Diagnostic and therapeutic aspects of β1-adrenergic receptor autoantibodies in human heart disease

Autoimmunity Reviews 13 (2014) 954–962

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Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev

Review

Diagnostic and therapeutic aspects of β1-adrenergic receptor autoantibodies in human heart disease Beatrice Bornholz a, Dirk Roggenbuck b, Roland Jahns c,d , Fritz Boege a,⁎ a

Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Med. Faculty, Duesseldorf, Germany Faculty of Natural Sciences, Brandenburg University of Technology, Cottbus–Senftenberg, Germany Comprehensive Heart Failure Center (CHFC), University Hospital of Wuerzburg, Germany d Interdisciplinary Bank of Biomaterials and Data Wuerzburg (IBDW), University Hospital of Wuerzburg, Germany b c

a r t i c l e

i n f o

Article history: Received 4 June 2014 Accepted 16 June 2014 Available online 20 August 2014 Keywords: Autoantibodies Beta1-adrenergic receptor Cardiomyopathy Diagnostic tests Heart failure Targeted therapy

a b s t r a c t Growing evidence indicates a cardio-pathogenic role of autoantibodies against β1-adrenergic receptors (β1AR). In particular autoantibodies stimulating β1AR-mediated cAMP-production (i.e. agonistic β1AR autoantibodies) play a paramount role in chronic heart failure. When induced by immunisation, such autoantibodies cause heart failure in rodents; when present in patients they negatively affect survival in heart failure. However, the true prevalence and clinical impact of agonistic β1AR autoantibodies in human heart disease are still unclear, as are the events triggering their production, and the inter-relationship between autoantibody level and disease activity. β1AR autoantibodies can be removed by extracorporeal absorption or neutralised by systemic administration of synthetic epitope mimics. Only patients bearing agonistic β1AR autoantibodies in their bloodstream will benefit from these approaches. Therefore, reliable detection of agonistic β1AR autoantibodies is a key prerequisite for the future implementation of these strategies. β1AR autoantibodies impact on conformation and down-stream signalling of the receptor by binding a conformational epitope, which is poorly represented by synthetic mimics and readily destroyed by fixation. Consequently, β1AR autoantibodies can reliably be detected only by assays utilising the native β1AR as a test antigen. To provide a sufficient basis for diagnostic predictions or therapeutic decisions, one must also determine whether β1AR autoantibodies stimulate the receptor, which again requires native, cell-based reporter systems. Translation of these procedures into versatile diagnostic tests fitting the requirements of general health care is a challenge for future development. Here, we will review the state of diagnostic and therapeutic efforts in the field of β1AR-directed autoimmunity, thereby aiming to furnish a conceptual frame for the further development of novel, more reliable diagnostic tools and more specific antibodytargeted therapeutic concepts. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC-BY license (http://creativecommons.org/licenses/by/3.0/).

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . The evidence of β1AR-directed autoimmune cardio-pathogenesis . . Impact of autoantibodies on β1AR conformation and function . . . . 3.1. Impact on base line receptor activity . . . . . . . . . . . . 3.2. Induction of conformational changes in the receptor molecule 3.3. Impact on receptor trafficking and maximal agonist response 3.4. A model explaining functional autoantibody effects . . . . . Mechanism of cardio-noxicity of β1AR autoantibodies . . . . . . . Therapeutic approaches . . . . . . . . . . . . . . . . . . . . . 5.1. Removal of autoantibodies by extracorporeal absorption . . 5.2. Systemic neutralisation of autoantibodies . . . . . . . . .

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Abbreviations: β1AR, β1 adrenergic receptor; CHF, chronic heart failure; DCM, dilated cardiomyopathy; ELISA, enzyme linked immune assay; FRET, fluorescence resonance energy transfer; GPCR, G-protein coupled receptor; IA, immune apheresis, extracorporeal absorption of circulating IgG; TSH-R, thyroid-stimulating hormone receptor. ⁎ Corresponding author at: Institute of Clinical Chemistry and Laboratory Diagnostics, Central Laboratory, University Hospital, Moorenstraße 5, 40225 Düsseldorf, Germany. Tel.: +49 211 8118290; fax: +49 211 8118021. E-mail address: [email protected] (F. Boege).

http://dx.doi.org/10.1016/j.autrev.2014.08.021 1568-9972/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC-BY license (http://creativecommons.org/licenses/by/3.0/).

B. Bornholz et al. / Autoimmunity Reviews 13 (2014) 954–962

6.

