GPCRs are involved in a variety of physiological and pathophysiological processes. So far, much effort has been made on anti-GPCR drug discovery with a focus on the development of small molecules and monoclonal antibodies for the treatment of cancer, infection, metabolic disorders or inflammatory diseases. Recently, functional autoantibodies directed against GPCRs were identified in various diseases associated with pathogenesis, uncovering a potential new field of therapeutic intervention. Thus, the aim of this journal is to combine the current knowledge on the role of GPCRs in different pathologies, their mode of action and state-of-the-art research techniques to identify common fundamental pathways that can be transferred to other disease entities with similar manifestations.
Organotropism of anti-CXCR3/4- and anti-ETAR/-AT1R-mediated leukocyte migration in inflammatory and autoimmune disease
ligands orchestrate a myriad of cellular events, among them cell trafficking and migration. Further, altered expression of GPCR and their ligands has been implicated in different autoimmune and inflammatory diseases. Apart from natural ligands, it has recently been shown that autoantibodies targeting GPCR might play a role in inflammatory and autoimmune diseases such as systemic sclerosis (SSc). Based upon previous findings it is assumed that high concentrations of anti-CXCR3 and anti-CXCR4 antibodies act in a protective manner, while high concentrations of anti-ETAR and anti-AT1R aggravate inflammatory mechanisms. We want to show those effect of antibodies against CXCR3, CXCR4, ETAR and AT1R in a murine model of atherosclerosis. Methods: We injected IgG preparations containing different levels of autoantibodies directed against CXCR3/4 or ETAR and AT1R from patients with SSc and from healthy controls into a passive murine model of atherosclerosis (C57BL/6, Apoe-/-, high fat diet). Using red oil staining of the atherosclerotic plaques in the aorta, flow cytometry, differential blood count via Vet abc plus, ELISA and immunohistology analyses, it will be examined if those IgG preparations are able to modify leukocyte migration into different tissues like lung and spleen in comparison to peripheral blood. Results: Preliminary findings of the ELISA indicates migration of leukocytes into the spleen modulated by the anti-GPCR IgG. More preliminary findings, like some results of the red oil staining and the flow cytometry analyses will be shown. Conclusion: We assume an interplay between anti-AT1R/anti-ETAR & anti-CXCR3/4 and their receptors mediates organotropism of leukocytes in inflammatory & autoimmune diseases. In addition, membrane extracts of CHO cells overexpressing CXCR3/4, ETAR and AT1R will be applied in the same murine model to compare passive and active immunization schemes.
Copyright (c) 2018 Laurence Johanson, Antje Müller, Zouhair Aherrahrou, Jennifer Hundt, Anja Kerstein, Gabriele Marschner, Silke Pitann, Sara Comdühr, Gesche Weppner, Harald Heidecke, Peter Lamprecht, Christiane Herden, Gabriela Riekemasten https://creativecommons.org/licenses/by/4.0
Patterns of 31 new autoantibodies against G-protein-coupled receptors and growth factors
Background: Systemic sclerosis (SSc) is a rare autoimmune disease with a significant disease burden. Scl70 and centromere ab are disease specific and prognostic antibodies. Functional ab against G protein-coupled receptors (GPCR) regulate immune function and were reported in the pathogenesis of various inflammatory diseases. Objectives: We analyzed 31 ab against GPCRs in a retrospective cohort of 71 SSc sera compared to 196 sera from healthy controls (HC). Ab levels were related to disease manifestations in order to hypothesize functional ab in SSc. Methods: SSc patients were retrospectively characterized by mRSS, organ involvement, lab tests, spirometry and imaging. 30/71 had active disease (EUSTAR activity score). Ab patterns were analyzed using new statistical approaches: factor analysis, principal component analysis (PCA), linear discriminant analysis (LDA), cluster analysis and biserial correlation. Results: SSc subgroups (diffuse/ limited cutaneous) differ in ab levels forming separate clusters (LDA method). Moreover, latent factors group ab and clinical disease manifestations. Factor analysis reveals VEGFR2 ab and the signaling molecule YBX1 ab to be more unique with the lowest communalities. The biserial correlation shows moderate associations between ab patterns and SSc symptoms. Compared to association of ETAR ab with Raynaud’s and skin sclerosis HGFR ab are inversely correlated. Conclusions: We describe 31 new ab against GPCR and growth factors in SSc. Ab levels can be clustered by latent factors. 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.
Copyright (c) 2018 A. Carvalho-Marques, H. Bittern, C. Fouodo, I. König, S. Schinke, O. Cabral-Marques, H. Heidecke, G. Riemekasten https://creativecommons.org/licenses/by/4.0
Anti-AT1R ab and as cause or contributor to skin fibrosis and interstitial lung disease
Introduction: Systemic sclerosis (SSc) is a complex connective tissue disease which is characterized by autoimmunity, vasculopathy and fibrosis. Recent studies have suggested that autoantibodies (ab) against angiotensin II receptor I (AT1R) might play a pathogenic role in the development of SSc, which need to be further verified by animal models in vivo. However, due to the difficulties in preparation of antigens with native conformational epitopes, induction of functional aab against surface-expressed receptors is a challenging task. Methods: We Immunized mice with membrane extracts derived from CHO cells overexpressing human AT1R (hAT1R) and evaluated the immunological and histological phenotypes in the immunized mice. Results: The hAT1R-immunized mice developed functional autoantibodies against AT1R which are able to bind to and to activate the native receptor. Furthermore, hAT1R-immunization induced SSc-like disease symptoms including vasculopathy and interstitial inflammation in the lung as well as perivascular inflammation and fibrosis in the skin. Importantly, the SSc-like disease can be partially transferred by the serum IgG of hAT1R-immunized mice. Conclusion: Our study provides a new animal model for SSc, which strongly supports the hypothesis of a pathogenic role of functional antibodies against AT1R in this disease.
