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Article ; Online: C-Reactive Protein (CRP) Blocks the Desensitization of Agonistic Stimulated G Protein Coupled Receptors (GPCRs) in Neonatal Rat Cardiomyocytes

Gerd Wallukat / Stephan Mattecka / Katrin Wenzel / Wieland Schrödl / Birgit Vogt / Patrizia Brunner / Ahmed Sheriff / Rudolf Kunze

Journal of Clinical Medicine, Vol 11, Iss 1058, p

2022 Volume 1058

Abstract: Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) ... ...

Abstract	Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) signaling by CRP can be observed by using CRP in combination with different GPCR agonists on spontaneously beating cultured neonatal rat cardiomyocytes. All used agonists (isoprenaline, clenbuterol, phenylephrine, angiotensin II and endothelin 1) affected the beat rate of cardiomyocytes significantly and after washing them out and re-stimulation the cells developed a pronounced desensitization of the corresponding receptors. CRP did not affect the basal beating-rate nor the initial increase/decrease in beat-rate triggered by different agonists. However, CRP co-incubated cells did not exhibit desensitization of the respective GPCRs after the stimulation with the different agonists. This lack of desensitization was independent of the GPCR type, but it was dependent on the CRP concentration. Therefore, CRP interferes with the desensitization of GPCRs and has to be considered as a novel regulator of adrenergic, angiotensin-1 and endothelin receptors.
Keywords	C-reactive protein ; adrenergic receptor ; desensitization ; GPCR signaling ; endothelin ; Medicine ; R
Subject code	571
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Antigen-free control wells in an ELISA set-up for the determination of autoantibodies against G protein-coupled receptors—a requisite for correct data evaluation

Haberland, Annekathrin / Gerd Wallukat / Johannes Müller / Katrin Wenzel

Analytical and bioanalytical chemistry. 2018 Aug., v. 410, no. 21

2018

Abstract: First functional acting autoantibodies against G protein-coupled receptors such as the beta2-adrenoceptor in e.g. asthmatic patients have already been discovered in the early 1980s of the last century using assays that show their functional activity. ... ...

Abstract	First functional acting autoantibodies against G protein-coupled receptors such as the beta2-adrenoceptor in e.g. asthmatic patients have already been discovered in the early 1980s of the last century using assays that show their functional activity. Today, almost 40 years later, the measurement of such autoantibodies is still a challenge. Bioassays able to show the functional activity of such autoantibodies against G protein-coupled receptors are still the ne plus ultra for their detection and also classification when additionally exploiting specific receptor blockers for the neutralisation of the effect. Bioassays based on living cells make specific demands on the laboratories and are, therefore not suitable for every routine laboratory. Routine diagnostics, therefore, ideally requires different assays based on e.g. solid-phase technology, such as enzyme-linked immunosorbent assay (ELISA) technology. Here, endeavours are going on, using either the exact epitopes of such autoantibodies, if known, for trapping the autoantibodies, or the complete receptor in biological or artificial membranes that are immobilised onto a plastic carrier (ELISA principle). Here, we question and discuss the outcome of such tests, especially, if no controls such as the non-coated plastic carrier or the corresponding receptor-free membrane coat is offered as control in parallel, in light of the manifold experiences already collected with even non-agonistic acting autoantibodies by Güven et al. (J Immunol Methods 403:26–36, 2014).
Keywords	artificial membranes ; autoantibodies ; beta-2 adrenergic receptors ; bioassays ; diagnostic techniques ; enzyme-linked immunosorbent assay ; epitopes ; neutralization ; patients
Language	English
Dates of publication	2018-08
Size	p. 5101-5105.
Publishing place	Springer Berlin Heidelberg
Document type	Article
ISSN	1618-2642
DOI	10.1007/s00216-018-1172-x
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology.

Katrin Wenzel / Sarah Schulze-Rothe / Johannes Müller / Gerd Wallukat / Annekathrin Haberland

PLoS ONE, Vol 13, Iss 2, p e

2018 Volume 0192615

Abstract: Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ... ...

Abstract	Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.
Keywords	Medicine ; R ; Science ; Q
Subject code	500
Language	English
Publishing date	2018-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Performance and in-house validation of a bioassay for the determination of beta1-autoantibodies found in patients with cardiomyopathy

Katrin Wenzel / Sarah Schulze-Rothe / Annekathrin Haberland / Johannes Müller / Gerd Wallukat / Hanna Davideit

Heliyon, Vol 3, Iss

2017 Volume 7

Abstract: Background: Autoantibodies specific for the adrenergic beta1-receptor were identified to be an essential factor for the pathogenesis of dilated cardiomyopathy. For the detection of these autoantibodies, a bioassay was developed and has been used, ... ...