Diagnostic issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. The problem of the polymorphic and labile native autoantibody-epitope 6.2. Routine assays for the detection of β1AR autoantibodies . . . . . . . . 6.3. Discrimination of stimulating and non-stimulating β1AR autoantibodies . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Autoantibodies that bind to G-protein coupled receptors (GPCRs) of neuro-endocrine transmitters have been demonstrated to possess the potential to cause pathological changes in humans. These autoantibodies alter base line activity and/or agonist responsiveness of the receptors and thereby exert toxic effects in target tissues. It is suggested to classify such clinical entities as GPCR-targeted agonistic autoantibody-diseases. The most firmly established example is Grave's disease (M. Basedow), which is caused by autoantibodies that stimulate the thyroid-stimulating hormone receptor (TSH-R) and thereby promote pathologically enhanced growth and endocrine dysfunction of the thyroid gland [1,2]. Over the past three decades, growing evidence has accumulated that a number of other aetiologies are probably based on the same pathogenic principle and therefore should also be recognised as agonistic GPCR-targeted autoantibody-diseases [3]. These aetiologies have in common (i) that they are associated with circulating autoantibodies targeting GPCRs involved in autonomic vegetative regulation, (ii) that these autoantibodies mimic receptor agonists, and (iii) that the pathology predominantly manifests in the cardiovascular system and the kidney (Table 1; for a more complete list please refer to [3]). The β1-adrenergic receptor (β1AR) appears to be a common target of several agonistic autoantibody-diseases that lead to chronic heart failure (Table 1). Most notably, agonistic β1AR autoantibodies are involved in the pathogenesis of dilated cardiomyopathy (DCM), a disease characterised by progressive ventricular dilation and dysfunction due to non-ischemic myocardial damage. A DCM sub-entity commonly classified as “idiopathic” [4] is strongly associated with agonistic β1AR autoantibodies. The majority of idiopathic DCM patients have agonistic β1AR autoantibodies in their circulation [5–7]. Clinical correlations [6,8] in conjunction with animal immunisation experiments [9–14] strongly suggest that these autoantibodies play a causal role in the pathogenesis of the disease. Agonistic β1AR autoantibodies are also present in roughly a third of ischemic cardiomyopathy cases [8,15], and they are frequently associated with arrhythmic complications also in other diseases primarily not affecting the heart [16]. It is estimated

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that agonistic β1AR autoantibodies could be involved in up to 10% of cardiac morbidity in western societies. This figure is probably even higher in South America, where β1AR autoimmunity is triggered by endemic infection with Trypanosoma cruzi [17]. However, these estimates remain highly speculative pending the results of an on-going prospective cohort study on the prevalence of agonistic β1AR autoantibodies in DCM and ischemic heart disease [18]. It must finally be noted that depending on the detection method deployed agonistic β1AR autoantibodies can be also determined in up to 12% of healthy individuals [8,19]. From these observations the postulate can be derived that patients, whose cardiac health is endangered by agonistic β1AR autoantibodies, should be identified, monitored and subjected to specific therapies counteracting the adverse effects of the autoantibodies on cardiac function. Various procedures have been developed to protect or cure patients from the adverse effects of agonistic β1AR autoantibodies either by removing the antibodies from the blood stream or by neutralising them in situ by systemic application of synthetic mimics of the epitope [20–23]. However, a targeted application of these concepts to the causal therapy or prevention of heart failure is currently hampered by diagnostic issues, as there are no available assays that are suitable for reliable quantification of β1AR autoantibodies in patient's blood samples or a standardised assessment of their impact on receptor function. Therefore, it is currently not readily possible to select patients for specific antibody-targeted therapies and to monitor such therapeutic regimen in a clinical setting. Here, we will review the current state of knowledge regarding these therapeutic and diagnostic aspects of the issue and try to give an outlook on further developments. 2. The evidence of β1AR-directed autoimmune cardio-pathogenesis DCM-associated auto-reactive IgG has the potential to stimulate cAMP production [24] and MAP-kinase signalling [25] via the β1/2AR, inhibit cAMP signalling via the M2-muscarinic receptor [26], and exert negative inotropic effects via Fcγ receptors [27]. These pleiotropic autoantibody effects have been discriminated by blockade with selective receptor antagonists and/or proteins presenting the respective binding

Table 1 Established and emerging diseases caused by, or associated with, autoantibodies against G-protein coupled receptors. Disease/Syndrome

Receptor type target

Effect on receptor function

References

Arterial hypertension (primary, malignant or refractory) Pulmonary arterial hypertension Preeclampsia Renal allograft rejection Dilated cardiomyopathy

α1-adrenergic α1-adrenergic endothelin-1 angiotensin I angiotensin II, type 1 β1-adrenergic β2-adrenergic M2-muscarinic acetylcholine β1-adrenergic β2-adrenergic M2-muscarinic acetylcholine β1-adrenergic β2-adrenergic M2-muscarinic acetylcholine β2-adrenergic β2-adrenergic M2-muscarinic acetylcholine

Stimulation Stimulation Stimulation Stimulation Stimulation

[87–89] [20] [90,91] [92] [8,26,28,30,34]

Stimulation

[31,33]

Stimulation

[16,93–95]

Inhibition Stimulation

[96] [97]

