Individuals with inherited chromosomally integrated human herpes virus 6 (ciHHV-6) have functionally active HHV-6 specific T-cell immunity.

To evaluate the human herpes virus 6 (HHV-6) -specific immune response in individuals with chromosomally integrated HHV-6 (ciHHV-6), we measured HHV-6-antigen-specific cytokine responses (interferon-γ, interleukin-2, tumour necrosis factor-α) in T cells by flow cytometry in 12 and 16 individuals with and without ciHHV-6, respectively. All individuals with ciHHV-6 showed HHV-6-specific T cells with higher frequencies of HHV-6-specific CD8(+) cells (0.03-14.93, median 2.15% of CD8(+) cells) compared with non-ciHHV-6 (0.0-10.67, median 0.36%, p 0.026). The observed increased HHV-6-specific functionally active responses in individuals with ciHHV-6 clearly disprove speculations on immune tolerance in ciHHV-6 and indicate clinical and immunological implications of ciHHV-6.

Aggravation of left ventricular dysfunction in patients with biopsy-proven cardiac human herpesvirus A and B infection.

BACKGROUND: Human herpesvirus 6 (HHV-6) A and B are lymphotropic viruses with life-long persistence, primarily associated with non-cardiac diseases, and discussed as a possible etiologic factor of myocarditis and cardiomyopathy. OBJECTIVE: To analyze the long-term spontaneous course of cardiac patients suffering from suspected inflammatory cardiomyopathy (CMi) with persisting HHV-6 A and B infections by follow-up biopsies. STUDY DESIGN: We prospectively evaluated patients (n=73) with biopsy-proven viral HHV-6 A and B infection in endomyocardial biopsies (EMBs), followed up by reanalysis of EMBs and left ventricular ejection fraction (LV-EF) measurements after a median period of 8.8 months (range 4-73 months). Beyond, we studied HHV-6 prevalence in isolated peripheral blood cells (PBCs) and HHV-6 species in EMBs. HHV-6 species-specific cellular infection sites within the myocardium were identified by immunohistochemistry (IHC). RESULTS: We identified 73 patients with cardiac HHV-6 A and B persistence or newly detected in follow-up EMB (95.0% B). Proof of HHV-6 in PBCs was primarily associated with A. Persistence of cardiac HHV-6 B genome was significantly associated with cardiac dysfunction at follow-up (LV-EF deteriorated from 58.2±16.0 to 51.8±17.2%, p<0.001), and LV improvement was observed when HHV-6 B persistence resolved (LV-EF increased from 54.9±15.4 to 60.7±13.1%, p<0.001). CONCLUSIONS: Persistence of cardiac HHV-6 B genomes was significantly associated with cardiac dysfunction, and hemodynamic parameters improved in association with HHV-6 B clearance.

Analyses of germline, chromosomally integrated human herpesvirus 6A and B genomes indicate emergent infection and new inflammatory mediators.

Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.

Improved diagnosis of idiopathic giant cell myocarditis and cardiac sarcoidosis by myocardial gene expression profiling.

AIMS: Improvement of clinical diagnostics of idiopathic giant cell myocarditis (IGCM) and cardiac sarcoidosis (CS), two frequently fatal human myocardial diseases. Currently, IGCM and CS are diagnosed based on differential patterns of inflammatory cell infiltration and non-caseating granulomas in histological sections of endomyocardial biopsies (EMBs), after heart explantation or postmortem. We report on a method for improved differential diagnosis by myocardial gene expression profiling in EMBs. METHODS AND RESULTS: We examined gene expression profiles in EMBs from 10 patients with histopathologically proven IGCM, 10 with CS, 18 with active myocarditis (MCA), and 80 inflammation-free control subjects by quantitative RT-QPCR. We identified distinct differential profiles that allowed a clear discrimination of tissues harbouring giant cells (IGCS, CS) from those with MCA or inflammation-free controls. The expression levels of genes coding for cytokines or chemokines (CCL20, IFNB1, IL6, IL17D; P < 0.05), cellular receptors (ADIPOR2, CCR5, CCR6, TLR4, TLR8; P < 0.05), and proteins involved in the mitochondrial energy metabolism (CPT1, CYB, DHODH; P < 0.05) were deregulated in 2- to 300-fold, respectively. Bioinformatic analyses and correlation of the gene expression data with immunohistochemical findings provided novel information regarding the differential cellular and molecular pathomechanisms in IGCM, CS, and MCA. CONCLUSION: Myocardial gene expression profiling is a reliable method to predict the presence of multinuclear giant cells in the myocardium, even without a direct histological proof, in single small EMB sections, and thus to reduce the risk of sampling errors. This profiling also facilitates the discrimination between IGCM and CS, as two different clinical entities that require immediate and tailored differential therapy.

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

miRNA as activity markers in Parvo B19 associated heart disease.