Copyright (c) 2018 Xinhua Yu https://creativecommons.org/licenses/by/4.0
Network-based analysis reveals signatures of IgG autoantibodies targeting G protein-coupled receptors in health and disease
Background: Functional antibodies (ab) against G protein-coupled receptors (GPCR) were found in various diseases and partially in healthy individuals. Their role in physiology and in pathophysiology remains to be identified. Methods: Sera from 952 patients with different rheumatic diseases were analyzed for the presence of ten different anti-GPCR ab by ELISAs. For deeper ab phenotyping, ab levels against 30 different autoantigens including GPCR, growth factors, and growth factor receptors were studied in sera from 491 healthy controls (HC), 84 patients with systemic sclerosis, 91 with Alzheimer's disease, and 207 with ovarian cancer by ELISAs. Spearman's rank correlation coefficients, hierarchical cluster analyses, network mapping and target interactions using STRING and gene ontology (GO) analyses were performed as well as transwell migration assays. Results: Specific signatures were identified in different autoimmune diseases and in HC. Ab correlations and clusters of correlations were identified in HC, which were specifically affected by age, sex, and autoimmune and non-autoimmune diseases. Network mapping and GO analysis of ab targets displayed multiple associations and a central role of the endothelin receptor type-A (ETAR) in cell migration and chemoattraction. Indeed, the ab were chemoattractive for immune cells, which could be ameliorated by ETAR blockers. Conclusions: Our data indicate a functional ab network, which is affected by age, sex, and diseases. The network could affect receptor functions and could guide immune cells or their targets via chemoattraction to organs.
Copyright (c) 2018 Otavio Cabral-Marques, Alexandre Marques, Lasse Melvær Giilb, Roberta de Vito, Barbara Elizabeth Engelhardt, Jalid Sehouli, Harald Heidecke, Gabriela Riemekasten https://creativecommons.org/licenses/by/4.0
mon and ranges between 20% – 70% of the patients. The CNS involvement is listed in the ARA criteria for SLE diagnosis. CNS-SLE is associated with more than 20 different autoantibodies (1). Yet, remarkable among them are the anti-P-ribosomal antibodies (anti-PR) (2). These autoantibodies directed mainly against the carboxy 22 amino acids of the PO, P1 P2 ribosomal phosphoproteins. They are capable of penetrating lived cells and inducing apoptotic changes as well as leading to inhibi-tion of specific cytokine secretion (3-5). The titer of the autoantibodies correlates with disease activity, kidney involvement and hepatitis (6-8). Anti-PR were first reported to associate and correlate with CNS-SLE and especially psychosis in 1987(9). Subsequent analyses could only partially repeat Bonfa's conclusions (2-3). The con-tradictory results may stem from technical discrepancies (2-3), multiplicity of autoantibodies causing CNS-SLE (1,10), and differences in the time of drawing the blood in relation to the time when the patient had the CNS involvement. We employed affinity purified anti-PR on a PR column and injected them directly into the brain ventricles of Balb/c mice. Irrelevant Igs were employed as controls. The analyses entailed a variety of neurological tests known to evaluate cognitive impairment, anxiety states, depres-sive conditions and motor competence of the mice. Mice injected with anti-PR clearly expressed depressive behavior, characterized mainly by floating pattern rather than swimming in an enforced swimming tests. These floating pattern could be reversed by a specific therapy with monoclonal anti-idiotypic antibody to anti-PR (11), by IVIG as well as Fluoxetine (Prozac) an anti-depression drug. No response was achieved with anti-psychotic drug. The control mice were not affected in their behavior following the injection of the irrelevant Ig (12). The human purified anti-P-R, were shown to bind to CNS structures consisting with the smell apparatus (i.e. amygdale, hypocampus and other parts of the limbic systems). Smell defects characterize bulbectomized (cutting the olfactory nerves) depressive mice (13), as well as depressive women (14). Such depression can be overcome by exposing mice and humans to citrus fragrance (15). Our studies show for the first time the active induction of a psychiatric condition (i.e. depres-sion) with a specific autoantibody, i.e. anti-PR (16). Furthermore, the results allude to a novel mechanism to explain the induction of CNS-SLE depression (i.e. involvement of the smell ap-paratus). These data may pave the road to a novel approach to depression in CNS-SLE and its therapy as well as in other CNS conditions (12,17,18-21).
Copyright (c) 2018 Yehuda Shoenfeld https://creativecommons.org/licenses/by/4.0
Lessons From Anti-TSH-Receptor Autoantibodies: Challenges And Pitfalls In Diagnostics
Objective: Autoantibodies to the thyrotropin hormone receptor (TSH-R) directly mediate Graves’ disease (GD) and orbital manifestations of Graves’ orbitopathy (GO). To explore their heterogeneous function, TSH-R autoantibody binding in immunoassays (TRAb) versus their functional activity; stimulators of cAMP in bioassay (TSIs or TSAbs) or inhibitors of the thyrotropic hormone (TSH) binding to TSH-R, detected by decreased cAMP in bioassay (TBAbs), is assessed. Methods: TRAb measurements by automated electrochemical luminescence immunoassays (ECLIA) and stimulating and blocking activity on Chinese hamster ovary cell (CHO) Mc4 bioassays (Thyretain ®, Quidel). The activity of patient immunoglobulins on ECLIA; M22 Mab competition (cobas elecsys, Roche) or TSH-R bridge assay (Immulite,Siemens) versus Mc4 TSI bioassay were correlated with GO clinical activity by meta-analysis of complete Pubmed-published data sets (n=5). Results: The automated ECLIA show less sensitivity than cell-based bioassay for detection of TRAbs (Fig 1A and 1B) and do not distinguish between stimulator MAbs; M22 and KSAb (Fig 1A and 1B) versus the blocker MAb K1-70 (Fig 1C). In contrast, Mc4 TSAb bioassay of K1-70 detected null stimulatory activity (Fig 1C), in contrast to strong cAMP inhibition in Mc4 TBAb blocker reporter bioassay (Fig 1D). Diagnostic performances of Mc4 TSAb bioassay and automated TRAb ECLIA in untreated GD show high sensitivities 97% (95-100) and specificities 98% (96-100%). Meta-analysis of GD and/or GO undergoing treatment reveal lower percent positive on Mc4 TSI Bioassay (56%, range 13-87%) than ECLIA TRAb immunoassays (98.5%, range 98-99%; OR=2.76, p=0.09). The Forest plot total random effects of correlation coefficients of autoantibody activity with GO clinical activity were greater on Mc4 bioassay (r=0.4, I2 =92% (95% CI 89-95), p<0.0.001) versus TRAb immunoassay (r=0.2, I2 =39% (95% CI 0-70%), p=0.0.8). Conclusion: Diagnostic distinction of TSAbs and TBAbs is of paramount clinical importance. Cell-based bioassays are the only method to distinguish between TSAbs and TBAbs. To avoid the pitfall of incorrect reporting on automated TRAb binding assays, TSI or TSAb is strongly recommended for results of bioassays; whereas the results of automated ECLIA should be reported as TRAbs (of undetermined functional significance).