Chagas' disease, cardiomyopathy, megacolon, megaesophagus

Tachyarhythmia, atrial fibrillation

Postural hypotension Complex regional pain syndrome

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sites [26,28–31]. It has become clear from these studies that autoantibodies enhancing the activity of the β1AR are of particular relevance for the development of DCM in humans [32]. Such autoantibodies are frequently found in serum of patients with chronic heart failure. Their prevalence is associated with Chagas' cardiomyopathy [33], DCM [8, 34] or ischemic heart disease [8], but not cardiomyopathies of other aetiology [15,35,36]. Epitopes most frequently targeted by agonistic β1AR autoantibodies are located in first and second extracellular loops of the receptor [28,29]. Human autoantibodies directed against these epitopes exert positive ino- and chronotropic effects on cardiomyocytes [8,35,37]. Similar effects were demonstrated for antibodies produced in rodents against peptides corresponding to the second extracellular β1AR loop, which cross-react with the native β1AR [35,38,39]. Moreover, such immunisations induced left ventricular dilation and dysfunction [9,10,40] among other effects compatible with chronic cardiac dysfunction [11–13]. These pathological effects were reversible upon removal of the antibodies [41], transferable via serum transfusions [10,13,42,43], and partially abrogated by β1AR antagonists [44] or synthetic mimics of the receptor epitope [45,46]. In DCM patients, prevalence [8] and cAMP stimulatory efficacy [24] of β1AR autoantibodies are correlated to a more reduced cardiac function [8], increased cardiac mortality [6], more severe ventricular arrhythmia [47], and sudden cardiac death [48,49]. Therefore, autoimmunity against the second extracellular loop of the β1AR is considered a cause or cofactor of chronic left ventricular dysfunction and a potential therapeutic target [22,41, 45,46,50–52]. Interleukine-6 seems to promote β1AR directed autoimmune cardiomyopathy through enhancing antibody production [53], but it is not known what initiates the generation of potentially pathogenic β1AR autoantibodies. Various viral and microbial candidate proteins have been proposed [54] to trigger bystander effects analogous to the ribosomal P2β protein of T. cruzi, which induces β1AR-directed autoimmune cardiomyopathy in Chagas' disease [54,55]. However, firm evidence of a microbial immunogenic agent that can cause DCM by raising a β1AR-directed humoral immune response is still lacking. Therefore, the only specific causal therapy conceivable to date is the removal or neutralisation of the autoantibodies [20–23,56]. 3. Impact of autoantibodies on β1AR conformation and function 3.1. Impact on base line receptor activity Many β1AR autoantibodies retrieved from patients with DCM or ischemic cardiomyopathy are “agonistic” in as much as they have the potency to moderately increase cAMP stimulation through the otherwise un-liganded receptor [24,34,57]. The occurrence of agonistic β1AR autoantibodies is a predictive factor of DCM mortality [6], and therefore of considerable diagnostic interest. Agonistic β1AR autoantibodies appear to fall into two classes regarding the impact on baseline receptor activity: About half of the autoantibodies found in DCM or ischemic heart disease are “high” activators that are almost half as efficient as full agonists such as isoprenaline, whereas the other half are “low” activators that reach only a third of the effect of the “high” activators. Interestingly, the effect of “low” activators on cAMP can be preferentially blocked by peptide homologs of the first extracellular loop of the β1AR, while “high” activators can be preferentially blocked by peptide homologs of the second extracellular loop of the β1AR, which indicates that the two classes of agonistic autoantibodies target different portions of the receptor [24]. It stands to reason that the heterogeneous impact of the autoantibodies on base line receptor activity is related to an equally heterogeneous impact on receptor conformation (see: 3.2), but this has not yet been systematically investigated. 3.2. Induction of conformational changes in the receptor molecule The molecular mechanisms by which autoantibodies activate the β1AR are not yet fully understood. Activation of βAR by agonistic ligands

involves a conformational switch enabling Gs-protein coupling [58,59]. It has been proposed that β1AR autoantibodies recognise conformational epitopes [8] prompting the hypothesis that they modulate β1AR conformation similar to agonists [57] and/or alter the stability of transient conformational receptor states that are spontaneously assumed or induced by agonists [32]. More recently, this hypothesis has been proven using a genetically engineered β1AR bearing a fluorescence resonance energy transfer (FRET) sensor for the activation-associated conformation switch. A similar change in FRET-efficiency was observed upon exposure to β1AR autoantibodies as upon exposure to an agonist [37], demonstrating that the autoantibodies indeed alter the conformation of the receptor molecule in an agonist-like fashion. However, there is no clear correlation between the impact of the autoantibodies on receptor conformation (as detected by a FRET sensor) and their impact on base line receptor activity (as detected by increased cAMP production in the absence of agonistic ligands), indicating that the autoantibodies induce and/or stabilise a range of different receptor conformations that are heterogeneous regarding their efficiency of coupling to the stimulatory G-protein and adenylate cyclase. Interestingly, β1AR autoantibodies isolated from DCM patients induce or stabilise receptor conformations that are as active as the ones induced by agonists, while β1AR autoantibodies isolated from healthy individuals mostly induce changes in receptor conformation not leading to cAMP stimulation [37]. In summary, these studies corroborate the notion that β1AR autoantibodies bind conformational epitopes transiently formed by a small domain encompassing first and second extracellular loops of the receptor. This particular interaction results in stimulation of the receptor to a varying degree by inducing or stabilising distinct conformations of that domain. 3.3. Impact on receptor trafficking and maximal agonist response Heart failure following chronic β1AR stimulation is typically associated with a reduction of β1AR-mediated regulatory amplitude due to an increase in baseline cAMP and a decrease in maximal chronotropic catecholamine response [60]. A similar situation is promoted by β1ARautoantibodies present in subpopulations of DCM patients, which increase cAMP baseline while attenuating maximal cAMP response to a β1AR agonist [57]. This feature appears to be associated with an additional property of the autoantibodies, namely the attenuation of agonist induced receptor internalisation. About half of DCM-associated agonistic autoantibodies studied in this respect inhibit receptor trafficking. As a consequence, maximal catecholamine-induced cAMP production is differentially affected by agonistic β1AR autoantibodies: Those autoantibodies that attenuate receptor internalisation dampen maximal catecholamine response, while those autoantibodies that have no effect on receptor trafficking increase maximal isoproterenol response [37]. The impact on receptor internalisation appears directly linked to the interaction of the autoantibodies with the receptor (as opposed to e.g. the coincident action of autoantibodies blocking clathrin), because the effect can be blocked by pre-adsorption with a peptide homolog of the second extracellular loop domain of the β1AR. Currently it is not clear what determines the distinct impact of agonistic β1AR autoantibodies on receptor trafficking and how this property is related to cardio-pathogenesis. 3.4. A model explaining functional autoantibody effects The data reviewed in this chapter strongly suggest that β1AR autoantibodies have heterogeneous effects on baseline activity, maximal agonist response and trafficking of the receptor. These effects are linked to the ability to bind conformational epitopes presented by a domain encompassing the first and second extracellular loops of the β1AR. It remains unclear how the impact on receptor conformation is mechanistically linked to the effect on receptor activity, since the receptor domain targeted by the autoantibodies is not identical and not even close to the ligand binding-site, as predicted from high resolution X-ray diffraction

