Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease‐associated protein chip

Abstract

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37 200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.

Probing the mRNA processing body using protein macroarrays and "autoantigenomics"

“…The bacteria are robotically transferred, at high density, to nitrocellulose membranes and are induced to express the cDNA in situ. This technique was used by other investigators to identify the targets of autoantibodies in patients with cardiac and dermatologic disorders (Lueking et al 2005;Horn et al 2006). In the present study, we used serum from patients with primary biliary cirrhosis and protein macroarray technology to identify potential P-body autoantigens and show that Y-box protein 1 (YB-1) is a novel component of these structures.…”

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays

“…Our laboratory has developed protein array based technologies and methodologies since first developing the hEx1 protein expression library [18] with which we have performed studies on the binding of autoantibodies to arrayed proteins in Alopecia areata and Dilated Cardiomyopathy [19,20] , binding of antibodies to proteins identified from tumour neovasculature in humans [21],context independent motif identification in the human proteome [22] and of identification of novel proteinprotein interaction networks [23,24]. We have employed this library screening method as part of an ovarian cancer research consortium, Discovary, performing a pilot study on autoantibody identification screening the hEx1 protein library with ovarian cancer serum samples from a well characterised patient cohort with stage I ovarian cancer of mixed histology, stage III serous papillary adenocarcinoma, primary peritoneal carcinoma and normal/healthy individuals.…”

Proteomics of the Heart

“…Several of the autoantobodies identified in the plasma of DCM patients are known to be associated with heart failure. 60 It is therefore clear that until these alternative approaches mature into robust techniques for quantitative protein expression profiling, 2-DE will remain the separation workhorse in many proteomic investigations. This technique has the capacity to support the simultaneous analysis of the changes in expression of hundreds to thousands of proteins, and, as such, it remains unrivaled as an "open"' protein expression profiling approach.…”

“…149 As discussed previously, using human protein arrays, the autoantibody repertoire of plasma from DCM patients has been profiled and novel DCM specific autoantigens identified. 60 Cardiac antigens that are associated with antibody responses after cardiac transplantation have also been characterized and may be involved in acute 150 and chronic 151 rejection. Recently, we used a proteomic approach to identify whether specific protective proteins are expressed in the hearts of long-term transplant patients who remain free of chronic rejection.…”