Parvovirus B19 is a frequent virus detected in endomyocardial biopsies of patients with clinically suspected myocarditis or dilated cardiomyopathy (DCM). Viruses often cause a more symptomatic disease with increased tissue injury if they become reactivated. A disease-specific differential expression of microRNAs (miRNAs) has been described in the regulation of replicating viruses. Analyzing patients with latent and reactivated B19V infection, we found 29 differentially regulated miRNAs and, in order to test whether predicted genes are differentially expressed, selected mRNAs were tested by TaqMan-QPCR.

[Quality analysis for treatment of heart muscle diseases]. / Qualitätsanalysen der Therapie von Herzmuskelerkrankungen.

Distinct examinations and resulting therapies are depending on the cardiomyopathy phenotype and the severity of the myocardial compromise. Specific treatment modalities for distinct cardiomyopathies, demand detailed diagnostic procedures in addition to the routine diagnostic workup. Quality analyses for causal treatment strategies of different cardiomyopathies are missing. Most diagnostic procedures are unable to detect the relevant pathophysiological changes that generally occur at the cellular and molecular level of myocardial tissue or individual cells. Therefore, often only additional biopsy-based tissue analyses can provide the necessary basis for causal treatment options.

The German Transregional Collaborative Research Centre 'Inflammatory Cardiomyopathy--Molecular Pathogenesis and Therapy'. Methods and baseline results from a 3-centre clinical study.

OBJECTIVES: This report focuses on the design and methods of the 3-centre clinical study of the Transregional Collaborative Research Centre 'Inflammatory Cardiomyopathy--Molecular Pathogenesis and Therapy', which aims to establish a comprehensive research registry on the diagnostics, therapy and disease outcomes of patients with inflammatory cardiomyopathy (CMi). The study goals are to investigate specific disease sub-entities and to develop standardised strategies for diagnostics and treatment. METHODS: All consecutive patients with clinically suspected CMi, post-myocarditic cardiomyopathy and acute myocarditis are included in the research registry. Cardiopulmonary functional tests, clinical and patient data are obtained at baseline and subsequent readmission appointments and are linked to allow for prospective follow-up. Co-morbidities, quality of life, health- related behaviour and sociodemographic variables are ascertained using uniform self-administered questionnaires. PRESENT STATUS: By May 2008, 2,061 cases had been included in the research registry (1,300 data-sets completed). At registration, 335 patients were diagnosed with CMi. The mean age was 50 +/- 13 years and the mean ejection fraction was 39.9 +/- 15.8%. CONCLUSIONS: The broad range of the acquired molecular-biological, histological, immunohistological, clinical and patient data makes this the most comprehensive research registry on patients with CMi to date.

Application of an asymmetric helical tube reactor for fast identification of gene transcripts of pathogenic viruses by micro flow-through PCR.

We have established a fast PCR-based micro flow-through process consisting of a helical constructed tube reactor. By this approach we can detect transcripts of measles and human papilloma virus (HPV) by continuous flow allowing for reverse transcription (RT) and amplification of cDNA. The micro reaction system consisted of two columnar reactors for thermostating the different reaction zones of the RT process and the amplification. The PCR reactor was built by asymmetric heating sections thus realizing different residence times and optimal conditions for denaturation, annealing and elongation. The system concept is based on low electrical power consumption (50-120 W) and is suited for portable diagnostic applications. The samples were applied in form of micro fluidic segments with single volumes between 65 and 130 nL injected into an inert carrier liquid inside a Teflon FEP tube with an inner diameter of 0.5 mm. Optimal amplification for template lengths of 292 bp (lambda-DNA), 127 bp (measles virus) and 95 bp (HPV) was achieved by maximal cycle times of 75 s.

Prevalence of erythrovirus genotypes in the myocardium of patients with dilated cardiomyopathy.

Parvovirus B19 (PVB19) is a member of the human erythrovirus family detected frequently in endomyocardial biopsies from patients with dilated cardiomyopathy. Human erythroviruses cluster into three genotypes 1-3 which share a high degree of homology between major structural proteins and may cause indistinguishable infections clinically and serologically. In human cardiac tissue erythrovirus genotypes other than PVB19 have not yet been reported. Three hundred seventeen consecutive patients with symptomatic dilated cardiomyopathy (median left ventricular ejection fraction: 28.6%, range 5-45%) who underwent endomyocardial biopsy for the elucidation of the etiology, were analyzed using a new consensus PCR assay designed for the detection of the three erythrovirus genotype sequences. Endomyocardial biopsies of 151 (47.6%) patients were erythrovirus-positive. Genotype 1 specific sequences were detected in 43/151 (28.5%) of positive biopsy samples, whereas genotype 2-specific sequences so far considered rare in human disease and not yet been described in human heart tissue was identified in 108/151 (71.5%) of virus-positive endomyocardial biopsies with a preference in patients above 50 years of age. In spite of younger age, systolic left ventricular dysfunction of genotype 1-positive patients was significantly reduced as compared to genotype 2-positive patients (24.4+/-10.4% vs. 31.0+/-9.5%, P=0.0001) at the initial presentation. The data show that two genetically distinct erythrovirus variants with a different age distribution are detectable in endomyocardial biopsies of patients with dilated cardiomyopathy. The erythrovirus genotype 2, not described previously in human heart tissue, is highly prevalent in the heart but the less prevalent genotype 1 is associated with more severe disturbed cardiac function.

