Uwe Kuehl

Enteroviral RNA Replication in the Myocardium of Patients With Left Ventricular Dysfunction and Clinically Suspected Myocarditis

BACKGROUND Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome. METHODS AND RESULTS We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand-positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26). CONCLUSIONS These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy.

Chromosomally integrated human herpesvirus 6: questions and answers

Chromosomally integrated human herpesvirus 6 (ciHHV‐6) is a condition in which the complete HHV‐6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV‐6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV‐6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV‐6 due to cell lysis and release of cellular DNA. High HHV‐6 DNA loads associated with ciHHV‐6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV‐6 may be at increased risk for bacterial infection and graft rejection. ciHHV‐6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV‐6 individuals are put at clinical risk by the use of drugs that have been associated with HHV‐6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV‐6. Little is known about whether individuals with ciHHV‐6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV‐6 in disease. Copyright

Endothelium-Dependent Flow-Mediated Vasodilation of Systemic Arteries Is Impaired in Patients With Myocardial Virus Persistence

Background—Myocardial virus persistence is frequently observed in patients with cardiomyopathy. Endothelial dysfunction in patients with cardiomyopathy is associated with inflammatory immunoresponses in myocardial biopsies. The aim of this study was to investigate the impact of myocardial virus persistence on endothelial function. Methods and Results—In 124 patients with suspected cardiomyopathy, myocardial biopsies were examined for virus persistence (by polymerase chain reaction) and inflammation (by immunohistology). Endothelial function of the radial artery was examined by high-resolution ultrasound. Diameter changes in response to reactive hyperemia (flow-mediated dilation [FMD]) compared with glycerol trinitrate (GTN-MD) were measured. Mean age of the patients (55 men, 69 women) was 45±13 years; ejection fraction was 57±17%. In 73 patients, adenovirus, enterovirus, parvovirus, or HHV6 virus (V) was detected; in 51, no virus was detected. FMD was significantly impaired in patients with myocardial virus persistence compared with control subjects (Co): FMD-V, 3.38±2.67%; FMD-Co, 7.34±3.44 (P<0.001). In 86 patients, myocardial inflammation was confirmed (Inf). Of those, 57 had virus, and 29 did not. FMD was significantly impaired in patients with virus compared with controls: FMD-Inf-V, 3.24±2.66%; FMD-Inf-Co, 6.07±3.00 (P<0.001). In 38 patients, immunohistology of the myocardial biopsies was normal (Co); of those, 16 had virus, and 22 did not. FMD was impaired in patients with virus compared with control subjects: FMD-Co-V, 3.88±2.72%; FMD-Co-Co, 9.00±3.32% (P<0.001). Endothelium-independent vasodilation (GTN-MD) was not significantly affected. Conclusions—Myocardial virus persistence is associated with endothelial dysfunction. Endothelial dysfunction in patients with myocardial virus persistence can occur independently of endothelial activation or myocardial inflammation but is more pronounced in patients with concurrent inflammation.

Analysis of HER2 and HER4 in human myocardium to clarify the cardiotoxicity of trastuzumab (Herceptin).

AbstractPurpose. When combined with anthracyclines, the humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin™) provides significant clinical benefit for women with HER2-overexpressing metastatic breast cancer. However, its use is limited by severe cardiotoxicity. To clarify whether myocardial HER2 and HER4 expression in response to anthracycline exposure and cardiac damage contributes to cardiotoxicity, we assessed expression of HER2 and HER4 in pathologically altered myocardium. Experimental design. Cardiac biopsies from 60 patients with severe heart disease and cardiac tissue from 35 patients with breast cancer were obtained. Twenty-five of the patients with breast cancer had previously received anthracyclines. Three of 10 anthracycline-naïve patients with breast cancer had received trastuzumab. Expression of HER2 and HER4 was analyzed immunohistochemically (HER2: HercepTest™/A0485 (Dako), Cy3 detection (Dianova); HER4: Ab-4 (NeoMarkers)). FISH analysis (Ventana) was used to assess HER2 gene amplification. Results. Immunohistochemistry revealed weak HER2 membrane staining in six cardiac biopsies, appearing as dotted staining of the whole cell membrane and intensified HER2 signal using fluorescent Cy3 labeling. No HER2 membrane staining was detected in the remaining 54 cardiac biopsies or in the myocardium of the 35 patients with breast cancer. HER2 gene amplification was not observed. All specimens showed the mild cytoplasmatic HER4 staining of normal myocardium. No strong HER4 expression was detected. Conclusions. Cardiac alterations are not associated with an strong increase in HER2 and HER4 levels. IHC detects potential low-level HER2 expression in some samples. However, a more sensitive technique may be needed for studies of the role of HER2 in cardiac tissue. These data do not exclude a role for inhibition of cardiac HER2 expression by trastuzumab in the onset of heart failure in trastuzumab-treated patients.

