RNA interference to treat enteroviral disease: current status and clinical perspectives.

Enteroviruses are common human pathogens involved in a wide spectrum of clinical outcomes ranging from mild or non-symptomatic illness to severe diseases with neurological and/or cardiac manifestation. Despite being responsible for significant morbidity and mortality especially in immunocompromised patients and infants, to date no effective vaccines or specific antiviral treatment modalities are available to prevent or treat non-polio enteroviral infections. The discovery of the endogenous RNA interference pathway as an innate defence mechanism conferring intracellular immunity against foreign genetic elements has provided exciting possibilities in the fight against so far intractable, enteroviral diseases. We and others have shown the encouraging potential of RNA interference to limit enteroviral infections, leading to significant suppression of viral replication and cytopathogenicity, in vitro as well as in vivo. Yet, considerable limitations concerning efficacy, stability, specificity as well as viral escape need to be addressed to translate the anti-enteroviral potential into a novel treatment modality.

Endocardial cushion defect in a patient with Crouzon syndrome carrying a mutation in the fibroblast growth factor receptor (FGFR)-2 gene.

Crouzon syndrome is an autosomal dominant disorder caused by mutation in the fibroblast growth factor receptor (FGFR)-2 gene. Recent findings from animal studies imply a critical role for FGFs in the regulation of cardiac development including cardiac cushion proliferation and valvulogenesis. We report on a 36-year-old woman, who required surgical closure for an atrial septal defect, a clinical feature that has not been previously reported in other patients with Crouzon syndrome. The findings suggest that cardiac investigations are warranted in patients with a diagnosis of Crouzon syndrome.

Cardiac involvement in Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a common form of muscular dystrophy frequently involving cardiac muscle, thus leading to dilated cardiomyopathy. Clinical outcome and prognosis is frequently determined by the involvement of the cardiac conduction system causing symptomatic bradyarrhythmias, as well as tachyarrhythmias and, if untreated, frequent sudden cardiac death. Typical features of the cardiac involvement of EDMD are presented, caused by a novel missense mutation in the splice receptor sequence of intron 6 of the LMNA gene on chromosome 1, encoding for the lamin A/C gene, consistent with the autosomal dominant form of EDMD.

Transcriptome analysis reveals a role of interferon-gamma in human neointima formation.

The most effective immediate cure for coronary stenosis is stent-supported angioplasty. Restenosis due to neointima proliferation represents a major limitation. We investigated the expression of 2435 genes in atherectomy specimens and blood cells of patients with restenosis, normal coronary artery specimens, and cultured human smooth muscle cells (SMCs). Of the 223 differentially expressed genes, 37 genes indicated activation of interferon-gamma (IFN-gamma) signaling in neointimal SMCs. In cultured SMCs, IFN-gamma inhibited apoptosis. