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Functional non-HLA antibodies (antibodies to non-human leukocyte antigens) targeting the G protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling (a regulator of cell growth) may represent a general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established. Here we hypothesize involvement of the versatile adaptor proteins, β-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair. To test this, human microvascular endothelial cells were treated with AT1R-/ETAR-antibodies isolated from patients with kidney transplant vasculopathy. These antibodies activated both mTOR complexes via AT1R and ETAR in a PI3K-dependent and ERK-independent manner. The mTOR inhibitor, rapamycin, completely abolished activation of mTORC1 and mTORC2 after long term treatment with receptor antibodies. Imaging studies revealed that β2- but not β1-arrestin was recruited to ETAR in response to ET1 and patient antibodies but not with antibodies isolated from healthy individuals. Silencing of β2-arrestin by siRNA transfection significantly reduced ERK1/2 and mTORC2 activation. Non-HLA antibodies impaired endothelial repair by AT1R and ETAR-induced mTORC2 signaling. Thus, we provide evidence that functional AT1R-/ETAR antibodies induce ERK1/2 and mTOR signaling involving β2-arrestin in human microvascular endothelium. Hence, our data may provide a translational rational for mTOR inhibitors in combination with receptor blockers in patients with non-HLA receptor recognizing antibodies.

The role of autoimmunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although clinicians observe a growing population of convalescent COVID-19 patients with manifestation of post-acute sequelae of COVID-19. We analyzed the immune response in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. The phenotyping of lymphocytes showed a significantly higher number of CD8+ T cells expressing the Epstein-Barr virus induced G protein coupled receptor 2, chemokine receptor CXCR3 and C-C chemokine receptor type 5 playing an important role in inflammation and migration in PASC patients compared to controls. Additionally, a stronger, SARS-CoV-2 reactive CD8+ T cell response, characterized by IFNγ production and predominant TEMRAphenotype but low SARS-CoV-2 avidity was detected in PASC patients compared to controls. Furthermore, higher titers of several autoantibodies were detected among PASC patients. Our data suggest that a persistent inflammatory response triggered by SARS-CoV-2 might be responsible for the observed sequelae in PASC patients. These results may have implications on future therapeutic strategies.

Age is a significant risk factor for the coronavirus disease 2019 (COVID-19) outcomes due to immunosenescence and certain age-dependent medical conditions (e.g., obesity, cardiovascular disorder, diabetes, chronic respiratory disease). However, despite the well-known influence of age on autoantibody biology in health & disease, its impact on the risk of developing severe COVID-19 remains poorly explored. Here, we performed a cross-sectional study of autoantibodies directed against 58 targets associated with autoimmune diseases in 159 individuals with different COVID-19 outcomes (with 71 mild, 61 moderate, and 27 severe patients) and 73 healthy controls. We found that the natural production of autoantibodies increases with age and is exacerbated by SARS-CoV-2 infection, mostly in severe COVID-19 patients. Multivariate regression analysis showed that severe COVID-19 patients have a significant age-associated increase of autoantibody levels against 16 targets (e.g., amyloid β peptide, β catenin, cardiolipin, claudin, enteric nerve, fibulin, insulin receptor a, and platelet glycoprotein). Principal component analysis with spectrum decomposition based on these autoantibodies indicated an age-dependent stratification of severe COVID-19 patients. Random forest analysis ranked autoantibodies targeting cardiolipin, claudin, and platelet glycoprotein as the three most crucial autoantibodies for the stratification of severe elderly COVID-19 patients. Follow-up analysis using binomial regression found that anti-cardiolipin and anti-platelet glycoprotein autoantibodies indicated a significantly increased likelihood of developing a severe COVID-19 phenotype, presenting a synergistic effect on worsening COVID-19 outcomes. These findings provide new key insights to explain why elderly patients less favorable outcomes have than young individuals, suggesting new associations of distinct autoantibody levels with disease severity.

To examine concentrations of circulating antibodies targeting C3a and C5a complement receptors in ANCA-associated vasculitis (AAV) and analyze their association with disease activity.Concentrations of antibodies against C3a and C5a complement receptors and plasma complement fragments C3a and C5a were determined in patients with AAV (n = 110; granulomatosis with polyangiitis [GPA, n = 82], microscopic polyangiitis [MPA, n = 28]), systemic lupus erythematosus as disease control (SLE, n = 36) and healthy donors (HD, n = 220). C3aR and C5aR expression by circulating neutrophils, monocytes and T cells was analyzed using flow cytometry. Clinical data were assessed at time of serum sampling and during follow-up for 60 months.In AAV, anti-C3aR and anti-C5aR antibodies were decreased (P = 0.0026 and ≤ 0.0001, respectively). In remission, anti-C3aR antibody concentrations rose to values comparable to HD, whereas anti-C5aR antibody concentrations did not. In GPA, C5a and anti-C5aR antibody concentrations inversely correlated with each other (r = -0.6831, P = 0.0127). In newly diagnosed GPA, decreased concentrations of anti-C5aR antibodies but not anti-C3aR antibodies were associated with disease activity (P = 0.0009). Moreover, low anti-C5aR antibodies were associated with relapse in GPA (HR: 3.54, P = 0.0009) and MPA (HR: 4.41, P = 0.0041). The frequency of C5aR-expressing cells within T cell populations was increased in GPA (CD4+ T cells: P = 0.0021; CD8+ T cells: P = 0.0118), but not in MPA.Low concentrations of anti-C5aR antibodies reflect disease activity and are associated with an increased risk for relapse in AAV. This article is protected by copyright. All rights reserved.

Background Recently, a novel humanized mouse model for systemic sclerosis (SSc) was established by transferring peripheral blood mononuclear cells (PBMC) from patients with SSc to Rag2−/−Il2rg−/− immunodeficient mice. Here, we aimed to investigate the role of T and B cells in this humanized mouse model. Methods T and B cells were depleted in vitro from freshly isolated PBMC using anti-CD3 and anti-CD19 magnetic microbeads, respectively. Subsequently, PBMC and T or B cell-depleted PBMC were transferred into Rag2−/−/Il2rg−/− mice via intraperitoneal injection. Twelve weeks after the transfer, mice were sacrificed and evaluated. Results Mice transferred with whole PBMC from SSc patients developed systemic inflammation in the lungs, kidneys, and liver, and 6 out of 11 mice died or had to be sacrificed during the experiment. By contrast, such inflammation and death were not observed in mice transferred with corresponding T or B cell-depleted PBMC. In line with this finding, transfer with whole PBMC restored the splenic white pulp composing of human T, B, and plasma cells and led to the production of a considerable amount of human autoantibodies in recipient mice, while those immunological features were rarely observed in mice that received T or B cell-depleted PBMC. In contrast to our previous findings demonstrating a transfer of the protective effect of a B cell therapy into the mouse, treatment of SSc patients with chemical immunosuppressive drugs did not affect the pathogenicity of PBMC. Conclusions This study demonstrates that both T and B cells are indispensable for the pathogenesis of the PBMC transfer-induced mouse model for SSc.

We hypothesized that prolonged or cumulative exposure to indoor air dampness microbiota in moisture-damaged buildings and daily exposure to wireless telecommunication devices would potentiate the risk of electromagnetic hypersensitivity (EHS), which is poorly defined. We performed a nested comparative analysis within an age- and sex-matched study of females who were exposed to dampness microbiota with self-reported complaints compatible with EHS (n=11). Their levels of autoantibodies towards 13 different autoantigens were measured. EHS presented as multiple chemical sensitivity, profound fatigue, memory disturbances in all subjects (11/11), and cognitive impairment in the majority (9/11). When comparing the patients to controls, no difference was detected between the levels of the following autoantibodies: angiotensin II type 1 receptor (AGTR1), endothelin receptor type A (ETAR), adrenergic receptors α1AR, α2AR, β1AR, β2AR and cholinergic muscarinic receptors m1AChR, m2AChR, m3AChR and m5AChR. In contrast, IgG levels towards m4AChR and fibroblast growth factor receptor 3 (FGFR3), and IgM autoantibodies against glycosylated moieties of heparan and heparan sulphate (TS-HDS) were significantly decreased in the study cohort, p=0.008; p=0.032, p

Scleroderma renal crisis is a rare complication of systemic sclerosis characterized by a rapid decline in kidney function due to acute renal vascular injury. Recently, activating autoantibodies targeting the angiotensin II type 1 receptor and the endothelin-1 type A receptor have been implicated in the pathophysiology of scleroderma renal crisis by sensitizing the angiotensin II type 1 receptor and endothelin-1 type A receptor in renal resistance arteries to their natural ligands. Here, we describe a cohort of 10 patients with scleroderma renal crisis refractory to standard treatment, including blockade of the renin-angiotensin system. Multimodal therapy was initiated, targeting at the removal of anti-angiotensin II type 1 receptor and anti-endothelin-1 type A receptor autoantibodies by plasma exchange and the reduction of vasoconstrictive activity. Further treatment options included angiotensin II type 1 receptor and endothelin-1 type A receptor blockade, iloprost, intravenous immunoglobulins, and immunosuppression. Six patients were hypertensive. On kidney biopsy, concentric intimal sclerosis was present in all patients, whereas acute vascular injury was evident in eight. Levels of anti-angiotensin II type 1 receptor and anti-endothelin-1 type A receptor autoantibodies were significantly reduced by multimodal treatment. Kidney function improved in three patients with histological signs of severe acute renal vascular damage. This report demonstrates that intensive multimodal therapy taking account of potentially pathogenic anti-angiotensin II type 1 receptor and anti-endothelin-1 type A receptor autoantibodies in concert with other vasodilatory interventions provides a salvage option for patients with refractory scleroderma renal crisis.

The SARS-CoV-2 infection is associated with increased levels of autoantibodies targeting immunological proteins such as cytokines and chemokines. Reports further indicate that COVID-19 patients may develop a wide spectrum of autoimmune diseases due to reasons not fully understood. Even so, the landscape of autoantibodies induced by SARS-CoV-2 infection remains uncharted territory. To gain more insight, we carried out a comprehensive assessment of autoantibodies known to be linked to diverse autoimmune diseases observed in COVID-19 patients, in a cohort of 248 individuals, of which171 were COVID-19 patients (74 with mild, 65 moderate, and 32 with severe disease) and 77were healthy controls. Dysregulated autoantibody serum levels, characterized mainly by elevated concentrations, occurred mostly in patients with moderate or severe COVID-19 infection, and was accompanied by a progressive disruption of physiologic IgG and IgA autoantibody signatures. A similar perturbation was found in patients with anosmia. Notably, autoantibody levels often accompanied anti-SARS-CoV-2 antibody concentrations, being both indicated by random forest classification as strong predictors of COVID-19 outcome, together with age. Moreover, higher levels of autoantibodies (mainly IgGs) were seen in the elderly with severe disease compared with young COVID-19 patients with severe disease. These findings suggest that the SARS-CoV-2 infection induces a broader loss of self-tolerance than previously thought, providing new ideas for therapeutic interventions.

ObjectiveTo determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such as systemic sclerosis (SSc).MethodsC57BL/6J mice were immunised with membrane-embedded human AT1R or empty membrane as control. Mice deficient for CD4+or CD8+T cells and B cells were immunised with membrane-embedded AT1R or an AT1R peptide proposed to be a dominant T cell epitope. A monoclonal (m)AT1R Ab was generated by hybridoma technique and transferred into C57BL/6J and AT1Ra/b knockout mice. The induced phenotype was examined by histology, immunohistochemistry, immunofluorescence, apoptosis assay and ELISA. In vitro, Abs responses towards AT1R were measured in cells of different origins and species.ResultsAT1R-immunised mice developed perivascular skin and lung inflammation, lymphocytic alveolitis, weak lung endothelial apoptosis and skin fibrosis accompanied by Smad2/3 signalling, not present in controls or mice deficient for CD4+T and B cells. The AT1R peptide 149–172 provoked lung inflammation. Application of the mAT1R Ab induced skin and lung inflammation, not observed in AT1Ra/b knockout mice. In vitro, AT1R Abs activated rat cardiomyocytes and human monocytes, enhanced angiotensin II-mediated AT1R activation in AT1R-transfected HEK293 cells via AT1R binding and mAT1R Ab-activated monocytes mediated the induction of profibrotic markers in dermal fibroblasts.ConclusionOur immunisation strategy successfully induced AT1R Abs, contributing to inflammation and, possibly, to fibrosis via activation of AT1R. Therefore, AT1R Abs are valuable targets for future therapies of SSc and other AT1R Ab-related diseases.

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an acquired complex disease with patients suffering from the cardinal symptoms of fatigue, post-exertional malaise (PEM), cognitive impairment, pain and autonomous dysfunction. ME/CFS is triggered by an infection in the majority of patients. Initial evidence for a potential role of natural regulatory autoantibodies (AAB) to beta-adrenergic (AdR) and muscarinic acetylcholine receptors (M-AChR) in ME/CFS patients comes from a few studies. Methods: Here, we analyzed the correlations of symptom severity with levels of AAB to vasoregulative AdR, AChR and Endothelin-1 type A and B (ETA/B) and Angiotensin II type 1 (AT1) receptor in a Berlin cohort of ME/CFS patients (n = 116) by ELISA. The severity of disease, symptoms and autonomic dysfunction were assessed by questionnaires. Results: We found levels of most AABs significantly correlated with key symptoms of fatigue and muscle pain in patients with infection-triggered onset. The severity of cognitive impairment correlated with AT1-R- and ETA-R-AAB and severity of gastrointestinal symptoms with alpha1/2-AdR-AAB. In contrast, the patients with non-infection-triggered ME/CFS showed fewer and other correlations. Conclusion: Correlations of specific AAB against G-protein-coupled receptors (GPCR) with symptoms provide evidence for a role of these AAB or respective receptor pathways in disease pathomechanism.

A subset of patients has long-lasting symptoms after mild to moderate COVID-19. In a prospective observational cohort study we analysed clinical and laboratory parameters in 42 patients (29 female/13 male, median age 36.5 years) with persistent moderate to severe fatigue and exertion intolerance six months following COVID-19 referred to as Chronic COVID-19 Syndrome (CCS). Most patients were moderately to severely impaired in daily live. 19 patients fulfilled the 2003 Canadian Consensus Criteria for myalgic encephalomyelitis/chronic fatigue syndrome referred to as CFS/CCS. Hand grip strength (HGS) was diminished in most patients. Association of several biomarker with key symptoms of physical and mental fatigue and post exertional malaise indicate low level inflammation and hypoperfusion as potential pathomechanisms.

Background: Silicone breast implants (SBIs) has been shown to be associated with an increased risk of autoimmune diseases. In the current study, we aimed to explore the potential association between circulating autoantibodies against the autonomic nervous system and cognitive impairment, memory deficit, and depressive symptoms reported by women with SBIs. Methods: ELISA assays were used to quantify anti-adrenergic receptors (α1, α2, β1, β2), anti-muscarinic receptors (M1-M5), anti-endothelin receptor type A, and anti-angiotensin II type 1 receptor titers in the sera of 93 symptomatic female subjects with SBIs and 36 age-matched healthy female controls. Results: A significant difference was detected in the level of autoantibodies against the autonomic nervous system receptors in women with SBIs who reported memory impairment, cognitive impairment, and sleep disturbance as compared with both women with SBIs who did not complain of these symptoms or with healthy individuals without SBIs. Conclusions: Clinical symptoms such as depression, cognitive impairment, and sleep disturbances were found to be associated with dysregulation of the levels of circulating autoantibodies targeting the autonomous nervous system receptors in women with SBIs. These autoantibodies may have diagnostic significance in diseases associated with breast implants.

Background. Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis (SSc). Autoantibodies (Abs) against endothelial cell antigens have been implicated in SSc and SRC. However, their detailed roles remain poorly defined. Pro-inflammatory cytokine interleukin-6 (IL-6) has been found to be increased in SSc, but its role in SRC is unclear. Here, we aimed to determine how the autoantibodies from patients with SSc and SRC affect IL-6 secretion by micro-vascular endothelial cells (HMECs). Methods. Serum IgG fractions were isolated from either SSc patients with SRC (n = 4) or healthy individuals (n = 4) and then each experiment with HMECs was performed with SSc-IgG from a separate patient or separate healthy control. IL-6 expression and release by HMECs was assessed by quantitative reverse transcription and quantitative PCR (RT-qPCR) and immunoassays, respectively. The mechanisms underlying the production of IL-6 were analyzed by transient HMEC transfections with IL-6 promoter constructs, electrophoretic mobility shift assays, Western blots and flow cytometry. Results. Exposure of HMECs to IgG from SSc patients, but not from healthy controls, resulted in a time- and dose-dependent increase in IL-6 secretion, which was associated with increased AKT, p70S6K, and ERK1/2 signalling, as well as increased c-FOS/AP-1 transcriptional activity. All these effects could be reduced by the blockade of the endothelial PAR-1 receptor and/or c-FOS/AP-1silencing. Conclusions. Autoantibodies against PAR-1 found in patients with SSc and SRC induce IL-6 production by endothelial cells through signalling pathways controlled by the AP-1 transcription factor. These observations offer a greater understanding of adverse endothelial cell responses to autoantibodies present in patients with SRC.

Background Angiotensin II type-1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) autoantibodies, in addition to allograft injury, can bind native endothelial cells and cause vascular vasoconstriction and fibrosis progression in nontransplanted organs. Therefore, we investigated long-term native renal function in liver transplant (LT) recipients with and without anti-AT1R-Abs and/or anti-ETAR-Abs present in serum. Methods Primary LT recipients at our single center from January 2000 to April 2009 had their prospectively collected pre-LT (1269 patients) and year 1 post-LT (795 patients) serum tested retrospectively for anti-AT1R-Abs and/or anti-ETAR-Abs. Anti-AT1R-Abs and anti-ETAR-Abs testing was accomplished with a standardized solid phase assay in which >10 U was considered positive. Results Pretransplant anti-AT1R-Abs and/or anti-ETAR-Abs did not change the median delta creatinine from pretransplant to 1 year post-transplant. In multivariable analysis controlling for diabetes (DM) and calcineurin inhibitor (CNI) use, anti-AT1R-Abs and/or anti-ETAR-Abs at 1-year remained statistically significantly associated with a decline in GFR (measured by Modification of Diet in Renal Disease-6) from years 1-5 post-LT (P = .04). In diabetic patients the association with a decline in renal function was more pronounced with (-9.29 mL/min) vs without (-2.28 mL/min) anti-AT1R-Abs and/or anti-ETAR-Abs at year 1, respectively (P = .004). Conclusion At 1-year post-LT, the autoantibodies anti-AT1R-Abs and/or anti-ETAR-Abs are associated in multivariable analysis with an increased risk of native renal function decline especially in diabetic patients.