Abstract	Background: Autoantibodies specific for the adrenergic beta1-receptor were identified to be an essential factor for the pathogenesis of dilated cardiomyopathy. For the detection of these autoantibodies, a bioassay was developed and has been used, measuring the positive chronotropic effect on spontaneously beating neonatal rat cardiomyocytes. In order to use this bioassay as an analytical tool to monitor the effectiveness of autoantibody neutralizing therapy in a regulated field, there is a need to assess its analytical performance and validate it according to current guidelines. Methods: Using standard autoantibody samples, the increased beat rate compared to the basal rate [delta beats/min] was recorded when investigating guideline required assay performance parameters. Results: The analytical specificity and sensitivity of the bioassay was demonstrated. The limit of detection and positivity cut-off level were determined to be 3.56 and 7.97 delta beats/min, respectively. The coefficient of variation (CV) of all tested single values (four technical replicates each) was ≤15.2%. The CV of precision within each measuring series did not exceed 20%. Furthermore, the sample stability under a variety of different storage conditions was assessed, as well as the robustness of the cardiomyocyte preparations, which were both given. Conclusion: This bioassay fulfilled guideline determined quality requirements and proved to be appropriate for its application in clinical trials.
Keywords	Medicine ; Cell biology ; Immunology ; Cardiology ; Health sciences ; Biological sciences ; Science (General) ; Q1-390 ; Social sciences (General) ; H1-99
Language	English
Publishing date	2017-07-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Doberman pinschers present autoimmunity associated with functional autoantibodies

Gerhard Wess / Gerd Wallukat / Anna Fritscher / Niels-Peter Becker / Katrin Wenzel / Johannes Müller / Ingolf Schimke

PLoS ONE, Vol 14, Iss 7, p e

A model to study the autoimmune background of human dilated cardiomyopathy.

2019 Volume 0214263

Abstract: Background Autoimmunity associated with autoantibodies against the β1-adrenergic receptor (β1-AAB) is increasingly accepted as the driver of human dilated cardiomyopathy (DCM). Unfortunately, there is a lack of animal models to extend the knowledge about ...

Abstract	Background Autoimmunity associated with autoantibodies against the β1-adrenergic receptor (β1-AAB) is increasingly accepted as the driver of human dilated cardiomyopathy (DCM). Unfortunately, there is a lack of animal models to extend the knowledge about β1-AAB autoimmunity in DCM and to develop appropriate treatment strategies. Objectives To introduce an animal model, we investigated the β1-AAB associated autoimmunity in Doberman Pinscher (DP) with dilated cardiomyopathy, which has similarities to human DCM. Materials and methods Eighty-seven DP with cardiomyopathy in terms of pathological ECG and echocardiography (DoCM) and 31 dogs (at enrollment) without DoCM (controls) were analyzed for serum activity of β1-AAB with a bioassay that records the chronotropic response of spontaneously beating cultured neonatal rat cardiomyocytes to the DP's IgG. To locate the receptor binding site of β1-AAB and the autoantibody's sensitivity to inhibition, competing experiments with related blockers were performed with the bioassay. In controls that developed DoCM during follow-up, β1-AAB were analyzed during progress. Results Fifty-nine (67.8%) DoCM dogs and 19 (61.3%) controls were β1-AAB positive. Of the controls that developed DoCM, 8 were β1-AAB positive (p = 0.044 vs. dogs remaining in the control group); their β1-AAB activity increased with the cardiomyopathy progress (p<0.02). To supplement DoCM group with the 9 animals which developed cardiomyopathy in the follow up, a more pronounced β1-AAB positivity became visible in the DoCM group (p = 0.066). Total and cardiac mortality were higher in β1-AAB positive DP (p = 0.002; p = 0037). The dogs' β1-AAB recognized a specific epitope on the second extracellular receptor and were sensitive to inhibition by drugs already successfully tested to inhibit the corresponding human autoantibody. Conclusions Doberman Pinschers presented β1-AAB associated autoimmunity, similar as in the pathogenesis of human DCM. Consequently, DP could compensate the lack of animal models for the ...
Keywords	Medicine ; R ; Science ; Q
Subject code	630
Language	English
Publishing date	2019-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Immunoadsorption for Treatment of Patients with Suspected Alzheimer Dementia and Agonistic Autoantibodies against Alpha1a-Adrenoceptor—Rationale and Design of the IMAD Pilot Study

Sylvia Stracke / Sandra Lange / Sarah Bornmann / Holger Kock / Lara Schulze / Johanna Klinger-König / Susanne Böhm / Antje Vogelgesang / Felix von Podewils / Agnes Föel / Stefan Gross / Katrin Wenzel / Gerd Wallukat / Harald Prüss / Alexander Dressel / Rudolf Kunze / Hans J. Grabe / Sönke Langner / Marcus Dörr

Journal of Clinical Medicine, Vol 9, Iss 1919, p

2020 Volume 1919

Abstract: Background: agonistic autoantibodies (agAABs) against G protein-coupled receptors (GPCR) have been linked to cardiovascular disease. In dementia patients, GPCR-agAABs against the α1- and ß2-adrenoceptors (α1AR- and ß2AR) were found at a prevalence of 50%. ...