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data of the human β2AR [58,59] and the turkey β1AR [61]. However, based on the aforementioned structural data, one can assume that the receptor domain targeted by the autoantibodies presents one series of transient conformations when the receptor idles and another slightly different series of transient conformations when it undergoes activation by an agonist. Individual cocktails of β1AR autoantibodies present in patients or healthy individuals will bind and stabilise distinct subsets of these conformations, thereby “freezing” the receptor in a unique transient state of activity/inactivity, and thus exerting a unique impact on its function, which provides a plausible explanation for the interindividual heterogeneity of the impact of β1AR autoantibodies on receptor functions. In general, β1AR autoantibodies present in the circulation of patients with chronic heart failure (CHF) appear to have higher levels/avidities and stimulate and/or sensitise the receptor (i.e. target conformational epitopes presented both by inactive receptors, which are then switched into an active conformation, and by active receptors, which are further stabilised). In contrast, β1AR autoantibodies present in the circulation of healthy individuals have lower levels/avidities and no significant effects on β1AR activity (i.e. target conformational epitopes not relevant for receptor activity/activation). Some autoantibodies even act as mild antagonists (i.e. target epitopes not altering conformation during receptor activation/inactivation). Thus, it is not the mere presence of β1AR autoantibodies that distinguishes DCM patients from healthy subjects, but a different impact of the autoantibodies on β1AR function most likely dependent on/related to the conformation of the targeted epitope. This fact needs to be taken into consideration when devising therapeutic and diagnostic strategies. 4. Mechanism of cardio-noxicity of β1AR autoantibodies It is to be expected that the continuous exposure to agonistic β1AR autoantibodies leads to desensitisation and ultimately down-regulation of cardiac β1AR [62], which is a common hallmark of CHF [60,63]. Rodents undergoing left ventricular dysfunction following immunisation with synthetic homologs of the second extracellular loop of the β1AR have indeed reproducibly shown alterations of cardiac signal transduction compatible with this putative pathogenic mechanism [9–11,64]. However, in vitro-exposures of cardiomyocytes or other cellular reporter systems of β1-adrenergic signal transduction to agonistic β1AR autoantibodies derived from human DCM patients have failed to induce receptor desensitisation or gradual attenuation of cAMP-accumulation over time [34], which may be connected to the potency of the autoantibodies to inhibit agonist-induced receptor internalisation [37]. Therefore, it cannot be excluded that mechanisms other than desensitisation of cardiac β1AR mediate the cardio-noxicity of the antibodies. It has been observed that agonistic β1AR autoantibodies stimulate apoptosis [13,65] and stress responses of the endoplasmic reticulum [42], suggesting that the pathogenic mechanism may involve direct cardiac cytotoxicity. Moreover, it has been shown that the autoantibodies induce MAP-kinase and β-arrestin signalling [25], which potentially interfere with β1-adrenergic signal transduction [66]. Furthermore, DCM-derived β1AR autoantibodies alter the function of cardiac L-type Ca2+ channels [38,67] and are suspected to modulate lateral mobility of the receptors through simultaneous interactions with Fcγ receptor IIa recently discovered on cardiomyocytes [27]. Finally it has been suggested that the antibodies induce homo-dimerisation of β1AR [3], which is known to play a role in cardiac signalling efficacy and contractility [68,69]. In summary, receptor desensitisation seems to be the major cardio-pathogenic mechanism of β1AR autoimmunity in immunised rodents, while cardio-noxicity of agonistic β1AR autoantibodies in humans could also be executed by a variety of other mechanisms. However, this discrepancy does not impinge on the principle desideratum that the primary interaction between receptor and agonistic autoantibody should be addressed by diagnostic and therapeutic measures, irrespective of the cardio-noxious mechanisms downstream thereof.

