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.

Betaferon in chronic viral cardiomyopathy (BICC) trial: Effects of interferon-ß treatment in patients with chronic viral cardiomyopathy.

BACKGROUND: Chronic viral infections of the heart are considered one antecedent event leading to progressive dysfunction of the myocardium, often with an impaired prognosis due to a virus- or immune-mediated myocardial injury. Symptomatic treatment does not influence the viral cause of heart failure, and the effect of antiviral treatment has not been determined, yet. METHODS AND RESULTS: In this phase II study 143 patients with symptoms of heart failure and biopsy-based confirmation of the enterovirus (EV), adenovirus, and/or parvovirus B19 genomes in their myocardial tissue were randomly assigned to double-blind treatment, and received either placebo (n = 48) or 4 × 10(6) (n = 49) and 8 × 10(6) IU (n = 46) interferon beta-1b (IFN-ß-1b) for 24 weeks, in addition to standard heart failure treatment. Patients with active myocarditis or other specific causes of heart failure were excluded. Compared to placebo, virus elimination and/or virus load reduction was higher in the IFN-ß-1b groups (odds ratio 2.33, p = 0.048), similarly in both interferon groups and both strata. IFN-ß-1b treatment was associated with favourable effects on NYHA functional class (p = 0.013 at follow-up week 12), improvement in quality of life (Minnesota Heart Failure score; p = 0.032 at follow-up week 24) and patient global assessment (follow-up week 12 to follow-up week 24; p = 0.039). The frequency of adverse cardiac events was not higher in the IFN-ß-1b groups compared to the placebo group. CONCLUSIONS: Immunomodulatory IFN-ß-1b treatment is a well-tolerated and safe treatment option, leading to effective virus clearance or reduction of the virus load in patients with chronic viral cardiomyopathy. Favourable clinical effects assess quality of life, NYHA functional class, and patient global assessment. ClinicalTrials.gov identifier: NCT001185250.

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.

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.

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.

Alterations in myocardial tissue factor expression and cellular localization in dilated cardiomyopathy.

OBJECTIVES: We investigated the myocardial localization and expression of tissue factor (TF) and alternatively spliced human tissue factor (asHTF) in patients with dilated cardiomyopathy (DCM). BACKGROUND: Tissue factor is expressed in cardiac muscle and may play a role in maintaining myocardial structure. METHODS: Myocardial biopsies were obtained from patients with a normal or mildly impaired ejection fraction (EF) (> or =50%) and moderate to severely reduced EF (<50%). Explanted DCM hearts were also examined. Myocardial TF expression level was assessed by real-time polymerase chain reaction, TF protein by enzyme-linked immunosorbent assay, and localization by immunohistochemistry. RESULTS: We report the identification of asHTF in the human myocardium: it was located in cardiomyocytes and endothelial cells. Quantification of myocardial TF messenger ribonucleic acid in DCM revealed a decrease in the TF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio (1.76 x 10(-1) +/- 6.08 x 10(-2) for EF > or =50% [n = 19] vs. 1.06 x 10(-1) +/- 5.26 x 10(-2) for EF <50% [n = 27]; p < 0.001) and asHTF/GAPDH ratio (13.91 x 10(-5) +/- 11.20 x 10(-5) for EF > or =50% vs. 7.17 x 10(-5) +/- 3.82 x 10(-5) for EF <50%; p = 0.014). Tissue factor isoform expression level was also decreased in explanted DCM hearts (p < 0.01; n = 12). Total TF protein was reduced by 26% in DCM (p < 0.05). The TF/GAPDH ratio correlated positively with the EF (r = 0.504, p < 0.0001). Immunohistochemistry showed TF localized to the sarcolemma and Z-bands of the cardiomyocytes in patients with normal EF, whereas TF was found in the cardiomyocytic cytosol around the nucleus in DCM. CONCLUSIONS: Tissue factor was down-regulated in the myocardium of DCM patients. The reduction in TF expression and change in localization may influence cell-to-cell contact stability and contractility, thereby contributing to cardiac dysfunction in DCM.

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

PURPOSE: 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.