Myalgic encephalomyelitis (ME) also known as ME/CFS (Chronic Fatigue Syndrome) or ME/SEID (Systemic Exertion Intolerance Disorder), is a disabling and often long-lasting disease that can drastically impair quality of life and physical/social functioning of the patients. Underlying pathological mechanisms are to a large extent unknown, but the presence of autoantibodies, cytokine pattern deviations and the presentation of cognitive and autonomic nervous system related symptoms provide evidence for ME being an immunological disorder with elements of autoimmunity. Increased levels of autoantibodies binding to adrenergic and muscarinic receptors in ME-patients have been reported. It is hypothesized that these autoantibodies have pathological significance and contribute to the ME-specific symptoms, however, these observations need to be validated. This study was designed to investigate potential differences in adrenergic and muscarinic receptor autoantibody levels in plasma and cerebrospinal fluid (CSF) samples between ME patients and gender and age-matched healthy controls, and to correlate the autoantibody levels to disease severity. We collected bodyfluids and health-related questionnaires from two Swedish ME cohorts, plasma and CSF from one of the cohorts (n ​= ​24), only plasma from the second cohort (n ​= ​24) together with plasma samples (n ​= ​24) and CSF (n ​= ​6) from healthy controls. All samples were analysed for IgG autoantibodies directed against Alpha- (α1, α2) and Beta- (β1-3) adrenergic receptors and Muscarinic (M) 1–5 acetylcholine receptors using an ELISA technique. The questionnaires were used as measures of disease severity. Significant increases in autoantibody levels in ME patients compared to controls were found for M3 and M4 -receptors in both cohorts and β1, β2, M3 and M4-receptors in one cohort. No significant correlations were found between autoantibody levels and disease severity. No significant levels of autoantibodies were detected in the CSF samples. These findings support previous findings that there exists a general pattern of increased antibody levels to adrenergic and muscarinic receptors within the ME patient group. However, the role of increased adrenergic and muscarinic receptor autoantibodies in the pathogenesis of ME is still uncertain and further research is needed to evaluate the clinical significance of these findings., Highlights • Myalgic Encephalomyelitis (ME) is a devastating disorder (with millions of patients worldwide) with unclear aetiology and no clear diagnostic biomarker available. • This study support the existance of a general pattern of increased antibody levels to adrenergic and muscarinic receptors within the ME patient group. • No evidence for intrathecal antibody production was found in cerebrospinal fluid. The role of increased autoantibodies in the pathogenesis of ME is still uncertain and further research is needed to evaluate their clinical significance.

Overactivation of renin-angiotensin system (RAS) is one of the main pathophysiological features in the evolution of chronic heart failure (CHF). The (pro)renin receptor ((P)RR) represents an important player in a tissue renin-angiotensin system (tissue RAS), which mediates tissue injury through fibrosis and hypertrophy of the affected organs in CHF patients. In our study we used plasma samples from 556 elderly subjects with CHF and 198 healthy participants in order to evaluate prognostic and diagnostic potential of s(P)RR in setting of CHF. The patients with CHF showed significantly higher plasma levels of s(P)RR than the healthy volunteers (p=0.0005). We observed association between higher s(P)RR plasma concentrations and lower left ventricular ejection fraction and higher degree of left ventricular dilatation on baseline echocardiography examination of the CHF patients. Elderly CHF patients with higher baseline s(P)RR plasma concentration were at same risk for death, stroke and hospitalization due to heart failure worsening at mean follow-up from forty-eight months in comparison to low s(P)RR counterparts.

Preeclampsia is associated with an increased cardiovascular risk later in life. Anti-GPCR autoantibodies have been shown to contribute to the development of cardiovascular disease. We investigated whether anti-GPCR autoantibodies are elevated in women with a history of early-onset preeclampsia 8-11 years postpartum, and whether they correlate with clinical outcomes. We investigated data from the Preeclampsia Risk EValuation in FEMales cohort, a retrospective matched case-control study. Anti AT1R-, beta1AR-, ETAR-, PAR1- and CXCR3- autoantibodies were determined in 485 samples by using commercially available ELISA. Women with the lowest combined levels of autoantibodies and a history of early preeclampsia had significantly higher SBP, DBP and MAP (all p

An autoimmune reaction directed against the cardiac b1-adrenergic receptor (beta1-ADR) leading to the generation of autoantibodies (AA) against this G-coupled receptor has been described in patients with heart failure (HF). Agonist-like beta1-ADR-AA are associated with morbidity in HF patients and even predict mortality. Standardised and valid diagnostic tools to detect beta 1-ADR-AA in clinical routine are lacking. We used a novel ELISA approach to investigate beta 1-ADR-AA in a cohort of 574 HF patients of the CIBIS-ELD trial with follow up. The CIBIS-ELD trial compared the titration of bisoprolol and carvedilol to recommended target doses in regard to BB tolerability in patients aged 65 years and older. Patient with left ventricular (LV) ejection fraction (EF) less than 50% or LV diameter end diastolic (DED) more than 55 cm showed significantly higher levels of beta1-ADR-AA. Although not yet fully validated, this ELISA allowed for a negative correlation of beta1-ADR-AA with the EF at baseline and at the follow up, beta1-ADR-AA further correlated positively with basal heart rate at follow up 12 weeks later. beta1-ADR-AA levels thus determined significantly increased under titration with beta-blockers (pless than 0.01). Changes in beta1-ADR-AA between F-Up and baseline were significantly higher in patients who used beta blockers (p=0.016) before study inclusion. The type of beta-blocker titrated in this study did not affect log beta1-ADR-AA levels at baseline (p=0.132), follow-up (p=0.058), nor the change (p=0.426). beta1-ADR-AA levels were estimated using a novel, commercially available ELISA. Although not yet fully validated, this ELISA allowed for pathophysiological insights: beta1-ADR-AA levels thus determined significantly increased under titration with beta-blockers (pless than 0.01), irrespective of type of BB. Higher levels of beta1-ADR-AA at baseline are associated with higher heart rates, lower ejection fraction and enlarged left ventricles. The relevance of the beta1-ADR-AA biomarker should be further evaluated.

Background Networks formed of numerous autoantibodies (aabs) directed against G-protein coupled receptors (GPCR) have been suggested to play important role in autoimmune disorders. In present study, we aimed to evaluate the association between anti-GPCR antibodies and primary Sjogren’s syndrome (pSS) to determine the potential pathogenic factors. Methods By applying a cell membrane-based ELISA technique, which is capable of detecting aabs against conformational epitopes within GPCR, serum levels of fourteen GPCR were determined in well-characterized patients with pSS (n = 52) and gender-matched healthy controls (n = 54). Comparisons between groups were analyzed by two-tailed Mann-Whitney U test, Bonferroni correction was applied for multiple comparisons. Spearman`s rank correlation coefficients were calculated between variables and visualized by heat map. Results Compared to healthy subjects, sera of patients with pSS showed significantly higher binding to β2AR and ETAR, but lower binding to C5aR1, C3aR1, CXCR3, and CXCR4. Autoantibodies against C5aR1, C3aR1, CXCR3, and CXCR4 were also decreased in patients with rheumatoid arthritis. In pSS patients, levels of anti-CXCR3 and anti-CXCR4 antibodies were negatively correlated with circulating lymphocyte counts. Furthermore, correlation signatures of anti-GPCR antibodies changed dramatically in the patients with pulmonary involvement. Conclusions This study demonstrates an association between pSS and autoantibodies recognizing GPCR, especially those functionally involved in immune cell migration and exocrine glandular secretion.

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.