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5. Therapeutic approaches 5.1. Removal of autoantibodies by extracorporeal absorption Numerous studies on the treatment of DCM by non-selective immune apheresis (IA) of circulating IgG or selective extracorporeal removal of the IgG3 subclass have largely shown positive effects on haemo-dynamics, NYHA classification of disease state, hospitalisation, bio-markers of cardiac function and other clinical parameters of disease activity (reviewed in: [20,21]). Interestingly, two small uncontrolled pilot studies using immobilised peptide homologs of the β1AR epitope for selective IA have provided similarly positive results [52,70], which suggests that among a multitude of cardiac autoantibodies potentially involved in the pathogenesis of DCM [71,72] β1AR autoantibodies appear to play a paramount role. Due to the very small case series of the two studies on β1AR-selective IA (n = 8) and the heterogeneity of follow-up parameters, the therapeutic efficacy of β1AR-selective and unselective IA cannot be compared based on existing data, and it remains to be evaluated, whether selective IA has a significant clinical benefit over the more firmly established non-selective IA protocols. It is also conceivable that new synthetic mimics of one or several autoantibody epitope(s) (see 5.2) can be employed to further improve the performance of selective IA. However, irrespective of the IA procedure utilised, DCM patients apparently profit most from it when β1AR autoantibodies are present at baseline [20], which is in line with the observation that the presence of agonistic β1AR autoantibodies at baseline predicts DCM morbidity and cardiovascular mortality [6,8,24,47–49]. In summary, IA is a valid and largely established therapy option for DCM, provided it is possible to select the subgroup of DCM patients that are positive for agonistic β1AR autoantibodies, and to monitor clearance and reappearance or persistence of the autoantibodies during follow-up. 5.2. Systemic neutralisation of autoantibodies Two approaches have been established to neutralise β1AR autoantibodies by the systemic application of ligands that mimic the targeted epitope. One type of such synthetic neutralising ligands has been made by affinity selection and single step exponential enrichment of a short single-stranded nucleotide sequence (aptamere) [73] that binds with high affinity to the epitope recognition site of β1AR autoantibodies. It has been shown that this aptamere can neutralise the ino- and chronotropic effects of DCM-derived β1AR autoantibodies on neonatal rat cardiomyocytes in vitro [45] and decrease the levels of agonistic β1AR autoantibodies in spontaneously hypertensive rats [74]. It remains to be demonstrated whether this compound is also able to reverse dilated cardiomyopathy in an immunisation model and, thus, may be suited for the further development of a systemic therapy in human heart failure [75]. The other approach consists in a peptide-based strategy aiming to specifically neutralise conformational β1AR autoantibodies directly in the circulating blood to prevent their harmful cardio-stimulatory effects. To this end, peptide-homologs mimicking the epitope(s) thought to represent the key target of agonistic β1AR antibodies, that is, the second extra-cellular β1AR loop were generated and cyclised in order to increase their stability in vivo. These peptides are referred to as β1ECII-CP or COR1. In pre-clinical studies β1AR antibody-positive sera from immunised rats served to assess the blocking capacity of β1ECII-CP both in vitro and in vivo [22]. In vitro pre-incubation of stimulating rat β1AR antibodies with β1ECII-CP efficiently blocked receptor signalling and reduced β1AR antibody-induced cAMP-production by more than 80%. Similarly, injection of β1ECII-CP into immunised rats resulted in a significant antibody-neutralising effect in vivo, and reduced the initially observed cAMP-response by about 60% (assays performed before and 24 h after β1ECII-CP injection) [49]. In addition, long-term injection of β1ECII-CP (every 4 weeks over 12 months) was found to revert cardiac dilatation

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and to normalise cardiac function in β1AR antibody-positive cardiomyopathic rats [76], indicating that β1ECII-CP might be beneficial also in the treatment of agonistic β1AR autoantibody-positive human heart failure. Recently, the first application of β1ECII-CP in man has been successfully accomplished [46], and a clinical phase IIa trial with this compound is currently under way. Neutralisation of β1AR autoantibodies by systemic application of synthetic epitope mimics could evolve into a more versatile, less invasive, less expensive and less hospitalisation-burdened alternative. However, like IA, this therapy option will require the selection of suitable DCM patients (that are positive for agonistic β1AR autoantibodies) and a monitoring of the levels of the circulating autoantibodies β1AR during therapy and follow up. Therefore, it will be necessary to move the assessment of β1AR autoantibodies from research to clinical diagnostics in order to develop suitable assays for agonistic β1AR autoantibodies that meet the requirements of standardisation and reliability of clinical diagnostic tests.

with the results of immunoassays based on native β1AR presented on intact cells [8]. It appears that peptide-based immunoassays are much less sensitive and specific than cell-based assays and only detect a subset of positive patients [37]. This probably explains, why studies on the prevalence of β1AR autoantibodies in cardiac diseases have largely diverging results. In general, much higher figures are reported using peptide-based immunoassays [49,78] as compared to native cell-based assays [8,15,24]. The discrepancy is currently blamed on a high rate of false positive results. Clinical correlations suggest that the figures derived from native cell-based assays are more valid. As a consequence, peptide-based immunoassays are currently not considered a valid tool for the detection of clinically relevant human β1AR autoantibodies [5, 32], and assays based on native (living) cells are still the main stay of clinical and pre-clinical studies [3,18,31,45,74]. However, the latter assays are clearly unsuited for routine clinical diagnostics.