Copyright (c) 2018 Simon D. Lytton https://creativecommons.org/licenses/by/4.0
Novel targets in the diagnosis of autoimmune and non-autoimmune diseases
Antibodies against GPCR are present in autoimmune and non-autoimmune diseases. Furthermore, CellTrend described for the first time antibodies against growth factors (GF, e.g. VEGF-A) and their related receptors (GFR, e.g. EGF-R). Elevated as well as decreased antibodies against GPCRs, GFs and GFRs are present in many diseases. There are a growing number of antibodies against different GPCR, GF and GFRs. The following ELISAs for the quantitative determination of AT1R-Ab, ETAR-Ab, ETBR-Ab, PAR1-Ab, PAR2-Ab, M1R-Ab, M2R-Ab, M3R-Ab, M4R-Ab, M5R-Ab, Complement-Receptor-5a-Ab, CXCR3-Ab, CXCR4-Ab, ?1-adr-R-Ab, ?2-adr-R-Ab, ?1-adr-R-Ab, ?2-adr-R-Ab, VEGF-A-Ab, VEGF-B-Ab, VEGFR1-Ab, EGF-Ab, EGFR-Ab and PlGF-Ab are available as kits. Some of these assays are registered as in vitro Diagnostica (IvD). In addition, antibodies against Serotonin-R, Dopamin-R and FGF are available as a CellTrend lab service. Current findings indicate the important role of anti-GPCR, anti-GF, anti-GFR antibody patterns as markers of diseases. Therefore, further studies determining the whole spectrum of antibodies targeting GPCRs, GFs and GFRs in healthy subjects and in different diseases are necessary. New mathematic models for the analysis of the antibody patterns are required, too.
Copyright (c) 2018 Harald Heidecke https://creativecommons.org/licenses/by/4.0
Signaling and GPCR biology using cell models
A wide range of untreatable or difficult to treat disease entities are caused by activating immunoglobulins G (IgG) directed against G-protein coupled receptors (GPCR). Hence, molecular architecture of the binding of these antibodies to the receptors and the functional consequences of changes in specific structural modules are of immense clinical relevance. Our laboratory has focused over the years on the Angiotensin II type 1 receptor (AT1R) and Endothelin-1 type A receptor (ETAR), which signal upon natural ligand Angiotensin II (Ang II) or Endothelin-1 (ET-1)-mediated and AT1R- or ETAR-agonistic IgG (AT1R-/ETAR-IgG)-mediated stimulation. Development of high-resolution methods allows for structural-functional relationship studies of specific receptor modules, including the extracellular domains, in the receptor activation. To distinguish natural peptide and IgG-mediated activation, we first developed a yeast model where human GPCR activation controls yeasts growth. These yeasts have been modified to express one single human GPCR that couples to a chimera between human and yeast G-protein. Each yeast strain is specific of a different G-protein. Point mutations can be introduced by site-directed mutagenesis in order to determine the influence of amino acids sequences on the receptor activation. To investigate further the link between conformation and activation of the receptor in a more complicated environment, we performed luciferase reporter assays in Human Microvascular Endothelial Cells (HMEC-1) after transfecting the cells with wild type or mutated receptors and the appropriate reporter plasmids. Comparison between wild-type and mutants, non-stimulated and stimulated cells allowed us to better characterize the influence of the conformation changes on activation of the receptors. We successfully created models allowing for structural and functional studies of molecular architecture modules appreciating GPCR receptor plasticity, which helped us to define the role of specific extracellular domains. Better understanding of the molecular mechanisms responsible for GPCR activation holds great potential for design of more specific drugs in autoantibody-mediated diseases.
Copyright (c) 2018 Philippe Aurélie https://creativecommons.org/licenses/by/4.0
Role of antibodies against PAR-1 and PAR-2 in cancer in the kidney transplant recipients
Background: Activated angiogenesis and impaired host immune response contribute to cancer in renal transplant recipients. Induction of VEGF is crucial for neoangiogenesis in tumors. Functional autoantibodies targeting GPCRs are able to induce endothelial dysfunction. We hypothesized that autoimmune GPCR targeting process may disturb VEGF induced angiogenesis. We identified in an in vitro model PAR-1 as novel activating autoantibody target and assessed the presence of this naturally occurring blocking antibody in 20 Kidney Transplant Recipients (KTR) with and 29 KTR without metastatic cancer. In further in vitro studies, we determined the influence of IgG derived from KTR on PAR-2-dependent G-protein activation Methods/Materials: Human endothelial cells were stimulated with IgG isolated from sera of kidney transplant recipients (KTR-IgG). Transcriptional regulation of VEGF was studied by promoter deletion assay. Transcription factor activation and binding was assessed by qRT-PCR, western blot, EMSA and cFOS knockdown. VEGF secretion was determined by ELISA. Tube formation on matrigel served to study endothelial neoangiogenic response. G-protein activation was measured by reporter assay. All 49 patients enrolled had sera for assessment of PAR antibodies (PAR-Ab) via ELISA in 2016 and at the time of transplantation. Results: Treatment with KTR-IgG reduced ERK1/2-dependent VEGF secretion and tube formation. VEGF secretion and endothelial tube formation could be restored by pretreatment with specific PAR-1 inhibitor. KTR-IgG contributed to deregulated neoangiogenesis via reduced VEGF promoter activity and increased cFos protein expression via its binding to the VEGF promoter. Mutation of extracellular loops of PAR2 receptor increased PAR2-Ab dependent Gq11 Signalling. Levels of PAR1-Ab were statistically significantly lower pre transplant and at diagnosis in those KTR who subsequently developed cancer compared to controls without cancer. Conclusion: We identified the PAR-1 receptor as a new target for functional antibodies in the context of kidney transplantation and tumor angiogenesis. PAR-1 regulated angiogenesis could offer new possibilities for treatment of kidney transplants to obviate tumor angiogenesis.
