Damaged myocytes as detected by the colocalization of DNA fragmentation and tissue transglutaminase and their prognostic significance in enterovirus-associated dilated cardiomyopathy.
ABSTRACT Apoptotic cardiac myocytes have been described in chronic heart failure, but no data exist on the relationship between these 'damaged' myocytes and myocardial detection of enterovirus RNA often associated with dilated cardiomyopathy (DCM).
In patients with idiopathic DCM, endomyocardial biopsy samples were studied for enteroviral RNA by one step reverse transcription-polymerase chain reaction (PCR) and a subsequent hybridization of the PCR product using a Southern blot technique. The endomyocardial biopsies were further investigated for markers of cell damage and apoptosis: DNA fragmentation and expression of tissue-transglutaminase (TTG) in the myocytes using the in-situ endlabelling method or an anti-TTG-staining, respectively. To assess the prognostic significance of these two markers the correlation between the percentage of myocytes positive both for DNA fragmentation and TTG (the index of damaged myocytes) and the hemodynamic course of the patients during a mean follow-up period of 15.9 +/- 6.2 months was investigated prospectively by echocardiography.
In 14 (45%) of the 31 patients with idiopathic DCM, enteroviral RNA was found in the endomyocardial biopsy samples, while 17 patients (55%) were enterovirus-negative. In enterovirus-positive patients, the index of 'damaged' myocytes was significantly lower (10.7 +/- 4.9% vs. 19.2 +/- 8.8%, P = 0.002) and the left ventricular ejection fraction (LVEF) improved significantly (P = 0.00017 vs. P = 0.13) during long-term follow-up. In addition, a weak negative correlation was seen between the index of damaged myocytes and the changes in LVEF in all patients during long-term follow-up (r = - 0.48, P = 0.004).
Our results favour the view that enterovirus-positive patients with DCM have less damaged myocytes and a better haemodynamic course than enterovirus-negative patients.
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ABSTRACT: The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected chi(2)=19.99, P<0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.Medical Microbiology and Immunology 05/2004; 193(2-3):109-14. · 3.55 Impact Factor
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ABSTRACT: For celiac disease (CD), screening a trend has recently emerged to measure tissue transglutaminase antibodies (tTGA) by immunoassays instead of the more laborious endomysial antibodies (EmA), as they recognize the same target, tissue transglutaminase (tTG). However, a high rate of false-positive results has been reported in some patient series with diseases known to be associated with CD. Moreover, tTG is a ubiquitous, multifunctional enzyme, overexpressed in experimental models of heart failure. Therefore, we assessed the specificity of tTGA assays in a large series of EmA-negative patients with end-stage heart failure. We studied 288 patients with end-stage heart failure and 60 blood donors. No subject had clinical evidence of CD or IgA deficiency, and all were EmA negative. Serum IgA and IgG tTGA were measured by means of commercial kits using as substrate, either guinea pig or recombinant human tTG. Blocking studies and Western blots were also performed using recombinant human tTG. All blood donor sera were IgA tTGA negative. IgA tTGA positivity was observed in 47.6% and 49.1% of patients with heart failure using, respectively, guinea pig tTG and recombinant human tTG as substrates. Preincubation of positive sera with recombinant human tTG resulted in 81% blocking of IgA tTGA in immunoassay. Western blot analysis confirmed the presence of antibodies against recombinant human tTG. IgA tTGA-positive sera were also IgG tTGA positive. IgA and IgG tTGA occur in a large number of EmA-negative patients with end-stage heart failure, and their presence is unlikely to be caused by concomitant CD.The American Journal of Gastroenterology 12/2002; 97(11):2850-4. · 7.55 Impact Factor
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ABSTRACT: Most individuals have viral infections at some point in their life, however, only few develop autoreactivity to cardiac myosin following infection suggesting a genetic predisposition. Population studies have shown that among all the genetic factors linked with autoimmune disease development, MHC class II genes are the most significant genetic factors. Experimental autoimmune myocarditis resembling human Dilated cardiomyopathy can be induced in susceptible mice by infection with coxsackie virus as well as immunization with purified foreign and murine cardiac specific a-myosin. We generated transgenic mice lacking endogenous class II molecules, HLA-DR3.Abo and HLA-DQ8.Abo transgenic mice in NOD and HLA-DQ8.Abo in B10 background, to study the role of MHC in spontaneous autoimmunity. The HLA molecules in these mice are expressed on cell surface and can positively select CD4+ T cells expressing various Vb T cell receptors. NOD.DQ8 female mice spontaneously developed myocarditis and dilated cardiomyopathy. Histopathology of heart revealed mononuclear infiltrate consisting of CD4 and Mac-1+ cells and myocyte necrosis. NOD.DQ8 mice showed cellular and humoral autoreactive response to self cardiac myosin.. Depletion of CD8 and CD4 + cells suggested that CD8 T cells may act as regulatory cells while CD4 cells are required as effector cells. NOD.DR3 and B10.DQ8 mice did not develop any cardiac pathology suggesting DQ8 is required for predisposition to the spontaneous autoreactivity while NOD background influences onset and progression of disease. Thus these mice provide powerful tools to understand the role of HLA class II molecules in predisposition and onset of human diseases and to develop immunotherapy.Journal of Autoimmunity 10/2009; 33(3-4):260-9. · 8.15 Impact Factor