BACKGROUND Endogenous antibodies to signaling molecules and receptors (Abs) are associated with Alzheimer's disease (AD). OBJECTIVES To investigate the association of 33 Abs to dopaminergic, serotoninergic, muscarinic, adrenergic, vascular, and immune receptors with cognitive, neuropsychiatric, and mortality outcomes. METHODS Ninety-one patients with mild AD were followed annually for 5 years with the Mini-Mental State Examination (MMSE) and the Neuropsychiatric Inventory (NPI; composite outcomes: "psychosis" (item 1 + 2), "mood" (item 4 + 5 + 7), and "agitation" (item 3 + 8 + 9)). Abs were quantified in sera obtained at baseline by ELISA and reduced to principal components (PCs). Associations between Abs and outcomes were assessed by a mixed model (MMSE decline), zero-inflated fixed effects count models (composite NPI scores), and Cox regression (mortality). The resulting p-values were adjusted for multiple testing according to a false discovery rate of 0.05 (Benjamini-Hochberg). RESULTS The measured levels of the 33 Abs formed four PCs. PC1 (dopaminergic and serotonergic Abs) was associated with increased mortality (Hazard ratio 2.57, p < 0.001), PC2 (serotonergic, immune, and vascular Abs) with decreased agitation symptoms (β - 0.19, p < 0.001), and PC3 (cholinergic receptor Abs) with increased mood symptoms (β 0.04, p = 0.002), over time. There were no associations between Abs and MMSE decline. CONCLUSION The associations between Abs, mortality, and neuropsychiatric symptoms reported in this cohort are intriguing. They cannot, however, be generalized. Validation in independent sample sets is required.

Naturally occurring antibodies are linked to inflammation, tissue injury and apoptosis, processes also linked to heart failure. Associations between antibodies, inflammation and myocardial damage, have not been elucidated in heart failure.We investigated if 25 antibodies to receptors expressed in the cardiovascular system were associated with troponin-T, biomarkers of inflammation and clinical measures of disease severity, in patients with heart failure.Antibodies in sera from patients (n=191) with ischemic (n=155) or non-ischemic (n=36) heart failure were measured with full-receptor sandwich enzyme-linked immunosorbent assays. All patients underwent coronary angiography with determination of left ventricular ejection fraction (LVEF) and left ventricular end-diastolic pressure (LVEDP). Measured biomarkers included troponin-T, C-reactive protein, erythrocyte sedimentation rate, fibrinogen and neopterin.Stabilin-1-antibodies correlated with troponin-T (β 0.23 p=0.008), soluble endoglin-antibodies with erythrocyte sedimentation rate (β 0.19, p=0.007) and fibrinogen (β 0.28, p0.001). Platelet-derived growth factor subunit β-antibodies were associated with neopterin (β 0.17, p=0.002). All antibodies were correlated (R 0.26 to 0.91) and formed 4 principal components (PCs). Patients with high CRP and high PC2 had higher NYHA class and patients with high troponin-T and high PC1 had lower LVEDP (interactions, all p0.05).Antibodies to receptors are correlated and are associated with biomarkers of inflammation and myocardial damage, which further modifies their association with disease severity in heart failure. Their functional activity and immunological function, remain undecided.

BACKGROUND Recent data have shown an increased risk for rejection, fibrosis progression, and death in liver transplantation (LT) recipients with preformed or de novo HLA donor-specific alloantibodies (DSA). However, the role of non-HLA autoantibodies and the interaction between HLA DSA and non-HLA autoantibodies remains uncharacterized. METHODS We analyzed 1269 primary LT recipients from 1 of 2000 to 4 of 2009 with known HLA DSA status for angiotensin II type-1 receptor and endothelin-1 type A receptor autoantibodies pre-LT, and year 1 post-LT. RESULTS Preformed non-HLA autoantibodies alone did not impact outcomes. In multivariable modeling, the combination of preformed non-HLA autoantibodies and HLA-DSA were associated with an increased risk for death (hazard ratio [HR], 1.66; P = 0.02) especially if the HLA DSA was of the IgG3 subclass (HR, 2.28; P = 0.01). A single de novo non-HLA autoantibody was associated with an increased risk for T cell-mediated rejection or antibody-mediated rejection (68% vs 41%, P = 0.01) and fibrosis progression (HR, 1.84; P = 0.02). Biopsies with de novo non-HLA autoantibodies revealed a new sinusoidal C4d staining pattern when compared with HLA DSA (71% vs 3%; P < 0.001). Liver sinusoidal endothelial cell activation and stellate cell activation was increased in patients with non-HLA autoantibodies in the location of C4d positivity. CONCLUSIONS A non-HLA autoantibody combined with a preformed HLA DSA is associated with an increased mortality risk. Isolated de novo anti-angiotensin II type-1 receptor and anti-endothelin-1 type A receptor autoantibodies are associated with an increased risk of rejection and fibrosis progression. The novel location of C4d staining in proximity to liver sinusoidal endothelial cell capillarization and stellate cell activation demonstrates allograft injury in proximity to non-HLA autoantibody binding.

Background: Alzheimer's disease (AD) is associated with several antibodies as well as signaling molecules and receptors. These may be detrimental in the presence of a disrupted blood-brain barrier (BBB). Objective: To investigate whether the levels of antibodies toward 33 signaling molecules involved in neurotransmitter, vascular, and immune functions were associated with AD and, within the AD group; cognitive function and mood. Methods: Antibodies in sera from patients with mild AD [(n = 91) defined as a Mini-Mental State Examination ≥ 20 or a Clinical Dementia Rating Scale≤1] and healthy controls (n = 102) were measured with enzyme-linked immunosorbent assays. Levels in AD and controls were compared by Mann-Whitney test. In the AD group, associations between antibodies and psychometric test scores were analyzed by robust regression. The false discovery threshold was set to 0.05. Results: Antibodies to serotonin receptors [5-HT2AR (effect size (r) = 0.21, p = 0.004), 5-HT2CR (r = 0.25, p = 0.0005) and 5-HT7R (r = 0.21, p = 0.003)], vascular endothelial growth factor receptor 1 [VEGFR1 (r = 0.29, p

Background Previous studies identified functional autoantibodies against the angiotensin receptor type-1 (AT1R) and the endothelin receptor type A (ETAR) in about 85% of the patients with systemic sclerosis (SSc, 1). The antibodies are cross-reactive, agonistic and functionally active by increasing the effects of the natural ligands as well as by specific activation of the receptors (2, 3). The levels of the antibodies are associated with clinical findings such as pulmonary arterial hypertension (PAH). Patients with highest antibody levels show worst prognosis and do not respond well to receptor blocker therapy (2-4). Several in vitro effects of the antibodies depend on the antibody levels and on the cell type bearing the receptors. Receptor expression of ETAR and AT1R was highest in early disease (3). Objectives To determine predictors of PAH, DU and clinical complications by measuring the antibody levels and the receptor expression on peripheral mononuclear cells in patients with systemic sclerosis. Methods The current study analyzed the serological levels of anti-AT1R and anti-ETAR antibodies and the extracellular and intracellular expression of AT1R, AT2R, ETAR and ETBR on circulating CD4pos T cells, CD8pos T cells, CD14pos Macrophages, CD15pos Granulocytes and CD19pos B cells in SSc (n=41) using sandwich ELISA and flow cytometry. Clinical data (PAH, history of digital ulcers, digital-ulcers score, mRSS, pulmonary fibrosis, therapy) and serological markers (ESR, CRP, NT-proBNP) were gathering at the time of serum sampling and every three-month up to 27month after baseline. Results Patients with PAH demonstrated a lower AT1R MFI and AT1R/AT2R MFI ratio on all PBMC. Levels of NT-proBNP correlated negatively with the AT1R MFI and AT1R/AT2R MFI ratio on all PBMC. The levels of anti-AT1R ab correlated with the NT-proBNP in SSc patients with levels of NT-proBNP Conclusion Expression of AT1R and AT2R on PBMC could be of diagnostic value identifying clinical progress and/or subgroups in SSc. Their role in the pathophysiology, e.g. their impact of endothelial damage, has to be further investigated in SSc. References [1] Riemekasten G. et al., Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis. 2011Mar;70(3):530-6. [2] Kill A. et al., Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis Res Ther. 2014Jan28;16(1):R29. [3] Becker MO. Et al., Vascular receptor autoantibodies in pulmonary arterial hypertension associated with systemic sclerosis. Am J Respir Crit Care Med. 2014Oct1;190(7):808-17. [4] Gunther J. et al., Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients. Arthritis Res Ther. 2014Mar11;16(2):R65. Acknowledgement We thank Actelion Pharmaceutical GmbH for their financial support. Disclosure of Interests Sebastian Klapa: None declared, Silke Pitann: None declared, Gabriela Marschner: None declared, Susanne Riepe: None declared, Andreas Koch: None declared, Antje Muller: None declared, Harald Heidecke: None declared, Peter Lamprecht: None declared, Gabriela Riemekasten Consultant for: Chugai, F. Hoffmann-La Roche, Speakers bureau: Chugai, F. Hoffmann-La Roche

Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan’s inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.