Copyright (c) 2018 R. Catar, R. Carroll, L. Chen, Q. Li, I. Schramm, M. Simon, O. Wischnewski, A. Kusch, A. Philippe, D. Dragun https://creativecommons.org/licenses/by/4.0
Impact of regulatory (auto-)antibodies against G-protein-coupled receptors on blood pressure in women ten years after early-onset preeclampsia
Introduction: Preeclampsia (PE) is associated with an increased cardiovascular risk in later life, especially in women after early-onset PE. We and others have shown that activating antibodies against angiotensin II receptor type 1 (AT1-) are present in women with PE, even after pregnancy. Further, regulatory (auto-) antibodies against G-coupled receptors (GPCR) have been shown to contribute to cardiovascular disease. Hypothesis: Regulatory autoantibodies’ titers are elevated in women with a history of early onset PE and correlate to physiological parameters and clinical outcomes. Methods: Autoantibodies against AT1-, ?1- adrenergic receptors, endothelin I receptor A (ETAR), protease-activated receptor 1 (PAR1) and C-X-C3 chemokine binding receptors (CXCR3) were determined by using commercially available ELISA (Celltrend, Luckenwalde, Germany) in women 10 years after after PE pregnancy or control pregnancy. We investigated samples from the PREVFEM cohort (PReeclampsia Risk EValuation in FEMales), a retrospective matched case-control study, which was performed in Zwolle, The Netherlands, from 2008 until 2010. All women registered in the early preeclampsia database as well as an equal number of age-matched females without preeclampsia from the regular obstetric database in the same time period (1991-2007) were invited to participate in the PREVFEM study. Results: Women with history of early PE had higher prevalence of hypertension than controls (42.7% vs 8.3%, p < 0.001). Women with the lowest combined levels of autoantibodies and a history of early PE had significantly higher SBP (mean difference = 11.54 mmHg, 95% CI [4.39; 18.70], p < 0.001), DBP (mean difference = 7.75 mmHg, 95% CI [2.42; 13.08], p < 0.001), and MAP (mean difference = 9.02 mmHg, 95% CI [3.24; 14.79], p < 0.001) compared to the controls. The titer levels of single regulatory AT1-, ?1-, ETAR-, PAR1- and CXCR3-autoantibodies were not different between control and former PE women 10 years after index pregnancy. Conclusions: Hypertension is a common problem after preeclamptic pregnancy in middle-aged women. Regulatory autoantibodies are not sufficient alone to cause hypertension or other pathologic conditions, but together with other risk factors such as pathological pregnancy might drive the whole balance towards pathophysiologic conditions. Further studies are needed to investigate the potential of regulatory autoantibodies against GPCR as presymptomatic biomarkers identifying women at particular cardiovascular risk.
Copyright (c) 2018 Anna Birukov, Hella Muijsers, Harald Heidecke, Nadine Haase, Kristin Kräker, Dominik N. Müller, Florian Herse, Angela Maas, Ralf Dechend https://creativecommons.org/licenses/by/4.0
Sialylated autoantigen-reactive IgG antibodies attenuate disease development in mouse models of lupus nephritis and rheumatoid arthritis
Pro- and anti-inflammatory effector functions of IgG antibodies (Abs) depend on their subclass and Fc glycosylation pattern. Accumulation of non-galactosylated (agalactosylated; G0) IgG Abs in the serum of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients reflects severity of the diseases. In contrast, sialylated IgG Abs are responsible for anti-inflammatory effects of the intravenous immunoglobulin (IVIG; pooled human serum IgG from healthy donors), administered in high doses (2 g/kg) to treat autoimmune patients. However, whether low amounts of sialylated autoantigen-reactive IgG Abs can also inhibit autoimmune diseases has been hardly investigated. Here, we explored whether sialylated autoantigen-reactive IgG Abs can inhibit autoimmune pathology in two mouse models (Bartsch et al., Front. Immunol. 2018; 9:1183). We found that sialylated IgG autoantibodies fail to induce inflammation and lupus nephritis in a B cell receptor (BCR) transgenic lupus model, but instead are associated with lower frequencies of pathogenic Th1, Th17 and B cell responses. In accordance, the transfer of small amounts of immune complexes containing sialylated IgG Abs was sufficient to attenuate the development of nephritis. We further showed that administration of sialylated collagen type II (Col II)-specific IgG Abs attenuated the disease symptoms in a model of Col II-induced arthritis and reduced pathogenic Th17 cell and autoantigen-specific IgG Ab responses. We conclude that sialylated autoantigen-specific IgG Abs may represent a promising tool for treating pathogenic T and B cell immune responses in autoimmune diseases.