6. Diagnostic issues

In principle there are three routes available that possibly lead to the development of standardised and valid routine assays for the detection of potentially cardio-pathogenic β1AR autoantibodies in human blood samples. One approach is to replace in solid phase immunoassays the linear peptide homologs with improved synthetic mimics of the target domain such as aptameres [3] or cyclic peptides (e.g. the COR-1 peptide [46]). To our knowledge this has not been thoroughly worked out/optimised or the so far obtained results were not satisfying. As a second approach, native cell-based assays could be reformulated in such a way that they can be subjected to normal storage and transport procedures and meet the requirements of standardisation and quality control in routine clinical diagnostics. One step in that direction is the recent development of a competition ELISA based on formaldehyde-fixed cells overexpressing the human β1AR and a competitive monoclonal antibody against the autoantibody target domain that specifically displaces human autoantibodies bound to that site. The assay was validated according to “good laboratory practice” and can serve as a companion bio-diagnostic assay for the development and evaluation of antibodydirected therapies in antibody-positive heart failure [39]. However, it remains to be seen whether this approach is suitable for the sensitive detection of β1AR autoantibodies in a broad range of human blood samples, given the limitations imposed on the sensitivity by cell fixation and the competing monoclonal antibody, which both reduce the scope of conformational epitopes represented by that assay. Another possible starting point is the determination of autoantibody binding to native cells overexpressing biofluorescent β1AR by means of quantitative fluorescence microscopy or fluorescence-activated flow cytometry. These

6.1. The problem of the polymorphic and labile native autoantibody-epitope The receptor domain targeted by β1AR autoantibodies associated with DCM and other cardiac diseases encompasses the first and second extracellular loops of the receptor [28,29], which are the only extracellular domains capable of inducing the production of antibodies exerting pharmacological effects on the receptor [77]. The three-dimensional structure of this domain as predicted from high resolution X-ray diffraction data of the human β2AR [58,59] and the turkey β1AR [61] is an extracellular α-helix formed by the second extracellular loop. This helix is linked via an S–S bridge to the more amorphous structure of the first extracellular loop. There is a strong argument that the entire domain changes its conformation during receptor activation/inactivation and in doing so presents a multitude of transient conformational epitopes, which are targeted by β1AR autoantibodies (see 3.2 and 3.4). As a consequence, the autoantibodies poorly cross-react with linear representations or denatured versions of the targeted receptor domain. In line with that notion, DCM-associated β1AR autoantibodies are unable to bind to denatured human β1AR presented on Western-blots, unless the blots are first subjected to extensive renaturation procedures [8]. Moreover, the binding of human autoantibodies to native β1AR presented on the surface of overexpressing cells is rapidly destroyed by all known fixation procedures (Fig. 1), supporting the notion that the antibodies indeed target an extremely labile conformational epitope [37]. Along the same lines, detection of β1AR autoantibodies by their binding to immobilised peptide homologs of the target domain is poorly correlated

native

Acetone

6.2. Routine assays for the detection of β1AR autoantibodies

50% MeOH

2% FA G FP Ig -Y d AR un β1 bo

Fig. 1. Disruption of IgG co-localisation with native biofluorescent β1ARs by cell fixation. Receptor- (top) and IgG-associated fluorescence (bottom) visualised by confocal fluorescence microscopy at 400-fold magnification in cells overexpressing YFP-fused human β1ARs and stained with human autoimmune IgG from a DCM patient. Prior to the incubation with autoimmune IgG, the cells were exposed (for 5 min at 4 °C) to PBS (left), 100% acetone (middle left), 50% methanol in PBS (middle right), or 2% formaldehyde in PBS (right). Image cited with the author's permission from [37].