Genomic expression profiling of human inflammatory cardiomyopathy (DCMi) suggests novel therapeutic targets.

The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage.

Inflammation, ECG changes and pericardial effusion: whom to biopsy in suspected myocarditis?

The role of endomyocardial biopsies in patients with clinically suspected acute myocarditis, myocarditis in the past, and dilated cardiomyopathy is discussed controversially. In fact, it is still under discussion whether information obtained from endomyocardial biopsies is relevant for further clinical decisions. Therefore this Critical Perspective will deal with the question, which patient should undergo endomyocardial biopsy investigations for an etiopathogenic differentiation of the disease and for the possible choice of immunomodulatory treatment strategies.

[Cardiomyopathies. I: classification of cardiomyopathies--dilated cardiomyopathy]. / Kardiomyopathien. Teil I: Klassifikation der Kardiomyopathien -- dilatative Kardiomyopathie.

Cardiomyopathies are common causes of heart failure and sudden cardiac death. According to the WHO classification, "specific" cardiomyopathies are differentiated from "idiopathic" cardiomyopathies. Thus, this classification is primarily based on pathophysiological characteristics. The diagnostic spectrum in cardiomyopathies comprises the entire spectrum of non-invasive and invasive cardiological examination techniques. The exact verification of certain cardiomyopathies necessitates additionally investigations. For example, immunohistological and molecular biological investigations of endomyocardial biopsies may confirm inflammatory cardiomyopathy, which is often induced by viruses. Several studies have shown that specific immunomodulatory treatment options can halt the progressive course of the disease. Several gene mutations have been identified in genetic/familial dilated cardiomyopathy. First-degree relatives should be screened for early stages. Primary prevention of sudden cardiac death shows increasing superiority of the implantable defibrillator compared with pharmacological approaches (i.e. amiodarone).

[Cardiomyopathies II. Hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy]. / Kardiomyopathien Teil II: Hypertrophe Kardiomyopathie, restriktive Kardiomyopathie, arrhythmogene rechtsventrikuläre Kardiomyopathie.

This review focuses on hypertrophic (HCM), restrictive (RCM) and arrhythmogenic right ventricular cardiomyopathies (ARVC). The clinical phenotype of HCM depends not only on the gene mutations involved, but also on "modifier genes". It is characterized by an asymmetrical hypertrophy. Investigations of endomyocardial biopsies (EMBs) typically reveal a disarray of the hypertrophied cardiomyocytes. Percutaneous septum ablation has gained relevance as the treatment of choice in hypertrophic obstructive cardiomyopathy. Myocardial and endomyocardial RCM-forms can be differentiated. Enlargement of the atria in concert with normal dimensions of the ventricles and almost normal systolic contractility as well as the dip-plateau phenomenon are characteristic findings in RCM. EMB diagnostics are pivotal to identify the causes underlying secondary RCM types. Treatment is directed at heart failure and specifically at the underlying disease. With ARVC, apoptosis, viral infection/inflammation and genetic dystrophy result in fibrofatty degeneration primarily of the right, and with further progression also of the left ventricle. The primary treatment goal in ARVC is prevention of sudden cardiac death. As for other cardiomyopathies, there is increasing evidence for the superiority of ICD compared with pharmacological approaches.

Diagnosis and monitoring of acute cytomegalovirus infection in peripheral blood of transplant recipients by nested reverse transcriptase polymerase chain reaction (RT-PCR).

The aim of this novel diagnostic approach is to monitor cytomegalovirus (CMV) infection in immunocompromised transplant recipients using early, sensitive, and specific predictors before and during antiviral therapy. The peripheral blood cells of 20 patients after transplantation (9 liver, 7 kidney, 4 simultaneous kidney-pancreas) were studied for an early diagnosis of acute infection. The mRNA and DNA of human CMV immediate-early antigen (IEA) were detected by nested-polymerase chain reaction (PCR) assay. Results of nested PCR were compared with the immunological detection of antigen pp65 and serological diagnosis of CMV infection. All data were correlated with clinical symptoms like leukopenia, thrombopenia, pneumonia, and allograft-rejection reaction. Of 20 transplant recipients, 12 were infected by CMV, and 9 suffered from a CMV-related disease. CMV mRNA were detected simultaneously with antigen pp65 and CMV DNA in all patients with symptomatic infection. Additionally, CMV mRNA was found over a longer period after ganciclovir treatment of infected recipients. Nested reverse transcriptase (RT)-PCR for CMV-IEA mRNA allows a sensitive and specific diagnosis of an acute CMV infection. CMV mRNA was found to be a good marker of acute viremia and could be a useful tool for CMV monitoring over the whole period of disease management, even during antiviral therapy.