits. In the postimmune studies, there were 22 sustained arrhythmias in 32 induction attempts, which occurred in all eight rabbits (P < 0.0001 for the independent effect of immunization). Of the 22 sustained arrhythmias postimmunization, 15 were sinus tachycardia compared with only two before immunization (P < 0.01 for the independent effect of immunization). Postimmune (but not preimmune) rabbit sera demonstrated specific binding to β1AR and induced significant β1AR activation in transfected cells in vitro. No cross-reactivity with β2AR was observed. In conclusion, in contrast with rabbits with β2AR-activating autoantibodies that demonstrate predominantly atrial tachycardias, enhanced autoantibody activation of β1AR in the rabbit leads to tachyarrhythmias mainly in the form of sustained sinus tachycardia.

Abstract 423: Angiotensin II Type 1 Receptor (AT1R) Activating Autoantibodies Contribute to the Pathophysiology of Primary Hyperaldosteronism

Objectives: Activating autoantibodies (AA) to the AT1R second extracellular loop (ECL2) are present in patients with primary aldosteronism (PA). We investigated whether AA from PA and from AT1R-ECL2 immunized rabbits activate AT1R and thereby contribute to hypertension by a direct contractile effect on the vasculature and stimulate adrenal aldosterone production. Methods: We studied 5 normal controls (NC) and 15 subjects with biochemically confirmed PA [4 APA, 4 IAH non- and 7 indeterminate (IPA)]. AT1R-AA activity in serum or IgG was analyzed in AT1R transfected CHO cells. Contractile effects were assayed in isolated perfused rat cremaster arteries. We determined the effects of an ARB and the L-aa AT1R-ECL2 target epitope on in vitro AT1R cremaster artery responsiveness to patient sera and to MAP-L-aa (AFHYESQ) peptide immunized rabbit serum. We examined the effect of IgG from 7 PA subjects on aldosterone production in HAC15 cells in vitro w/wo 1.0 and 10 nM Ang II; and w/wo ARB. Results: Serum from PA significantly (p&lt;0.05) increased Ang II-equivalent activity in AT1R-CHO cells in IAH 199± x pM, n=4; APA 177±y pM, n=3; IPA 187±z pM, n=7; vs NC 83±a pM, n=5 and was blocked by 10 μM losartan. IgG from PA decreased perfused rat arteriole diameter in IAH by 22±8(SD)% of a 100% (normalized) baseline, n=4; APA 14±0.8%, n=3; IPA 21±6%, n=7; and NC 7±3.5%, n=5 and was blocked by 10 μM losartan. IgG from 7 PA subjects increased HAC15 aldosterone (aldo) 1.8 fold over baseline (p&lt;0.01) and markedly increased 1nM and 10 nM Ang II-induced aldo by 3.5 fold (p&lt;0.01); and was blocked by ARB. BP in hi Na rabbits rose from 80/60 to 140/90 6 weeks after immunization with AT1R MAP-L-aa AFHYESQ peptide. Arteriole contraction rose from 5% (preimmune) to 30% (post-immunization). Preincubation with the L-aa 2 nd ECL target reduced contractility to 8%; not different from baseline. Conclusions: AA from PA and AT1R-immunized rabbits activates AT1R transfected CHO cells, increases in vitro arterial contractility and stimulates basal and Ang II induced HAC15 aldosterone secretion. ARBs and decoy peptides were effective in blocking AA effects in vitro . These AA have important etiological and therapeutic implications.

Serum or breast milk immunoglobulins mask the self‐reactivity of human natural IgG antibodies

B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

THU0159 Incidence and prevalence of major central nervous system (CNS) involvement in systemic lupus erythematosus (SLE): A 3 year prospective evaluation and clinical correlations in 370 patients

BackgroundIncidence and prevalence of CNS involvement in SLE is highly variable in all published series. Minor and non-specific symptoms such as headaches, mild cognitive dysfunction or depression, collectively labeled neuropsychiatric...

THU0056 A Small Chemical Compound Identified as an ACE2 Activator Prevented Pulmonary Arterial Hypertension Induced by Monocrotaline

BackgroundPulmonary arterial hypertension (PAH) is a lethal clinical symptom characterized by progressive increase in pulmonary arterial pressure and causes right ventricular hypertrophy (RVH) with RV failure. Complications of PAH are...

Is There a Role for the Detection of Autoantibodies in the Clinical Practice of Treating Infants with Bullous Pemphigoid? A Case Report

We present a case of infantile bullous pemphigoid (BP) triggered by primary infection with varicella zoster virus and we analyze the correlation between autoantibody levels and disease activity. With this report we suggest that serum autoantibody titers may not necessarily mirror the clinical course of the disease or represent a helpful tool in guiding therapeutic decisions in infantile BP.

Unraveling the IVIG mystique

Unraveling the <scp>IVIG</scp> mystique

Plasma levels of osteopontin identify patients at risk for organ damage in systemic lupus erythematosus

IntroductionOsteopontin (OPN) has been implicated as a mediator of Th17 regulation via type I interferon (IFN) receptor signaling and in macrophage activity at sites of tissue repair. This study assessed whether increased circulating plasma OPN (cOPN) precedes development of organ damage in pediatric systemic lupus erythematosus (pSLE) and compared it to circulating plasma neutrophil gelatinase-associated lipocalin (cNGAL), a predictor of increased SLE disease activity.MethodscOPN and cNGAL were measured in prospectively followed pSLE (n = 42) and adult SLE (aSLE; n = 23) patients and age-matched controls. Time-adjusted cumulative disease activity and disease damage were respectively assessed using adjusted-mean SLE disease activity index (SLEDAI) (AMS) and SLICC/ACR damage index (SDI).ResultsCompared to controls, elevated cOPN and cNGAL were observed in pSLE and aSLE. cNGAL preceded worsening SLEDAI by 3-6 months (P = 0.04), but was not associated with increased 6-month AMS. High baseline cOPN, which was associated with high IFNalpha activity and expression of autoantibodies to nucleic acids, positively correlated with 6-month AMS (r = 0.51 and 0.52, P = 0.001 and 0.01 in pSLE and aSLE, respectively) and was associated with SDI increase at 12 months in pSLE (P = 0.001). Risk factors for change in SDI in pSLE were cOPN (OR 7.5, 95% CI [2.9-20], P = 0.03), but not cNGAL, cumulative prednisone, disease duration, immunosuppression use, gender or ancestry using univariate and multivariate logistic regression. The area under the curve (AUC) when generating the receiver-operating characteristic (ROC) of baseline cOPN sensitivity and specificity for the indication of SLE patients with an increase of SDI over a 12 month period is 0.543 (95% CI 0.347-0.738; positive predictive value 95% and negative predictive value 38%).ConclusionHigh circulating OPN levels preceded increased cumulative disease activity and organ damage in SLE patients, especially in pSLE, and its value as a predictor of poor outcome should be further validated in large longitudinal cohorts.