Abstract	Background: agonistic autoantibodies (agAABs) against G protein-coupled receptors (GPCR) have been linked to cardiovascular disease. In dementia patients, GPCR-agAABs against the α1- and ß2-adrenoceptors (α1AR- and ß2AR) were found at a prevalence of 50%. Elimination of agAABs by immunoadsorption (IA) was successfully applied in cardiovascular disease. The IMAD trial (Efficacy of immunoadsorption for treatment of persons with Alzheimer dementia and agonistic autoantibodies against alpha1A-adrenoceptor) investigates whether the removal of α1AR-AABs by a 5-day IA procedure has a positive effect (improvement or non-deterioration) on changes of hemodynamic, cognitive, vascular and metabolic parameters in patients with suspected Alzheimer’s clinical syndrome within a one-year follow-up period. Methods: the IMAD trial is designed as an exploratory monocentric interventional trial corresponding to a proof-of-concept phase-IIa study. If cognition capacity of eligible patients scores 19–26 in the Mini Mental State Examination (MMSE), patients are tested for the presence of agAABs by an enzyme-linked immunosorbent assay (ELISA)-based method, followed by a bioassay-based confirmation test, further screening and treatment with IA and intravenous immunoglobulin G (IgG) replacement. We aim to include 15 patients with IA/IgG and to complete follow-up data from at least 12 patients. The primary outcome parameter of the study is uncorrected mean cerebral perfusion measured in mL/min/100 gr of brain tissue determined by magnetic resonance imaging with arterial spin labeling after 12 months. Conclusion: IMAD is an important pilot study that will analyze whether the removal of α1AR-agAABs by immunoadsorption in α1AR-agAAB-positive patients with suspected Alzheimer’s clinical syndrome may slow the progression of dementia and/or may improve vascular functional parameters.
Keywords	Alzheimer’s clinical syndrome ; dementia ; immunoadsorption ; autoantibodies ; α1-Adrenergic receptor ; Medicine ; R
Subject code	616 ; 610
Language	English
Publishing date	2020-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Alpha1A-adrenergic receptor-directed autoimmunity induces left ventricular damage and diastolic dysfunction in rats.

Katrin Wenzel / Gerd Wallukat / Fatimunnisa Qadri / Norbert Hubner / Herbert Schulz / Oliver Hummel / Florian Herse / Arnd Heuser / Robert Fischer / Harald Heidecke / Friedrich C Luft / Dominik N Muller / Rainer Dietz / Ralf Dechend

PLoS ONE, Vol 5, Iss 2, p e

2010 Volume 9409

Abstract: Background Agonistic autoantibodies to the alpha(1)-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. Methods/principal findings We immunized Lewis rats with the second extracellular- ...

Abstract	Background Agonistic autoantibodies to the alpha(1)-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. Methods/principal findings We immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A)-adrenergic receptor and maintained them for one year. Alpha(1A)-adrenergic antibodies (alpha(1A)-AR-AB) were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A)-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max) demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min). Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang) II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats. Conclusions/significance We show that alpha(1A)-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A)-AR-AB could contribute to cardiovascular endorgan damage.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2010-02-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Potential relevance of alpha(1)-adrenergic receptor autoantibodies in refractory hypertension.

Katrin Wenzel / Hannelore Haase / Gerd Wallukat / Wolfgang Derer / Sabine Bartel / Volker Homuth / Florian Herse / Norbert Hubner / Herbert Schulz / Marion Janczikowski / Carsten Lindschau / Christoph Schroeder / Stefan Verlohren / Ingo Morano / Dominik N Muller / Friedrich C Luft / Rainer Dietz / Ralf Dechend / Peter Karczewski

PLoS ONE, Vol 3, Iss 11, p e

2008 Volume 3742

Abstract: Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target.To pursue ... ...

Abstract	Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target.To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had alpha(1)-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate alpha(1)-adrenergic receptor antibodies (alpha(1)-AB). Patient alpha(1)-AAB and rabbit alpha(1)-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human alpha(1A)-adrenergic receptor were incubated with patient alpha(1)-AAB and rabbit alpha(1)-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient alpha(1)-AAB and rabbit alpha(1)-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca(2+) in cardiomyocytes and induce mesentery artery segment contraction.Patient alpha(1)-AAB and rabbit alpha(1)-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for alpha(1)-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2008-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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