Copyright (c) 2018 Johann Rahmöller, Yannic C. Bartsch, Alexei Leliavski, Gina-Maria Lilienthal, Janina Petry, Marc Ehlers https://creativecommons.org/licenses/by/4.0
Autoantibodies targeting complement receptors 3a and 5a are decreased in ANCA-associated vasculitis
Introduction: Necrotizing crescentic glomerulonephritis (NCGN) is a pathogenic hallmark of full-blown ANCA-associated vasculitis (AAV). In AAV, NCGN is characterized by scantly deposited immune complexes and involvement of the complement system. The relevance of the latter in AAV is reflected by elevated amounts of circulating C5a and, foremost, by protection from NCGN through blockade of C5aR1. In contrast, little if any is known about autoantibodies recognizing complement receptors 3a and 5a in AAV patients. Methods: To examine the presence of autoantibodies directed against complement receptor C3a (C3aR) and C5a (C5aR1) in AAV, sera of patients with AAV [granulomatosis with polyangiitis (GPA), n=49 and microscopic polyangiitis (MPA), n=10] were measured by Elisa in comparison to healthy controls (HC, n=127). In addition, amounts of circulating C3a and C5a were determined by Elisa. Clinical parameters (BVAS, VDI, therapy) and disease marker (ESR, CRP, creatinine, anti-PR3, anti-MPO) were gathered at the time of serum sampling. Results: Unlike MPA, in GPA lower titers of anti-C3aR were observed in comparison to HC. In addition, the concentration of anti-C5aR1 was decreased in both, GPA and MPA when compared to HC. In particular, GPA patients without a history of therapy using cyclophosphamide and/or rituximab exhibited a negative correlation between the concentration of anti-C5aR1 and the disease activity (BVAS). Conclusion: The results may give a hint that anti-C3aR and anti-C5aR1 antibodies play distinct roles in GPA and MPA. Further, anti-C5aR1 might be of diagnostic value to monitor the clinical progress of GPA.
Copyright (c) 2018 Sebastian Klapa, Andreas Koch, Anja Kerstein, Christian Karsten, Harald Heidecke, Markus Huber-Lang, Peter Lamprecht, Gabriela Riemekasten, Antje Müller https://creativecommons.org/licenses/by/4.0
The pathomechanisms leading to Alzheimer´s Disease (AD) are not well understood. Despite several robust hypotheses the nature of the disease seems to be more complex than initially thought. The role of vascular components of AD have been underestimated so far. In this novel treatment approach we perform immunoadsorption over 5 days to remove agonistic autoantibodies (AAB) against ?1A-adrenoceptor from the plasma of AD patients. These AAB are thought to alter the functional properties of small blood vessels leading to malfunctions in the supply and clearance processes of the brain. We will present the first 5 patients with AD who underwent immunoabsorption and who completed a 12-months follow up. Clinical, cognitive and MRI data as well as peripheral endothelial function data and data on AAB clearance will be presented. The study is funded by Fresenius Medical Care.
Copyright (c) 2018 Hans Jörgen Grabe https://creativecommons.org/licenses/by/4.0
First results of the prospective ETiCS-study: acute (microbial-associated) inflammation rather than (infarction-associated) myocardial ischemia triggers the development of cardiac GPCR autoantibodies
countries. In the past decade evidence for a clinical relevance of GPCR-autoimmunity in human HF has substantially increased. Stimulating autoantibodies targeting the second extracellular loop (ECII) of the cardiac beta1-adrenoceptor (beta1-aabs) have been claimed to be involved in the pathogenesis of HF and to increase the risk of cardio-vascular death by three-fold. Still, the events triggering the formation of beta1-aabs and their impact on HF-progression are unknown. Methods: Based on the rationale that presentation of beta1-adrenoceptors and other cardiac membrane antigens following inflammation or necrosis may induce the formation of beta1-aabs, in total 13 University Hospitals (12 in Germany, 1 in Serbia) prospectively recruited 226 patients with a first acute myocardial infarction (FAMI), and 140 pts with acute (biopsy- or cMRI-proven) myocarditis (AMitis) into the Etiology, Titer-Course and effect on Survival of cardiac autoantibodies-study (ETiCS-study). At baseline (BL) and three follow-up visits (Fup1-3) blood was sampled to follow the time-course of various GPCR-aabs. Beta1-aabs were assessed by using Dynabeads® M-270-Epoxy coated with increasing amounts of beta1-ECII-peptides (2.5-100 ?g/ml). As control, beads were coated with a peptide comprising same amino-acids, but in a scrambled order. After reacting with the beads (15min on ice) the samples were measured on a FACScan flow-cytometer. Data were analyzed using FlowJo (Treestar); when half-maximal binding was calculable the serum was classified beta1-aab-positive. Results: From n=366 pts (226 FAMI/140 AMitis) recruited into the ETiCS-study 45 pts had to be excluded because of unperformed cMRI’s; 46 pts stopped the study before Fup-1 (month 3). Only 180/226 FAMI- and 98/140 AMitis-pts had complete Fup1-3 (after 3, 6, and 12 months, including clinical evaluation, echocardiograms, and cMRI’s at BL & Fup-3). All valid ETiCS-pts (197 FAMI-/123 AMitis-pts) were assessed for the formation of beta1-aabs, and then compared focussed on the development of echo-LVEF. In the course of the study relevant (high-affinity) beta1-aab-titres were detected in ~31% (37/123) of the AMitis-pts compared to only ~21% (42/197) of the FAMI-pts. In aab-positive AMitis-pts echo-LVEF did not recover and was always significantly inferior to aab-negative AMitis-pts (BL: 38 vs. 49% LVEF; Fup-3: 49 vs. 64% LVEF) whereas such a difference was not noted in FAMI-pts. In addition, aab-positive AMitis-pts had higher NT pro-BNP-, renin-, and aldosterone-levels than aab-negative AMitispts. Conclusion: These first results from the ETiCS-study suggest that acute microbial-induced rather than post-infarction myocardial inflammation triggers the formation of clinically relevant beta1-aabs. AAb-positive AMitis-patients might profit from early intensification of standard HF-therapy (including early beta-blockade) and/or novel antibody-directed experimental therapies which are currently under development.