Adiponectin expression in patients with inflammatory cardiomyopathy indicates favourable outcome and inflammation control

AIMS Circulating adiponectin (APN) is an immunomodulatory, pro-angiogenic, and anti-apoptotic adipocytokine protecting against acute viral heart disease and preventing pathological remodelling after cardiac injury. The purpose of this study was to describe the regulation and effects of APN in patients with inflammatory cardiomyopathy (DCMi). METHODS AND RESULTS Adiponectin expression and outcome were assessed in 173 patients with DCMi, 30 patients with non-inflammatory DCM, and 30 controls. Mechanistic background of these findings was addressed in murine experimental autoimmune myocarditis (EAM), a model of human DCMi, and further elucidated in vitro. Adiponectin plasma concentrations were significantly higher in DCMi compared with DCM or controls, i.e. 6.8 ± 3.9 µg/mL vs. 5.4 ± 3.6 vs. 4.76 ± 2.5 µg/mL (P< 0.05, respectively) and correlated significantly with cardiac mononuclear infiltrates (CD3+: r(2)= 0.025, P= 0.038; CD45R0+: r(2)= 0.058, P= 0.018). At follow-up, DCMi patients with high APN levels showed significantly increased left ventricular ejection fraction improvement, decreased left ventricular end-diastolic diameter, and reduced cardiac inflammatory infiltrates compared with patients with low APN levels. A multivariate linear regression analysis implicated APN as an independent prognostic factor for inhibition of cardiac inflammation. In accordance with these findings in human DCMi, EAM mice exhibited elevated plasma APN. Adiponectin gene transfer led to significant downregulation of key inflammatory mediators promoting disease. Mechanistically, APN acted as a negative regulator of T cells by reducing antigen specific expansion (P< 0.01) and suppressed TNFα-mediated NFκB activation (P< 0.01) as well as release of reactive oxygen species in cardiomyocytes. CONCLUSION Our results implicate that APN acts as endogenously upregulated anti-inflammatory cytokine confining cardiac inflammation and progression in DCMi.

Adaptive immune responses against parvovirus B19 in patients with myocardial disease

BACKGROUND Parvovirus B19 (B19V)-DNA is frequently detected in endomyocardial biopsies (EMBs) from patients with acute myocarditis (AMC) and dilated cardiomyopathy (DCM), but also in various healthy tissues. The clinical relevance of this DNA-persistence is unclear. OBJECTIVES To investigate potential pathogenic influences of B19V-DNA in EMBs, we analyzed B19V-specific adaptive immune responses in AMC/DCM patients and healthy controls. STUDY DESIGN 15 AMC/DCM patients with detectable B19V-DNA in EMBs and 51 controls were analyzed for signs of acute B19V-infections and virus-specific immune responses by PCR, ELISA, Western line, and ELISpot-assays. RESULTS Productive B19V-infection was determined in three patients. Slightly lower levels of B19V-specific T-cells were observed in patients as compared to the controls, no differences were observed in virus-specific serology. Viral DNA-load in EMBs could not be correlated to the number of B19V-specific T-cells. No differences in T-cell response, viremia and/or serological markers indicative for viral pathogenesis were observed in patients with inflammatory cardiomyopathy. CONCLUSIONS Discrepancies in B19V-specific adaptive immunity were not observed in AMC/DCM patients as compared to controls. The data indicate that the exclusive detection of B19V-DNA in EMBs is not sufficient to associate B19V with AMC/DCM but should be complemented with additional virological and immunological parameters in further studies.

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

SummaryThe 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.

Enteroviral and immune mediated myocarditis in SCID mice.