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procedures were successfully set up to discriminate levels/avidities of β1AR autoantibodies from DCM patients and healthy subjects in a quantitative manner [37] and could be further developed into a clinical test if the problems of storage and transport can be solved. Digital fluorescence microscopy with modern pattern recognition algorithms could be a promising option for standardisation of such native cell-based assays in a clinical setting [79,80]. A third approach could be to produce semi-synthetic targets for the autoantibodies that consist of native β1ARs solubilised from the cell membranes and reconstituted in synthetic lipid vesicles. Such procedures have been developed and successfully used for the functional reconstitution of β1-adrenergic signal transduction from purified components [81]. β1AR thus reconstituted in their native conformation are known to remain fully functional upon prolonged storage at −20 °C and therefore could be used in immune assays as a durable test antigen that presents the full scope of native conformational epitopes targeted by β1AR autoantibodies. It should finally be noted that there remains an unsolvable problem of standardisation and validation that applies to all three approaches. The analyte (that is, the β1AR autoantibody) represents a heterogeneous mixture with respect to the target epitope. Therefore it will be almost impossible to formulate standards and calibrators that are representative of the entirety of β1AR autoantibody variants occurring in patients (and probably also in some healthy subjects). As a consequence, the performance of all these approaches can only be validated by clinical correlation of the prevalence of test results in blood samples of controlled study cohorts (e.g. the ETiCS study [18]). This may eventually pose a problem for the certification and accreditation of these diagnostic tools for an application in routine health care. 6.3. Discrimination of stimulating and non-stimulating β1AR autoantibodies DCM patients and healthy subjects are not discriminated by the mere presence of β1AR autoantibodies, but by a different impact of their autoantibodies on β1AR function (see chapter 3). Only the prevalence of agonistic β1AR autoantibodies is clearly correlated with increased morbidity and mortality in DCM [6]. Therefore the mere demonstration of β1AR-binding IgG in the blood of a patient or healthy individual does not provide a sufficient basis for diagnostic predictions or therapeutic decisions. It needs to be discriminated whether the autoantibodies stimulate the receptor or not. Currently this distinction is often based on the impact of the purified IgG fraction on beating rate and contractility of spontaneously beating cardiomyocytes isolated from neonatal rats [82]. Alternatively, this assay can be performed with spontaneously beating human embryonic cardiomyocytes generated by in vitro differentiation from induced human adult stem cells [37]. Both bio-assays are not suited for clinical diagnostics because they are constituted of primary cells that must be freshly prepared each time the assay is to be performed. Moreover, the readout is difficult to standardise even when employing automated monitoring of cell impedance [83]. An alternative more suited to routine clinical application are engineered cellular reporter systems of β1-adrenergic signal transduction that are based on immortalised cells. The most elegant of these assays is a cell line overexpressing human β1AR in the cell membrane together with a cytosolic FRET-reporter of cAMP concentration. This system has already been evaluated in a large cohort of patients. It enables a clear distinction of agonistic and non-stimulating β1AR autoantibodies and even discriminates between strong and weak agonistic β1AR autoantibodies [24]. From the point of view of availability of analytical platforms, it would be more convenient to combine β1AR overexpression with a light-generating cAMP-reporter gene assay. Such systems have been successfully established for various GPCRs [1,84–86] and are currently FDA-approved for clinical diagnostics of TSH-R autoantibodies in Grave's disease [1]. In principle it is conceivable to engineer even more versatile semisynthetic reporter assays by reconstitution of purified components in lipid vesicles [81].

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7. Conclusions By now, conformational agonistic β1AR autoantibodies are clearly recognised as playing a pivotal role in human heart failure, either as a disease-causing agent or as a “negative” disease-modifier. Preliminary clinical data clearly suggest that the presence of agonistic β1AR autoantibodies significantly worsens the prognosis of patients suffering from idiopathic DCM. A broader and more complete picture of pathophysiologic and prognostic issues in β1AR autoantibody-mediated heart disease will emerge from the result of prospective diagnostic studies currently planned or under way (e.g. the ETiCS-study), which are expected to provide new insights into cardiac events capable of triggering β1AR-directed autoimmune processes and the contribution of such processes to the initiation and/or progression of CHF. In particular, we might learn and better understand whether – and if so, then to which extent – the specific target epitope(s) of a conformational β1AR autoantibody, but also its respective titre and biological activity, and its respective kinetics (that is, antibody-persistence or -clearance over time) relate to the complex process of cardiac remodelling and dilation (and its eventual reversal). Different β1AR autoantibodies might have distinct propensities to induce a certain cardiac phenotype, and studies such as ETHiCS will allow for a differentiation of such features in a prospective manner. Already there exist a variety of specific therapy options for β1AR autoantibody-mediated heart disease. These include non-selective IA of total IgG and selective IA of IgG3, which are largely established therapy options, and selective IA of β1AR autoantibodies, which may have some advantages over the other IA options pending corroborative clinical studies of larger case series. An alternative to IA that is potentially more versatile, less invasive, less expensive and less burdened with hospitalisation is the neutralisation and clearance of β1AR autoantibodies by systemic application of synthetic epitope mimics. However, preclinical evaluation of such concepts has not yet been completed. A roll out of any of these therapeutic approaches into routine health care will eventually require (i) the selection of patients likely to profit from the therapy because they are positive for agonistic β1AR autoantibodies and (ii) a monitoring of the circulating levels of these autoantibodies during therapy and follow up. The assays currently in use for the detection of β1AR autoantibodies and the determination of their impact on receptor function are unfit for routine health care, because they employ primary cardiomyocytes or cell lines overexpressing the target receptor. The readout of such live cell reporter systems is difficult to standardise — even when automated. It poses a major challenge for future development to move the assessment of circulating agonistic β1AR autoantibodies from research procedures to clinical diagnostics, and to develop assays that accomplish the requirements of standardisation and reliability of clinical diagnostic testing. While developing such diagnostic tests one needs to keep in mind that the assays must (i) detect autoantibodies recognising a conformational epitope so far only found properly represented in the native β1AR, and (ii) discriminate autoantibodies that bind and stimulate the β1AR from autoantibodies that bind the β1AR without affecting its function. Possibly, that dual diagnostic goal will be best reached by a staged strategy. It is the firm belief of the authors that valid, standardised diagnostic tests for agonistic β1AR autoantibodies will substantially promote further development and evaluation of personalised therapeutic strategies specifically directed at clearance or neutralisation of distinct β1ARautoantibody species that prove to be cardio-noxious in a given patient or disease-entity.

Conflict of interest Dirk Roggenbuck is a shareholder of GA Generic Assays GmbH and Medipan GmbH. The remaining authors declare that they have no competing financial interests.