Copyright (c) 2018 Valérie Boivin-Jahns, Chistina Zechmeister, Claudia Schuetz, Niklas Beyersdorf, Stefan Stoerk, Roland Jahns https://creativecommons.org/licenses/by/4.0
The aptamer BC 007 completely neutralizes agonistic autoantibodies directed against ?1-adrenoceptors
Background: 80 percent of patients with non-ischemic heart failure (HF) and reduced ejection fraction have autoantibodies against ?1- adrenoceptors (?1-AAB), which cause and/or sustain HF. The removal of ?1-AAB by immunoadsorption (IA) leads to an improvement of cardiac function and significantly reduced mortality. With the aptamer BC 007, a new drug is under study that neutralizes autoantibodies against G-Protein-coupled receptors (GP-AAB) including ?1-AAB. Aptamers are often referred to as "chemical antibodies" and offer a number of advantages over e.g. peptides and biologics when used for treatment. Based on in vitro studies followed by animal testing, BC 007 was recently step-by-step transferred to humans. We present here a Phase I study investigated the safety, tolerability, and pharmacological effect of BC 007. Methods: In a randomized, double blind, placebo controlled single ascending dose study, 3 cohorts of 8 male healthy subjects each (age 18 - 45 yrs) were dosed with 15, 50 and 150 mg of BC 007 by i. v. infusion over 20 min. Additional 8 elderly healthy subjects of both sexes (55 - 70 yrs) received 150 mg BC007 or placebo. Subsequently, in an open labelled study part with 5 cohorts of 6 elderly volunteers each (55 - 75 yrs), who showed evidence for GP-AAB dosages of 50, 150, 300, 450, and 750 mg were tested. Infusion duration was 20 min in the 50 and 150 mg and 40 min in the 300, 450 and 750 mg cohort. Subjects safety was carefully monitored during the study (ECG, blood pressure, lab, injection site reactions, VS, physical examination, adverse events). 12 blood samples were taken from before the infusion began until 24 h thereafter for clinical chemistry, hematological and pharmacokinetic analysis. Urine was collected. Results: BC 007 was extremely well tolerated and without clinically relevant adverse events. GP-AAB neutralization has been seen 24 h after BC 007 infusion in 1 of 6 individuals in the 150 mg cohort, in 2/6 in the 300 and 400 mg and 5/6 in the 750 mg cohort. A slightly elevated aPTT (max 49 s) was observed in 5 subjects of the 300, 450, and 750 mg cohort which quickly normalized after end of infusion. AUC increased by rising dosage from 21 to 556 ?g*min/mL, Cmax increased from 1610 to 15832 ng/mL. The half-life was short at appr 4 min. Conclusion: BC 007 is a powerful new drug for the neutralization of ?1-AAB with a favorable side-effect profile which will be tested as the first causative acting drug for patients with ?1-AAB induced heart failure. Derived from IA data, a huge number of patients with HF will profit from this new drug.
Copyright (c) 2018 Niels-Peter Becker, Johannes Mueller, Annekathrin Haberland, Katrin Wenzel, Gerd Wallukat, Peter Goettel, Sarah Schulze- Rothe, Ingolf Schimke, Georg Golor, Matthias Grossmann, Angela Sinn, Martina Steiper, Tuba Yilmaz, Anne Wallukat, Hanna Davideit, Susanne Becker https://creativecommons.org/licenses/by/4.0
Agonistic antibody diseases = Autoimmune diseases?
In recent years, the number of diseases in which autoantibodies (AAB) against G Protein-coupled receptors (GPCR) have been detected has continued to increase. Because they act in a bioassay similar to the physiological ligands of GPCR, the term "agonistic autoantibodies" (agAAB) was established early on. The proportion of patients with a clinically diagnosed disease in whom agAAB has been detected in the blood can be very different. Currently agAAB can be determined against 8 different GPCRs. The probably most important ones are directed against the adrenoceptors (AR) ?, ß1, ß2 and the angiotensin2 receptor type 1 (AT1-R). The AAB binding epitopes are located on the extracellular 1st or 2nd loop and are consistently constant. For a few diseases such as dilated cardiomyopathy (DCM, ß1-AR, ?-AR), pre-eclampsia (AT1-R) and vascular necrotic kidney transplant rejection (AT1-R), antibodies could be induced in animal experiments by immunisation with the extracellular loop peptides and subsequently produce a disease pattern similar to that of humans. In the development of vascular M. Alzheimer's disease (VAD), animal experimental findings according to agAAK against ?-AR could also play a role in cerebral vascular obliteration. For most of the diseases associated with agAAB, the pathological significance of the proven agAAB has not yet been clarified. They may be an epiphenomenon, but can also be pathologically amplifying bystander or even cause the disease. The above agAABs often occur in typical combinations, and we do not yet know anything about their possible interference or parallel involvement in the respective disease. The distribution patterns of agAAK in diseases such as DCM, VAD, diabetes type 2 (DM2), primary open-angle glaucoma (POAG), thromboangiitis obliterans (TAO) or chronic fatigue syndrome (CFS) observed so far suggest that there are pathophysiological similarities between the very different diseases in the clinical symptoms. This is particularly noticeable in diseases with vascular contribution. Up to now, agAAB's participation in a disease beyond the classics such as DCM has not been taken into account therapeutically. The abundance of data on the prevalence of agAAB in a wide variety of diseases makes it almost necessary to search for new terms to take these conditions into account. One possible approach would be to label patients with different diagnoses and positive antibody status with the additional diagnosis"agonistic antibody disease". In specific individual cases, new therapeutic options may then become apparent.
Copyright (c) 2018 Rudolf Kunze https://creativecommons.org/licenses/by/4.0
Autoantibodies in serum of systemic scleroderma patients: peptide-based epitope mapping indicates increased binding to cytoplasmic domains of CXCR3
Introduction: Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Methods: We used peptide ELISA with serum samples in combination with a hierarchical Bayesian model for mapping of epitopes on CXCR3. For in silico prediction of epitopes on CXCR3, we used the ABCpred online tool. Results: To identify linear epitopes of anti-CXCR3 aabs, we used a peptide array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence for ELISA to compare the binding pattern of SSc patient sera (N=32) and healthy controls (N=30). We found increased binding of SSc patient sera to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced in Ssc patients. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N=31) from normal healthy controls (N=47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85.