Endothelin 1 (ET-1) is a potent vasoactive peptide hormone produced by endothelial cells, macrophages and vascular smooth muscle cells (VSMCs). Elevated ET-1 plasma levels have been reported in patients with ischaemic complications in giant-cell arteritis (GCA), a systemic vasculitis predominantly affecting large vessels and their branches.1 In temporal artery biopsy specimens, increased expression of ET-1 and the G protein-coupled vasoconstrictive endothelin receptor A (ETAR) has been found.2 ETAR activation induces focal adhesion kinase-mediated morphological changes and increased motility of VSMC potentially contributing to intimal hyperplasia and vascular occlusion in GCA.3 VSMC migration can be blocked by specific ETAR antagonists.3 Notably, disease-specific anti-G protein-coupled receptor autoantibody (aab) signatures have been found in different autoimmune diseases.4 5 Anti-ETAR aabs are increased and associated with pulmonary arteriolar occlusion and hypertension in systemic sclerosis.5 6 Anti-ETAR aabss induce vascular adhesion molecules, interleukin (IL)-8 and chemokine ligand CCL-18 productions and exhibit chemotactic activity by mediating neutrophil and T cell migration.5 6 To address …

Importance and objectives: There is unmet medical need to understand the pathogenic mechanism of the panoply of clinical manifestations associated with silicone breast implants (SBIs) such as severe fatigue, widespread pain, palpitations, dry mouth and eyes, depression, hearing loss etc. We aimed to determine whether autoantibodies against the autonomic nervous system receptors can explain the enigmatic and subjective clinical manifestation reported by women with SBIs. Results: Circulating level of autoantibodies against G protein-coupled receptors (GPCRs) of the autonomic nervous system (adrenergic, muscarinic, endothelin and angiotensin receptors) have been evaluated in symptomatic women with SBIs using an ELISA method. These women with SBIs addressed our clinic due to various subjective and autonomic-related manifestations such as chronic severe fatigue, cognitive impairment, widespread pain, memory loss, sleep disorders, palpitations, depression, hearing abnormalities etc. We report for the first time, a significant reduction in the sera level of anti-β1 adrenergic receptor (p < 0.001), anti-angiotensin II type 1 receptor (p < 0.001) and anti-endothelin receptor type A (p = 0.001) autoantibodies in women with SBIs (n = 93) as compared with aged matched healthy women (n = 36). Importantly, anti-β1 adrenergic receptor autoantibody was found to significantly correlate with autonomic-related manifestations such as: sleep disorders and depression in women with SBIs. Conclusions: Chronic immune stimulation by silicone material may lead to an autoimmune dysautonomia in a subgroup of potentially genetically susceptible women with SBIs. The appearance of autoantibodies against GPCRs of the autonomic nervous system serve as an explanation for the subjective autonomic-related manifestations reported in women with SBIs.

Background:The morbidity and mortality of systemic sclerosis (SSc) are largely determined by vascular and fibrotic pathologies. Levels of autoantibodies (ab) against G protein-coupled receptors (GPCR), growth factors (GF) and growth factor receptors (GFR) are altered in SSc compared to healthy controls (HC) 1. Thus, higher angiotensin II receptor type 1 - (AT1R) and endothelin receptor type A - (ETAR) ab levels are associated with severe disease and SSc-related mortality 2. CXC motiv chemokine receptor 3 - (CXCR3) and 4 - (CXCR4) ab have predictive value for deterioration of pulmonary fibrosis (PF) 3.Objectives:We used statistical methods to identify associations between disease manifestations and 28 new ab directed against GPCR, GF and GFR in SSc.Methods:Ab against the following targets were measured in sera from SSc patients (n = 177) and HC (n = 88): Adrenoceptors alpha-1 (ADRA1), alpha-2 (ADRA2), beta-1 (ADRB1), beta-2 (ADRB2); muscarinoceptors 1-5 (M1R - M5R); AT1R, ETAR, endothelin B receptor (ETBR); CXCR3, CXCR4; complement receptors 3a (C3aR) and 5a (C5aR); protease-activated receptors 1 (PAR1) and 2 (PAR2); vascular endothelial growth factor A (VEGFA) and its receptors 1 (VEGFR1) and 2 (VEGFR2), epithelial growth factor (EGF)/ - receptor (EGFR); hepatocyte growth factor (HGF)/ - receptor (HGFR), platelet-derived growth factor-AA (PDGFAA), placental growth factor (PlGF).The organ involvement (PF, cardiac involvement, PAH, gastrointestinal tract) and quantitative markers (modified Rodnan skin score, SSc activity score, pulmonary function, cardiac enzymes and echocardiography, routine laboratory, autoimmune diagnostics) as well as demographic data were recorded retrospectively at the time of sample collection. Statistical analysis was performed using the Mann-Whitney U test (MWU), Pearson correlations, ROC analysis, and age-adjusted logistic regression models.Results:In SSc 20 of 28 measured ab levels are significantly altered compared to HC. According to the Pearson correlation matrix, the ab-levels are highly correlated and build a network that differs between HC and SSc. Furthermore, altered network signatures are formed in the differentiated analysis of several disease manifestations of SSc such as SSc-subtype or PF. Based on ROC analysis, FGF-ab, ADRB1-ab and PlGF-ab are well suited to predict SSc (Figure 1).In addition, limited cutaneous SSc (lSSc) patients displayed lower levels of most ab than diffuse cutaneous SSc patients, whereas cardiac and pulmonary involvement are associated with higher ab levels. In the logistic regression lSSc is associated with lower levels of ab against M1R, M2R, C5aR, ETAR, AT1R, PAR1, EGFR. Higher levels for ab against M1R, M2R, ETBR, C5aR are associated with PF, higher levels of ab against complement receptors, adrenoreceptors and EGF with NT-proBNP elevation.Conclusion:The newly described antibodies against GPCR, GF and GFR are highly correlated. Associations with morbidity- and mortality-determining organ involvement indicate their possible functional relevance and novel pathophysiological mechanisms. As new biomarkers, some of the ab have prognostic value for SSc; for other manifestations, their value should be evaluated in further studies.References:[1]Cabral-Marques, O., Marques, A., Giil, L.M. et al. GPCR-specific autoantibody signatures are associated with physiological and pathological immune homeostasis. Nat Commun9, 5224 (2018). https://doi.org/10.1038/s41467-018-07598-9[2]Riemekasten G, Philippe A, Näther M, et al. Involvement of functional autoantibodies against vascular receptors in systemic sclerosis Annals of the Rheumatic Diseases 2011;70:530-536.[3]Weigold, F., Günther, J., Pfeiffenberger, M. et al. Antibodies against chemokine receptors CXCR3 and CXCR4 predict progressive deterioration of lung function in patients with systemic sclerosis. Arthritis Res Ther 20, 52 (2018). https://doi.org/10.1186/s13075-018-1545-8Disclosure of Interests:Kristina Sterner: None declared, Césaire J. K. Fouodo: None declared, Inke König: None declared, Axel Künstner: None declared, Hauke Busch: None declared, Harald Heidecke Shareholder of: Owner of CellTrend, Anja Schumann: None declared, Antje Müller: None declared, Gabriela Riemekasten: None declared, Susanne Schinke Grant/research support from: UCB sponsors EULAR registration fees

Women with preexisting or gestational diabetes mellitus have an increased risk for developing preeclampsia. Diabetes and pregnancy are both characterized by very high prorenin levels and renin-angiotensin system activation. Prorenin bound to the (pro)renin receptor has enzymatic activity. We hypothesized that soluble (pro)renin receptor levels are elevated in high-risk pregnancies. Third trimester maternal blood samples from complicated pregnancies (n = 165), (preeclampsia [n = 76], diabetes mellitus [type I diabetes, n = 35; type II diabetes, n = 11; gestational diabetes mellitus, n = 43]), and healthy pregnancies (n = 49) were analyzed for prorenin, renin, and soluble (pro)renin receptor. There were no significant differences in prorenin or renin levels between the study groups in a multivariate model. In the group of women with gestational diabetes, soluble (pro)renin receptor concentrations were significantly higher compared with healthy pregnancies or preeclampsia. Soluble (pro)renin receptor did not correlate with renin or prorenin levels for any of the study groups. Our results show that soluble (pro)renin receptor is dysregulated in pregnancies affected by diabetes mellitus, but not in preeclampsia. Alterations in circulating soluble (pro)renin receptor are unrelated to renin/prorenin in pregnancy, but may be of pathophysiological relevance in diabetic pregnancies in a renin-angiotensin system-independent manner.