Severe combined immune deficiency (SCID) mice have been used as an animal model to study both the direct cytopathic effect of enteroviruses on the heart in the absence of an effective immune system and to investigate the role of immune mediated processes in the pathogenesis of human myocarditis.The infection of SCID mice with coxsackievirus B3 resulted in severe myocarditis with very high titers of the virus in the myocardium and severe necrosis of myocytes. This direct cytopathic effect caused an impairment of the myocardial function and resulted in a high mortality rate of the infected animals.For the study of the immune mechanisms in human myocarditis, peripheral blood leukocytes of patients with myocarditis, having an impaired left ventricular function without viral persistence in the myocardium, were transferred into SCID mice. As controls peripherals blood leukocytes of normal donors were used. At 60 days after transfer, human immunoglobulines could be demonstrated in the peripheral blood of the SCID mice, however, human autoantibodies against the adenine nucleotide translocator, a myocardial autoantigen, were only present in the animals receiving peripheral blood leukocytes from patients with myocarditis. Cellular infiltrates of human leukocytes in the myocardium and an impaired left ventricular function were also only observed in animals reconstituted with peripheral blood leukocytes from patients. These effects were T cell dependent as shown by differential transfer.These results are of interest for the treatment of human myocarditis, suggesting the avoidance of an immunosuppressive therapy in acute or chronic myocarditis with viral persistence to prevent a direct cytophatic effect in the absence of an effective immune system. However, in the setting of a chronic, (auto-)immunological myocarditis with the proven absence of entero- or adneoviral sequences an immunomodulatory therapy seems to be effective and safe.ZusammenfassungDie Bedeutung einer enteroviralen Infektion sowie einer resultierenden Immunreaktion ist immer noch Gegenstand der Forschung in der Pathogenese der humanen Myokarditis. Mäuse mit einer kombinierten Immundefizienz (“severe combined immundeficiency”, SCID-Mäuse), die keinen eigenen funktionstüchtigen peripheren B- und T-Lymphozyten besitzen, wurden im Tiermodell verwendet, um den direkten zytopathischen Effekt von Enteroviren auf Herzmuskelzellen in der Abwesenheit eines effektiven Immunsystems zu untersuchen. Außerdem kann dieses Tiermodell verwendet werden, um die Rolle von immunologischen und autoimmunologischen Prozessen in der Pathogenese der humanen Myokarditis zu analysieren.Die Infektion von SCID-Mäusen mit Coxsackie-B3-Viren führt zu einer ausgeprägten Myokarditis bei sehr hohen Virustitern im Myokardgewebe und schweren Myokardzellnekrosen. Dieser direkte zytopathische Effekt verursacht eine Einschränkung der myokardialen Pumpfunktion und führte zu einer hohen Mortalitätsrate in den infizierten Tieren, während immunkompetente Mäuse praktisch keine Mortalität aufweisen.Zur Untersuchung der Immunmechanismen bei der humanen Myokarditis wurden periphere Blutleukozyten von Patienten mit Myokarditis, die eine eingeschränkte linksventrikuläre Funktion, jedoch keine Viruspersistenz im Myokard aufwiesen, auf SCID-Mäuse übertragen. Als Kontrollen dienten die peripheren Blutleukozyten von gesunden Personen. 60 Tage nach dem Transfer fanden sich humane Immungloboline im peripheren Blut der SCID-Mäuse. Humane Autoantikörper gegen den Adenin-Nukleotid-Translokator, ein myokardiales Autoantigen, fanden sich hingegen nur bei den Tieren, die periphere Blutleukozyten von Patienten mit Myokarditis erhalten hatten. Zelluläre Infiltrate von humanen Leukozyten im Myokard und eine Einschränkung der linksventrikulären Funktion (verminderte Druckanstiegsgeschwindigkeit) lagen ebenso nur bei den SCID-Mäusen vor, die periphere Blutleukozyten von Patienten mit Myokarditis erhalten hatten. Die Tiere wiesen jedoch keinen Anhalt für eine Graft-versus-Host-Reaktion auf, wie die Untersuchung von verschiedenen anderen Geweben ergab. Der Transfer der humanen Myokarditis in SCID-Mäusen ist ein T-Zell-abhängiger Prozess, wie ein entsprechender differenzieller Transfer von nur CD4-positiven oder CD4-depletierten peripheren Blutleukozyten zeigte.Diese Untersuchungen dürften für die Therapie der humanen Myokarditis von Bedeutung sein. Diese Experimente legen nahe, dass im Rahmen der Myokarditis des Menschen vor allem im Stadium der akuten Erkrankung auf eine immunsuppressive Therapie verzichtet werden sollte, um eine Schwächung des Immunsystems und damit eine erhöhte Virämie und somit auch eine Verstärkung des direkten zytopathischen Effektes auf die Herzmuskelzellen zu verhindern. Aber auch im chronischen Stadium der Erkrankung sollte eine Viruspersistenz durch Entnahme von Myokardbiopsien mit entsprechender molekularbiologischer Analyse (Polymerasekettenreaktion und/oder In-situ-Hybridisierung für Entero- bzw. Adenoviren) untersucht werden, bevor eine immunsuppressive Therapie erwogen wird. In diesem Stadium der Erkrankung sollte bei nachgewiesener Viruspersistenz vielmehr möglicherweise eine Therapie mit subkutanen Gaben von Interferon initiiert werden, wie Untersuchungen unserer eigenen Arbeitsgruppe zwischenzeitlich nahelegen.Zum anderen sprechen die Befunde dieses Tiermodells auch dafür, dass es sich bei der Myokarditis des Menschen im chronischen Stadium der Erkrankung bei Ausschluss einer Viruspersistenz um einen chronischen autoimmunologischen Prozess handelt, der einer immunmodulatorischen Therapie zugeführt werden sollte. Nur durch eine differenzierte histologische, immunhistologische und molekularbiologische Diagnostik ist es jedoch möglich, eine Differenzierung des akuten virologischen Stadiums der Erkrankung von einem chronischen, immunologisch bedingten Stadium vorzunehmen und dementsprechend eine sinnvolle kausale Therapie durchzuführen.