Copyright (c) 2018 Andreas Recke, Ann-Katrin Regensburger, Florian Weigold, Antje Müller, Gabriele Marschner, Harald Heidecke, Gabriele Marschner, Christoph M Hammers, Ralf J. Ludwig, Gabriela Riemekasten https://creativecommons.org/licenses/by/4.0
Detection and functional characterization of angiotensin receptor Type 1 autoantibodies: establishment and clinical translation
Introduction: Circulating AT1R autoantibodies (AT1R-aabs) directed against the ECL2 of the AT1R with agonist-like activity are supposed to play a pathophysiological role in diseases associated with vascular and renal damage, such as preeclampsia and malignant hypertension (HT), but they are also thought to be involved in heart failure and primary hyperaldosteronism (PHA). Methods: High-throughput screening assays aiming at a reliable detection of AT1R-aabs in sera from patients with HT and PHA were established. The agonist-like activity of AT1R-aabs was assessed by changes in intracellular calcium levels using Fura2-QBT dye and AlphaLISA SureFire Ultra Assay was used to assess induction of ERK1/2 phosphorylation in stably transfected AT1R-HEK-cells. Results: IgG purified from sera of n=60 patients with PHA and n=97 with hypertensive heart disease (HT) were screened for their capacity to increase [Ca2+]i or to activate ERK1/2. Ten out of 60 PHA patients increased [Ca2+]i compared to none of the HT-patients, whereas in both disease-entities we detected AT1R-aabs inducing ERK1/2 activation with similar prevalence (PHA: 38%, HT: 31%), indicating the existence of differentially acting AT1R-aabs. PHA-patients positive for ERK1/2 activating AT1R-aabs have lower serum potassium- and renin-levels together with an increased aldosterone concentration concordant with the disease phenotype. Similarly, higher BP values (syst/diast) are observed in AT1R-aab positive HT patients. In addition, ERK1/2-activation induced by either angiotensin II, a mouse AT1R-Mab, or by IgG isolated from patients with PHA or HT could be differentially blocked by the use of various signaling inhibitors. Whereas clinically used AT1R-blockers as e.g. Losartan, Olmesartan, or Candesartan fully block Ang II-mediated ERK activation, they are less efficient in blocking the effects of human AT1R-aabs, with Losartan having almost no effect. Conclusion: As hypothesized, the detection of AT1R-aabs directed against the native AT1R based on antigen binding only results in a high number of misleading results. By contrast, functional assays based on AT1R-activation ([Ca2+]i & ERK1/2-phosphorylation) are able to detect AT1R-aabs in 31% or 38% of patients with HT or PHA, respectively.
Copyright (c) 2018 Valérie Boivin-Jahns, Chistina Zechmeister, Claudia Schuetz, Martin J. Lohse, Roland Jahns, Xinhua Yu, Frank Petersen, Stefanie Hahner, Martin Fassnacht https://creativecommons.org/licenses/by/4.0
Autoantibodies targeting G-protein-coupled receptors in RA and SLE
Introduction: Previous studies have suggested a role of autoantibodies against G-protein-coupled receptors (GPCR) in autoimmune diseases. In this study, we aimed to determine the association of anti-GPCR autoantibodies with RA and SLE. Methods: The discovery cohort consisted of 10 patients with rheumatoid arthritis (RA), 10 patients with systemic lupus erythematosus (SLE), and 10 sex- and age-matched healthy donors (HD). Serum levels of autoantibodies against 14 GPCRs were measured for the discovery cohort by ELISA (Celltrend, Germany). Autoantibodies with significant difference between patients and controls were further investigated in a validation cohort containing 40 patients with RA, 14 patients with SLE, and 40 HD. For autoantibodies confirmed in the validation cohort, bioinformatic analysis was performed to explore possible functional interactions among them, and their diagnosis values were evaluated by ROC analysis. Results: For RA, levels of autoantibodies against 8 GPCRs were significantly different between patients and controls, 7 of which were confirmed in the validation cohort, including C5aR, C3aR, CXCR3, CXCR4, M3R, ?1-adrenergic receptor and AT1R. For SLE, levels of autoantibodies against 2 GPCRs, ?2-adrenergic receptor and ETAR, were significantly different between patients and controls, both were confirmed in the validation cohort. For the autoantibodies that were significantly changed in RA patients, bioinformatic analysis revealed that they were functionally related to chemotaxis. The ROC analysis showed good diagnostic values for all of those autoantibodies which were significantly changed in RA or SLE compared with controls. Conclusion: Levels of autoantibodies against GPCRs are significantly changes in RA and SLE compared to healthy controls, suggesting that they might be associated with the development of diseases.
Copyright (c) 2018 Xiaoyang Xiaoyang, Qiaoniang Huang, Fengyuan Deng, Juan Chen, Harald Heidecke, Gabriela Riemekasten, Frank Petersen, Xinhua Yu https://creativecommons.org/licenses/by/4.0
New insights into the diagnostics of autoantibodies against G-protein coupled receptors
Introduction: The knowledge about the impact of functionally acting autoantibodies which target G-protein coupled receptors (GPCR-AABs) is steadily growing. GPCR-AABs activate receptors; physiological counter-regulation, as known from natural ligands, does, however, not occur. This leads to pathological consequences such as heart failure (here AABs against the beta1-adrenoceptor, beta1-AABs) or other diseases. The problem with the detection of these GPCR-AABs is A) their low titers and B) their functional activity. A) For autoantibodies against the TSH-receptor (Grave’s disease) a bioassay (cAMP cascade) revealed titers between 0.0005 and 0.006% of the IgG fraction [Nakatake et al., Thyroid. 2006;16: 1077]. For most of the other GPCR-AABs almost nothing is known about the titer. B) With respect to the functional activity, binding without receptor activation has already been described [Bornholz et al., Cardiovasc Res. 2013;97: 472]. We here tested, if such factors influence on the GPCR-AAB detection. Methods: A bioassay [Wenzel et al., Heliyon. 2017;3: e00362] and a previously published ELISA [Nagatomo et al., J Am Coll Cardiol. 2017;69: 968] for the detection of the functional activity and the binding of beta1-AABs, respectively, were exploited. Results: Using the bioassay, beta1-AAB samples were identified and assigned to their epitopes on the receptor using mapping peptides. The samples were also applied onto an ELISA which was specifically developed and previously exploited for a large study investigating “Myocardial Recovery in Patients With Systolic Heart Failure and Autoantibodies Against ?1-Adrenergic Receptors” [Nagatomo et al.]. The comparison revealed that the ELISA results did not match the bioassay outcome. Conclusion: Since the existence of GPCR-AABs is increasingly gaining clinical importance and plays a decisive role in therapeutic decisions, erroneous measurements can have an enormous impact on patient therapy decisions. That is the reason why reliable tests have to be established which are able to identify the functional active GPCR AABs.