Background:Activation of the alternative and final common pathways have been shown in ANCA-associated vasculitis (AAV) (1). Circulating titers of C5a are elevated and correlate with disease activity in AAV. Binding to the corresponding G protein-coupled receptor (GPCR) C5aR1 enhances the influx of neutrophils, leading to ROS generation and severe necrotizing of vascular walls (2). Moreover, subsequent interaction of C5a with C5aR1 may represent a proinflammatory amplification loop (3). Blocking of the receptor is protective in a murine model in AAV (4). In humans, avacopan, a C5aR1-inhibitor showed promising results as glucocorticoid-sparing agent in two randomized phase II and one ongoing phase III clinicals trials in AAV (NCT02994927). Notably, disease-specific anti-GPCR autoantibody (aab) signatures have been found in different autoimmune diseases (5).Objectives:The aim of the present study was to examine whether (patho)physiological anti-C3aR and anti-C5aR1 aabs correlate with clinical findings in AAV, and whether this is linked to the clinical outcome.Methods:Sera and plasma of AAV patients [granulomatosis with polyangiitis (GPA), n=64; microscopic polyangiitis (MPA), n=26; eosinophilic granulomatosis with polyangiitis (EGPA), n=11] were measured by Elisa for circulating autoantibodies against complement receptors C3a (anti-C3aR aab) and C5a (anti-C5aR1 aab) and plasma levels of C3a and C5a. Expression of C3aR and C5aR1 on T-cells was determined using flow cytometry. Clinical data were assessed at the time of serum sampling and during follow-up for 48 monthsResults:GPA displayed low titers of anti-C3aR aab (GPA:5.33±2.54vs. HD:6.47±2.61, P=0.0031). Anti-C5aR1 aab were decreased in AAV, especially in GPA (GPA:1.02±1.07vs. HD:6.63±2.91, P=vs. HD:3.44±0.68%, P=0.0021; CD8+C5aR1+T-cells GPA:9.74±2.10%vs.HD:4.11±0.92%, P=0.0198). Reduced titers of anti-C5aR1 aab Conclusion:As potential diagnostic marker, anti-C5aR1 aab titer may additionally be useful to monitor disease activity in AAV.References:[1]Chen M et al.Complement deposition in renal histopathology of patients with ANCA-associated pauci-immune glomerulonephritis.Nephrol Dial Transpl. 2009;24:1247-1252[2]Schreiber A et al.C5a receptor mediates neutrophil activation an ANCA-induced glomerulonephritis.J Am Soc Nephrol. 2009; 20:289-298[3]Lamprecht P et al.: Pathogenetic and clinical aspects of Anti-Neutrophil Cytoplasmic Autoantibody-associated vasculitides.Front Immunol.2018 Apr 9;9-680[4]Xiao H et al.C5a receptor (CD88) blockade protects against MPO-ANCA GN.J Am Soc Nephrol. 2014;25(2):225-31[5]Klapa S et al. Decreased endothelin receptor A autoantibody levels are associated with early ischaemic events in patients with giant-cell arteritis.Ann Rheum Dis2019 Oct;78(19):1443-1444Disclosure of Interests:Sebastian Klapa Grant/research support from: Actelion, Consultant of: Pfizer, Abbvie, Antje Müller: None declared, Andreas Koch: None declared, Anja Kerstein-Staehle: None declared, Wataru Kaehler: None declared, Harald Heidecke Shareholder of: Cell Trend GmbH, Employee of: Cell Trend GmbH, Speakers bureau: Cell Trend GmbH, Susanne Schinke Speakers bureau: Pfizer, Markus Huber-Lang: None declared, Martin Nitschke: None declared, Silke Pitann: None declared, Christian Karsten: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared

Chronic fatigue syndrome, postural orthostatic tachycardia syndrome, complex regional pain syndrome and silicone implant incompatibility syndrome are a subject of debate among clinicians and researchers. Both the pathogenesis and treatment of these disorders require further study. In this paper we summarize the evidence regarding the role of autoimmunity in these four syndromes with respect to immunogenetics, autoimmune co-morbidities, alteration in immune cell subsets, production of autoantibodies and presentation in animal models. These syndromes could be incorporated in a new concept of autoimmune neurosensory dysautonomia with the common denominators of autoantibodies against G-protein coupled receptors and small fiber neuropathy. Sjogren's syndrome, which is a classical autoimmune disease, could serve as a disease model, illustrating the concept. Development of this concept aims to identify an apparently autoimmune subgroup of the disputable disorders, addressed in the review, which may most benefit from the immunotherapy.

G protein-coupled receptors (GPCRs) form a most diverse family of integral membrane proteins that mediate homeostatic and pathological processes, most notably by orchestrating cell distribution throughout the body, their infiltration, and time of presence in inflamed tissues. Here we discuss loss-of-orientation-effects in GPCR-mediated cell trafficking and migration and their impact on the phenotype of autoimmune diseases. In this context, we provide a systemic and integrative view of the contribution of abnormal GPCR expression as well as the levels of natural ligands and functional autoantibodies to the phenotype of autoimmune diseases. Currently, several studies propose that functional autoantibodies (including those targeting GPCRs) constitute an exclusively pathogenic or pathognomonic phenomenon. Here we reinforce the need of revising this point of view, and suggest that functional autoantibodies primary play a role in normal human physiology, while dysregulation of their functions causes autoimmune disease. Because patients with autoimmune diseases still suffer from severe morbidity and mortality rates, we consider expanding our knowledge on (patho)physiological roles of GPCR as a prerequisite for the development of novel specific therapeutic modalities.

Background: Anti-beta-1-adrenergic receptor antibodies (anti-β1AR Ab) are associated with ischemic cardiomyopathies (ICM). Evidence continues to emerge supporting an autoimmune component to various cardiac diseases. This study compares anti-β1AR Ab concentrations in patients with different entities of acute coronary syndromes (ACS) to asymptomatic non-ACS patients with positron-emission computed tomography (PET/CT)-proven atherosclerosis, and healthy controls.Methods: Serum anti-β1AR Ab IgG concentrations were measured in 212 ACS patients, 100 atherosclerosis patients, and 62 controls using ELISA. All ACS patients underwent coronary angiography. All 374 patients participating completed a structured questionnaire regarding traditional cardiovascular risk factors. ACS patients were followed up for 6 months.Results: Patients with ACS exhibited lower anti-β1AR Ab levels compared to patients with atherosclerosis or healthy controls (both p < 0.001). No differences in the ab levels were evident between healthy controls and patients with atherosclerosis. In the ACS groups, lower concentrations were found in patients with ST-elevation myocardial infarction (STEMI) (0.67 μg/ml) compared to patients with angina pectoris (AP) and non-ST elevation myocardial infarction (NSTEMI) (both 0.76 μg/ml, p = 0.008). Anti-β1AR Ab levels ≤ 0.772 μg/ml were predictive for death and reinfarction (AUC 0.77, p = 0.006). No significant correlations between anti-β1AR Ab levels and atherosclerotic burden or traditional cardiovascular risk factors were identified.Conclusions: Lower anti-β1AR Ab concentrations appear to characterize ACS phenotypes and could serve as diagnostic and prognostic markers independent from traditional risk factors for atheroscle. The prognostic predictive value of anti-β1AR Ab in ACS remains to be confirmed in larger studies.