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Take-home messages • Conformational agonistic β1AR autoantibodies play a pivotal role in human heart failure. • Specific therapy options already exist for heart disease mediated by these autoantibodies. • Roll out of these therapeutic approaches into routine health care will require (i) the selection of patients positive for agonistic β1AR autoantibodies and (ii) the monitoring of circulating autoantibody levels during therapy and follow up. • However, valid assays currently available for the detection of agonistic β1AR autoantibodies are unfit for routine health care, as they are based on live cell models. • It poses a major challenge for future development to develop assays that (i) detect autoantibodies recognising a conformational epitope so far only found properly represented in the native β1AR, (ii) discriminate autoantibodies that bind and stimulate the β1AR from autoantibodies that bind the β1AR without affecting its function, and (iii) accomplish the requirements of standardisation and reliability of clinical diagnostic testing. Acknowledgements FB receives public funding from/by the Deutsche Forschungsgemeinschaft (DFG), SFB 612, and the Bundesministerium für Wirtschaft und Energie (BMWi), Zentrales Innovationsprogramm Mittelstand (ZIM) (Grant nos. KF2728001MD0 and KF2728002FR2). RJ receives public funding from the German Ministry for Education and Research (Bundesministerium für Bildung und Forschung, ETiCSstudy, BMBF Project number 01ES0816). The ETiCS study has been acknowledged by the German Competence Network Heart Failure (CNHF, Subproject 6b), and has equally been associated to the BMBF-funded Comprehensive Heart Failure Centre, University Hospital of Würzburg (CHFC, Subproject C4). DR receives public funding from the Bundesministerium für Wirtschaft und Energie (BMWi), Zentrales Innovationsprogramm Mittelstand (ZIM) 2087804FR2. All authors had full access to the data and have read and approved the final article. References [1] Lytton SD, Kahaly GJ. Bioassays for TSH-receptor autoantibodies: an update. Autoimmun Rev 2010;10:116–22. [2] Zophel K, Roggenbuck D, Schott M. Clinical review about TRAb assay's history. Autoimmun Rev 2010;9:695–700. [3] Wallukat G, Schimke I. Agonistic autoantibodies directed against G-protein-coupled receptors and their relationship to cardiovascular diseases. Semin Immunopathol 2014;36:351–63. [4] Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, et al. Contemporary definitions and classification of the cardiomyopathies: an American Heart Association Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation 2006;113:1807–16. [5] Jahns R, Boivin V, Lohse MJ. Beta(1)-adrenergic receptor function, autoimmunity, and pathogenesis of dilated cardiomyopathy. Trends Cardiovasc Med 2006;16:20–4. [6] Stork S, Boivin V, Horf R, Hein L, Lohse MJ, Angermann CE, et al. Stimulating autoantibodies directed against the cardiac beta1-adrenergic receptor predict increased mortality in idiopathic cardiomyopathy. Am Heart J 2006;152:697–704. [7] Dandel M, Wallukat G, Potapov E, Hetzer R. Role of beta(1)-adrenoceptor autoantibodies in the pathogenesis of dilated cardiomyopathy. Immunobiology 2011;217: 511–20. [8] Jahns R, Boivin V, Siegmund C, Inselmann G, Lohse MJ, Boege F. Autoantibodies activating human beta1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure. Circulation 1999;99:649–54. [9] Buvall L, Bollano E, Chen J, Shultze W, Fu M. Phenotype of early cardiomyopathic changes induced by active immunization of rats with a synthetic peptide corresponding to the second extracellular loop of the human beta-adrenergic receptor. Clin Exp Immunol 2006;143:209–15. [10] Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G, et al. Direct evidence for a beta(1)-adrenergic receptor-directed autoimmune attack as a cause of idiopathic di-

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Associated immunological disorders and cellular immune dysfunction in thymoma: a study of 87 cases from Thailand Several immune disorders are often associated with thymoma. The aim of this study was to analyze the correlation between clinicopathological features of Thai patients with thymoma and concomitant immune-mediated diseases. Thus, Thongprayoon C, et al. (Arch Immunol Ther Exp (warsz) 2013;61:85-93) reviewed retrospectively the medical records of 87 patients diagnosed with thymoma during a 10-year period. Peripheral blood T cell subsets along with cytokine responses in 15 thymoma patients and 15 healthy controls were comparatively analyzed. The results demonstrated that thymoma type AB and B2 were the most common types among patients diagnosed with thymoma. The most common presentation was incidentaloma, followed by local chest symptoms and autoimmune diseases. The prevalence of autoimmune diseases, immunodeficiency states, and secondary neoplasms was 34.5, 10.3, and 10.3 %, respectively. Autoimmune diseases were most frequently found in thymoma type B2 and sometimes associated with clinical immunodeficiency, although classic Good's syndrome was rare. Patients with thymoma had significantly lower percentage CD4(+ve) T cells and interferon γ response, but higher percentage regulatory T cells than those in healthy controls. This study indicated that the aberrant immunologic disorders comprising autoimmune diseases, immunodeficiency states, and secondary neoplasms were found in almost 40 % of Thai patients with thymoma and possibly related to defective cytokine responses and altered T cell subsets.