Differential Cardiac MicroRNA Expression Predicts the Clinical Course in Human Enterovirus Cardiomyopathy

Background—Investigation of disease pathogenesis confined to protein-coding regions of the genome may be incomplete because many noncoding variants are associated with disease. We aimed to identify novel predictive markers for the course of enterovirus (CVB3) cardiomyopathy by screening for noncoding elements influencing the grossly different antiviral capacity of individual patients. Methods and Results—Transcriptome mapping of CVB3 cardiomyopathy patients revealed distinctive cardiac microRNA (miR) patterns associated with spontaneous virus clearance and recovery (CVB3-ELIM) versus virus persistence and progressive clinical deterioration (CVB3-PERS). Profiling of protein-coding genes and 754 miRs in endomyocardial biopsies of test cohorts was performed at their initial presentation, and those spontaneously eliminating the virus were compared with those with virus persistence on follow-up. miR profiling revealed highly significant differences in cardiac levels of 16 miRs, but not of protein-coding genes. Evaluation of this primary distinctive miR pattern in validation cohorts, and multivariate receiver operating characteristic curve analysis, confirmed this pattern as highly predictive for disease course (area under the curve, 0.897±0.071; 95% confidence interval, 0.758–1.000). Eight miRs were strongly induced in CVB3-PERS (miRs 135b, 155, 190, 422a, 489, 590, 601, 1290), but undetectable in CVB3-ELIM or controls. They are predicted to target multiple immune response genes, and 2 of these were confirmed by antisense-mediated ablation of miRs 135b, 190, and 422a in the monocytic THP-1 cell line. Conclusions—An immediate clinical application of the data is cardiac miR profiling to assess the risk of virus persistence and progressive clinical deterioration in CVB3 cardiomyopathy. Patients at risk are eligible for immediate antiviral therapy to minimize irreversible cardiac damage.

The forkhead transcription factor Foxo3 negatively regulates natural killer cell function and viral clearance in myocarditis

Aims Foxo3 is a transcription factor involved in cell metabolism, survival, and inflammatory disease. However, mechanistic insight in Foxo3 effects is still limited. Here, we investigated the role of Foxo3 on natural killer (NK) cell responses and its effects in viral myocarditis. Methods and results Effects of Foxo3 on viral load and immune responses were investigated in a model of coxsackie virus B3 myocarditis in wild-type (WT) and Foxo3 deficient mice. Reduced immune cell infiltration, viral titres, and pro-inflammatory cytokines in cardiac tissue were observed in Foxo3-/- mice 7 days post-infection (p.i.). Viral titres were also attenuated in hearts of Foxo3-/- mice at Day 3 while interferon-γ (IFNγ) and NKp46 expression were up-regulated suggesting early viral control by enhanced NK cell activity. CD69 expression of NK cells, frequencies of CD11b+CD27+ effector NK cells and cytotoxicity of Foxo3-/- mice was enhanced compared to WT littermates. Moreover, microRNA-155 expression, essential in NK cell activation, was elevated in Foxo3-/- NK cells while its inhibition led to diminished IFNγ production. Healthy humans carrying the longevity-associated FOXO3 single nucleotide polymorphism (SNP) rs12212067 exhibited reduced IFNγ and cytotoxic degranulation of NK cells. Viral inflammatory cardiomyopathy (viral CMI) patients with this SNP showed a poorer outcome due to less efficient virus control. Conclusion Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.