Background Since the time when Paul Ehrlich conceived the term “horror autotoxicus”, autoantibodies have been associated with the development of autoimmune diseases. However, several works have recently shown the presence of autoantibodies in sera from healthy subjects (n=489), who do not develop autoimmune diseases. Objectives Here, we report a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCRs), growth factors and growth factor-related molecules in sera from healthy subjects. Methods Autoantibody levels in sera were assessed using ELISA. Autoantibody network was analysed by exploratory factor analysis (EFA), dandelion plot method, hierarchical clustering, and multi-study factor analysis (MFA). We also reverse engineered autoantibody functions through in silico evaluation of autoantibody target interactions using STRING and gene ontology (GO). To test the autoantibodies functionality we assessed the in vitro production of IL-8 by PBMCs and neutrophil migration in response to IgG from healthy subjects as well as ETAR-immunised and control mice. Leukocyte cellularity to secondary immune organs was analysed comparing ETAR-immunised with control mice. Results Gender, age and the presence of pathological conditions (systemic sclerosis n=84, Alzheimer’s disease n=91 and ovarian cancer n=207) changed correlations between the autoantibodies and their hierarchical clustering distribution. Notably, subjects at age below and above 65 years or with pathological conditions exhibited particular autoantibody hierarchical clustering signatures. In addition, females at age above 65 years, representing the group of subjects with higher risk to develop SSc, displayed the closest link to SSc in terms of autoantibody hierarchical clustering. Finally, autoantibody directed against the endothelin receptor type A (ETAR) showed an essential role in the autoantibody network by orchestrating neutrophil trafficking in vitro and in ETAR-immunised mice. Conclusions Our data provide a framework for the existence of a physiological network of autoantibodies and reveal a new paradigmatic view on these physiological molecules. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is a complex connective tissue disease which is characterised by autoimmunity, vasculopathy and fibrosis1. Our recent study showed that the progression of SSc was strongly associated with the autoantibodies against angiotensin II receptor I (AT1R), suggesting a role of autoimmunity to AT1R in the pathogenesis of the disease 2 Objectives In this study, we aimed to investigate the role of AT1R in the pathogenesis of SSc in mice. Methods C57BL/6J mice were immunised with membrane extract (ME) of CHO cell overexpressing human AT1R or with ME of CHO cells as control. Serum, lung and skin samples were collected and assessed 63 days after immunisation for autoantibody production, inflammation and fibrosis, which are hallmarks for SSc. Results Immunisation with hAT1R induced the production of autoantibodies against the receptor in mice, and autoantibody deposition was found in the lung. Histologically, mice immunised with hAT1R showed a SSc-like disease, including perivascular infiltrates and fibrosis in the skin as well as pulmonary inflammation. The inflammation in the skin and the lung were characterised by infiltration of T- and B-cells. Furthermore, transfer of immune cells from hAT1R-immunised mice into C57BL/6J mice induced inflammation in the lung. Conclusions This study demonstrates that immunisation with hAT1R can induce a SSc-like disease, thus showing a pathogenic role of autoimmunity to AT1R and providng a novel mosue model for the diseases. Furthermore, this study also introduces a new immunisation strategy to generate functional autoantibodies against receptors on the cell membrane. References [1] LeRoy EC, Black C, Fleischmajer R, et al. Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol. 1988;15(2):202–205. [2] Riemekasten G, Philippe A, Nather M, et al. Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis. 2011;70(3):530–536. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is a rare autoimmune multisystemic disease with a significant disease burden and impact on life quality and survival. Disease specific, diagnostic and prognostic antibodies (ab) are known such as Scl70 and centromer (ACA) ab1 or recently endothelin or angiotensin receptors.2 Functional ab can bind G protein-coupled receptors (GPCR) regulating immune function and were reported in the pathogenesis of various inflammatory and non-inflammatory diseases.3 Objectives We analysed 31 ab against GPCRs and growth factors in a retrospective cohort of 71 SSc patients compared to 196 sera from healthy controls (HC). Ab levels were related to disease manifestations such as sex, age, SSc phenotype in order to hypothesise functional ab and new pathogenic targets in SSc. Methods The retrospective clinical characterisation of 14 male and 57 female SSc patients (26–82 years) included mRSS, organ involvement assessed by laboratory tests, spirometry and imaging such as CT-scan or echocardiography. 30/71 had active disease (EUSTAR activity score). Ab were measured by ELISA and normalised to a standard serum. Median ab levels from SSc were compared to HC (Mann Whitney Test). Ab patterns were analysed using different statistical approaches (factor analysis, principal component analysis (PCA), linear discriminant analysis (LDA), cluster analysis and biserial correlation. Results Clinical SSc subgroups (diffuse/limited cutaneous, male/female) differ in ab levels and form separate clusters (LDA method). Moreover, 5 resp. 7 latent factors group ab and clinical disease manifestations. Factor analysis reveals VEGFR2 and YBX1 ab to be more unique with the lowest communalities. The biserial correlation shows moderate associations between ab patterns and SSc specific symptoms such as Raynaud’s, calcinosis or akroosteolysis but also unspecific symptoms such as polyneuropathy. Compared to association of ETAR ab with Raynaud’s and skin sclerosis HGFR ab are inversely correlated. In HC most ab levels against GPCR and growth factors are higher than in SSc except for YBX1 which has the highest ab levels in SSc patients. In HC ab levels against YBX1 ab are associated with male sex and family history of rheumatic diseases. Yet, ADRB2 ab are linked to the absence of GI symptoms or depression and ab against ENG, ETAR, PAR2, PAR1 with normal troponine levels (absence of heart involvement). Conclusions We describe 31 new ab against GPCR and growth factors in SSc. Ab as well as SSc disease manifestations could be clustered by latent factors. Most ab titers in SSc were lower than in HC. Some ab were linked to the absence of SSc manifestations. Thus, we postulate that a dysbalance of functionally protective autoantibodies, that can be found in healthy individuals, and the appearance of SSc specific ab such as Scl70 contribute to its pathogenesis. Considering the preliminary character of our data, the functional impact of ab against GPCR and growth factors has to be validated in vitro and statistical correlations to be confirmed in a prospective independent patient cohort. References [1] Herrick AL, et al. ARD2018(0):1–8. [2] Kill A, et al. Curr Rheumatol Rep2015;(17):34. [3] Cabral-Marques O, et al. Nat. Rev2017;(13):648–656. Disclosure of Interest None declared

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer’s disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system., Autoantibodies are implicated in autoimmunity, but may also be present in healthy individuals. Here the authors find that the autoantibody specificity signatures against various G protein-coupled receptors are associated with multiple parameters, including disease states, to imply a physiological function in maintaining immune homeostasis.

G-protein-coupled receptors (GPCRs) are the largest family of receptors in humans. GPCRs are seven-transmembrane receptors that are activated by the binding of a ligand to the extracellular domain. In addition to the endogenous ligands, auto-antibodies (aab) can also bind to the GPCRs. They can activate different and specific cellular pathways which contribute to various diseases. In this review, the authors summarize the knowledge about antibodies targeting GPCRs and their effects and relevance in the pathogenesis of various diseases and their use in clinical diagnostics. We highlight the role of different activating anti-GPCR aab in solid organ transplantations, stem cell transplantations, systemic sclerosis, preeclampsia, chronic fatigue syndrome, cardiovascular diseases, Alzheimer's disease, and cancer.

AIM This study aimed to analyze the predictive, prognostic and diagnostic value of autoantibodies to coagulation factor II thrombin receptor (F2R; protease-activated receptor 1, PAR1) (PAR1-AB) in patients with primary epithelial ovarian cancer (EOC). MATERIALS AND METHODS A total of 197 patients with primary EOC and 200 healthy female blood donors were included in the study. Enzyme-linked immunosorbent assay was applied to determine PAR1-AB levels in blood sera taken preoperatively. Correlation of PAR1-AB with clinicopathological outcome, progression-free (PFS) and overall (OS) survival was analyzed and patients were compared with controls. RESULTS PAR1-AB was significantly negatively correlated with histological grading (p=0.008) and was significantly lower in the patient group compared to healthy controls (p

Background The chemokine receptors CXCR3 and CXCR4 are involved in the pathogenesis of fibrosis, a key feature of systemic sclerosis (SSc). It is hypothesized that immunoglobulin (Ig)G antibodies (abs) against these two receptors are present in patients with SSc and are associated with clinical findings. Methods Anti-CXCR3 and anti-CXCR4 ab levels were measured in 449 sera from 327 SSc patients and in 234 sera from healthy donors (HD) by enzyme-linked immunosorbent assay (ELISA). In SSc, ab levels were compared with clinical data in a cross-sectional and longitudinal setting. Protein expression of CXCR3 and CXCR4 on peripheral blood mononuclear cells (PBMCs) was analyzed in 17 SSc patients and 8 HD by flow cytometry. Results Anti-CXCR3 and anti-CXCR4 ab levels were different among SSc subgroups compared with HD and were highest in diffuse SSc patients. The ab levels strongly correlated with each other (r = 0.85). Patients with SSc-related interstitial lung disease (SSc-ILD) exhibited higher ab levels which negatively correlated with lung function parameters (e.g., r = −0.5 and r = −0.43 for predicted vital capacity, respectively). However, patients with deterioration of lung function showed lower anti-CXCR3/4 ab levels compared with those with stable disease. Frequencies and median fluorescence intensities (MFI) of CXCR3+ and CXCR4+ PBMCs were lower in SSc patients compared with HD and correlated with the severity of skin and lung fibrosis. They correlated with the severity of skin and lung fibrosis. Conclusions Anti-CXCR3/4 abs and their corresponding receptors are linked with the severity of SSc-ILD. Antibody levels discriminate patients with stable or decreasing lung function and could be used for risk stratification. Electronic supplementary material The online version of this article (10.1186/s13075-018-1545-8) contains supplementary material, which is available to authorized users.

Introduction Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) point to an autoimmune disease directed against neurotransmitter receptors. We had observed elevated autoantibodies against s2 adrenergic receptors, and muscarinic 3 and 4 acetylcholine receptors in a subset of patients. Immunoadsorption (IA) was shown to be effective in removing autoantibodies and improve outcome in various autoimmune diseases. Methods 10 patients with post-infectious CFS/ME and elevated s2 autoantibodies were treated with IA with an IgG-binding column for 5 days. We assessed severity of symptoms as outcome parameter by disease specific scores. Antibodies were determined by ELISA and B cell phenotype by flow cytometry. Results IgG levels dropped to median 0.73 g/l (normal 7–16 g/l) after the 4th cycle of IA, while IgA and IgM levels remained unchanged. Similarly, elevated s2 IgG antibodies rapidly decreased during IA in 9 of 10 patients. Also 6 months later s2 autoantibodies were significantly lower compared to pretreatment. Frequency of memory B cells significantly decreased and frequency of plasma cells increased after the 4th IA cycle. A rapid improvement of symptoms was reported by 7 patients during the IA. 3 of these patients had long lasting moderate to marked improvement for 6–12+ months, 2 patients had short improvement only and 2 patients improved for several months following initial worsening. Conclusions IA can remove autoantibodies against s2 adrenergic receptor and lead to clinical improvement. B cell phenotyping provides evidence for an effect of IA on memory B cell development. Data from our pilot trial warrants further studies in CFS/ME.