Copyright (c) 2018 Annekathrin Haberland, Johannes Müller, Gerd Wallukat, Katrin Wenzel https://creativecommons.org/licenses/by/4.0
The mosaic of autoimmunity: Why we develop autoimmune diseases
Background: Autoimmune diseases are conditions in which the immune system damages normal components of the individual. Initially it was thought that autoimmune disease was the inevitable outcome of the presence of clones of lymphocytes with receptors that recognize self-antigens. Thus tolerance to self, the state of non-autoimmunity, was due to the absence of self-recognizing lymphocytes, the 'forbidden' culprits of autoimmune disease. Autoimmune diseases were found to be multifactorial in their etiology. For practical reasons these factors are classified into four categories: Genetic, which entail the MHC class I, II, and III. A case in point will be the haplotypes of HLA-DRB1 which are prevalent in many classical diseases. Immune deficiencies: C1q C2, C4 and IgA deficiencies are among the most common defects associated with diverse autoimmune conditions. Hormonal state, most autoimmune diseases are detected in females at the child bearing ages. The role of estrogens will be delineated. In addition, other hormones play a role i.e. prolactin. Environmental causes: Those are the most important as a trigger factors determining the time and type of disease. They entail infectious agents, chemicals, adjuvants, drugs and even vaccines. We will discuss smoking, different types of food (i.e. salt, coffee, chocolate, spicy food, etc.) and their effect on the microbiome. The type of disease in an individual, in an autoimmune prone family, will be determined by the specific combination of the different factors mentioned above. A special emphasis will be put on the component of parasites.
Cardiovascular diseases, including coronary artery disease (CAD), are considered as killer number one worldwide. The pathology of CAD is caused by atherosclerosis, an inflammatory and immunological disorder. CXCL12 has been reported to be associated with CAD by genome-wide association studies (GWAS) and thus the CXCL12 / CXCR4 axis is determinant in atherosclerosis. CXCR4 belongs to the G proteins-coupled receptors (GPCR), which modulate different cellular functions. Although GPCRs are well established to function in immunity and inflammation, a little is known about their exact role in atherosclerosis. Recent studies pinpointed their therapeutic potential for atherosclerosis and CAD, however strong pre-clinical experimental evidence is still missing to understand whether targeting each of those molecules is protective or detrimental for atherosclerosis. Here we will provide a short update on the so far published data with regard to the potential implication of both receptors CXCR3 and 4 as well their ligands CXCL9-12 in the context of atherosclerosis. Recently a high anti-CXCR3/4 antibodies in patients with systemic sclerosis (SSc) along with other GPCRs was reported. An attempt to treat atherogenic mice with IgGs from systemic sclerosis (SSc) patients and healthy controls (HC) is in progress. Some of the so far generated preliminary results will be presented and discussed. This update will help us to further extend our better understanding on targeting GPCRs in the context of atherosclerosis.
Copyright (c) 2018 Zouhair Aherrahrou, Laurence Johanson, Jeanette Erdmann, Gabriela Riemekasten https://creativecommons.org/licenses/by/4.0
?1-adrenergic receptor autoantibody levels are higher in patients with Graves’ hyperthyroidism than in matched healthy controls and decrease after anti-thyroid treatment
Background: Graves’ disease (GD) is driven by autoantibodies targeting the thyroid-stimulating hormone receptor which belongs to the G-protein-coupled receptor (GPCR) superfamily. These autoantibodies (TRAb) result in hyperthyroidism, which entails symptoms from many organs, including the heart with risk for tachyarrhythmia, atrial fibrillation and heart failure. In previous studies of GD patients with heart complications, antibodies targeting cardiovascular GPCRs including the ?1-adrenergic receptor (B1R) were shown to be frequent. However, most of these studies were relatively small and used non-standardised peptide based ELISA methods or complex cell assays for antibody assessment. In this study, we used a standardised ELISA assay (CellTrend) to analyse antibodies directed against conformational B1R epitopes (B1RAb) to investigate if such antibodies are increased in GD patients compared to healthy controls, are influenced by antithyroid treatment and correlate with TRAb levels or heart related symptoms. Methods: This is a preliminary report of results from GD patients in an ongoing longitudinal observational prospective study. Premenopausal women in profound hyperthyroidism were included and symptom scores, TRAb and B1RAb were measured at baseline and after 7.5 months of treatment. We here present results from the first enrolled 43 patients and matched controls. Results: B1RAb levels were higher in patients at baseline compared to healthy controls (median 1.9 vs 0.9 ?g/ml, P<0.0001). B1RAb levels decreased after 7.5 months of treatment (median 1.6 ?g/ml, P=0.001), but remained higher than in controls (P=0.0008). B1RAb levels did not correlate with TRAb levels or heart related clinical symptoms. Conclusion: B1R autoantibodies were increased in GD patients at study inclusion and decreased with treatment. Continued studies include analysis of antibodies against other GPCRs expressed in the cardiovascular system and the relation to biomarkers of heart function.
Copyright (c) 2018 Anna Lundgren, Karin Tammelin, Mats Holmberg, Helena Filipsson Nyström https://creativecommons.org/licenses/by/4.0