Background There is growing evidence that nervous and immune system communicate with each other through soluble mediators.1 Immune cells such as neutrophils express muscarinic acetylcholine receptors (mAChR), which are neuroimmune receptors and highly prevalent in the nervous system.2 Aberrant neutrophil functioning plays an important role in various autoimmune diseases. Dysregulation of neutrophil immune responses such as oxidative burst and migration is one of the key mechanisms leading to tissue damage in autoimmune diseases.3 However, the impact of mAChR activation on neutrophils remains contradictory. Objectives We aimed to determine effects of muscarinic receptor activation on development and functions of neutrophils. Methods Neutrophils were isolated from peripheral blood of healthy donors by dextran sedimentation. After one hour in the absence or presence of the natural ligand acetylcholine (Ach) (10nM-100μM) or the muscarinic agonist oxotremorine-m (oxo-m) (10nM-100μM), neutrophil respiratory burst was analyzed by dihydrorhodamine (DHR) flow cytometry assay and migration assessed by transwell assay in response to N-formylmethionyl-leucyl-phenylalanine (fMLP). Cells that migrated were quantified by flow cytometry. To analyze the effects mAChR activation on the development of neutrophils, HL-60 cells were incubated in the presence of DMSO (1%), oxo-m (100μM) or DMSO plus oxo-m. After 6 days, cells were harvested and expression of maturation markers (CD15, CD63 and CD16) as well as mAChR (M1-M5) were measured by flow cytometry. Results We observed no effects of mAChR activation on the respiratory burst of neutrophils. However, both ACh and oxo-m inhibited neutrophil migration in a dose-dependent manner, but with peculiar differences. By increasing acetylcholine concentrations, we observed a reduction of neutrophil migration in a directly proportional manner. On the other hand, while the lowest dose (10nM) of oxo-m inhibited migration most effectively, the increase of oxo-m showed inversely proportional effects on neutrophil migration. Thus, we aimed to investigate, if the highest dose of oxo-m has a different effect on neutrophils ontogeny. In agreement with the results obtained with neutrophils, the incubation of HL-60 cells with the highest dose of oxo-m showed no effect on oxidative burst and migration and induced no changes in the expression of mAChRs (M1-M5), CD16 and CD63 in HL-60 cells. However, we observed that it resulted in significantly increased surface levels of the neutrophilic lineage marker CD15. Conclusions Our data indicate a differential activation of mAChR affecting different steps of neutrophil ontogeny. Considering this finding, abnormalities in the activation of muscarinic receptors as have been observed in autoimmune diseases might contribute to neutrophil dysfunction and need further investigation. References Blalock, J. E. The syntax of immune-neuroendocrine communication. Immunol Today 15, 504–511 (1994). Milara et al. Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients. Respiratory Research (2016) 17:145. Wright, H. L. et al. The multifactorial role of neutrophils in rheumatoid arthritis Nat Rev Rheumatol 10 593–601 (2014). Disclosure of Interest None declared

Background Endothelin-1 type A receptor (ETAR) antagonists (e.g. ambrisentan) are currently approved by the U.S Food and Drug administration, representing a well-tolerated treatment of pulmonary arterial hypertension (PAH)[1] for patients with connective tissue diseases such as systemic sclerosis (SSc). Noteworthy, increased numbers of infiltrating neutrophils have been associated with worse clinical outcome in PAH patients. In another context, several studies have reported that endothelin-1 and its receptor ETAR also play a central role in the development of tumour cell invasion and metastasis. However, the effects of ETAR antagonists on migration of neutrophils and tumour cells remain to be determined. Objectives The objective was to analyse the effects of two ETAR antagonists on migration of neutrophils and tumour cell lines. Methods The migratory ability of peripheral neutrophils from healthy donors (HD) and different tumour cell lines (myeloid leukaemia HL60 cells and human pancreatic adenocarcinoma COLO357 cells) was analysed in response to N-Formylmethionyl-leucyl-phenylalanine (FMLP) or Protease-activated receptor 2 (Par-2) agonist. Because it has been shown before [2], IgGs from HD and SSc patients were used as additional stimulus for migration. Neutrophils and HL60 cells were preincubated (1h) with sitaxentan or ambrisentan, respectively, before being tested for migration (1h) using the Transwell assay. COLO357 cells were incubated (48h) in the presence of sitaxentan and migration was tested in the Oris Pro Cell assay. Migration was analysed by automatic cell counting or digital photo analysis and a migration index was calculated. Results Sitaxentan and/or ambrisentan significantly blocked the migration of neutrophils and tumour cell lines. In more detail, neutrophil migration in response to FMLP, being set to 100%, was completely inhibited by sitaxentan (0,46%). Further, neutrophil migration in response to IgGs from HD and SSc patients was induced equally, again being set to 100%. In the presence of sitaxentan migration was reduced to 60%, respectively. In DMSO-differentiated HL60 cells the migratory capacity in response to FMLP (100%) was reduced to 66% by ambrisentan and to 14% by sitaxentan. Moreover, in the presence of sitaxentan and a Par-2 agonist the migratory ability of COLO357 cells was significantly decreased to 89% compared to Par-2 agonist only, being set to 100%. Conclusions Our results suggest a pivotal and non-redundant role of ETAR in cell migration, which needs further clarification in order to repurpose the use of ETAR inhibitors. Therapeutic switching of ETAR antagonists from PAH to cancer therapies is a promising adjuvant therapy. References Waxman AB. A review of sitaxsentan sodium in patients with pulmonary arterial hypertension. Vasc Health Risk Manag 2007;3:151–7. Kill A, Tabelin C, Undeutsch R, et al. Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis R&T, 2014, 16:R29. Disclosure of Interest None declared

Rationale: Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin (ET) 1 and angiotensin (Ang) II via endothelin receptor type A (ETAR) and Ang receptor type-1 (AT1R) activation are implicated in PAH pathogenesis.Objectives: We hypothesized that stimulating autoantibodies (Abs) targeting and activating AT1R and ETAR may contribute to SSc-PAH pathogenesis, and tested their functional and biomarker relevance.Methods: Anti-AT1R and -ETAR Abs were detected by ELISA in different cohorts of patients and tested in vitro and in an animal model for their pathophysiological effects.Measurements and Main Results: The Abs were significantly higher and more prevalent in patients with SSc-PAH (n = 81) and connective tissue disease–associated PAH (n = 110) compared with other forms of PAH/pulmonary hypertension (n = 106). High anti-AT1R and anti-ETAR Abs predicted development of SSc-PAH and S...

Objectives: Preeclampsia is a state of antiangiogenesis, with high levels of maternal circulating sVEGFR-1 (soluble vascular endothelial growth factor receptor 1, also named sFlt1) and low levels of PlGF (placenta growth factor). Various autoantibodies have been detected in preeclamptic patients. We hypothesize that circulating autoantibodies against VEGF-A (AA-VEGF-A), VEGFR-1 (AA-VEGFR-1) and PlGF (AA-PlGF) are present in preeclamptic women, with different levels from pregnant women with normotensive pregnancies. Secondly, we wanted to analyze if autoantibody levels are associated to sFlt1 or PLGF levels. Study design: Retrospective cross sectional study of 88 women with singleton pregnancies who delivered at Oslo University Hospital of whom 46 had preeclampsia and 42 had uncomplicated normotensive pregnancies. Novel immunoassays for IgG-autoantibodies against VEGFA, VEGFR-1 and PlGF were developed and serum samples were assayed. Main outcome measures and results: AA-VEGF-A, AA-VEGF-R1 and AA-PlGF were significantly lower in preeclamptic pregnancies (n = 42) compared to normotensive pregnancies (n = 46) (p < 0.05). On unadjusted analysis, only AA-VEGFA and AA-VEGFR-1 were predictors of PE, but none were independent predictors after adjusting for BMI (body mass index) and parity. In the subgroup of normotensive and PE women with overlapping sVEGFR-1/PlGF-ratios, AA-VEGF was a significant predictor of PE with AUC: 0.735. Conclusion: IgG autoantibodies against VEGF-A VEGFR-1 and PlGF can be found in pregnant women. They are dysregulated in preeclampsia. The roles of these autoantibodies are unknown, but this study suggests they play a protective role in pregnancy. The levels of AA against VEGF-A, VEGFR-1 and PlGF might be important factors contributing to anti-angiogenesis regulation.

The cold shock Y-box binding protein-1 (YB-1) fulfills important roles in regulating cell proliferation and differentiation. Overexpression occurs in various tumor cells. Given the existence of extracellular YB-1 we set out to determine the diagnostic, predictive and prognostic role of serum YB-1/p18 for patients with primary epithelial ovarian cancer (EOC).The protein fragment YB-1/p18 was quantified by sandwich ELISA in serum samples from 132 healthy female volunteers and 206 patients with histological diagnosis of primary EOC. The ELISA sensitivity and specificity to detect EOC were calculated using receiver operating curves. Survival data were calculated using Kaplan Maier curves.Median age at the time of diagnosis was 60years and follow-up ended with a mean of 44.8month. 188 (91%) patients were diagnosed at advanced stages (FIGO III/IV) and 188 patients (91%) suffered from high-grade serous ovarian carcinoma. YB-1/p18 levels were significantly decreased in older patients (p=0.021). Significantly lower serum levels of YB-1/p18 were detected in the EOC cohort when compared to the control group (p0.0001, AUC=0.827; 95% CI, 0.787-0.867). Using the expression of serum YB-1/p18 in early stages I and II cases these could be differentiated from control cases (p0.0001, AUC=0.816; 95% CI 0.704-0.929). No other significant associations between clinical prognostic factors and YB-1/p18 serum levels were detected. Immunoblotting results with serum samples suggest that masking of epitopes by the YB-1/p18 fragment in multiprotein-complexes under non reducing conditions leads to the observed reduced ELISA readings in the EOC cohort.The quantification of fragment YB-1/p18 derived from cold shock protein YB-1 in serum samples could be useful for the early diagnosis of EOC.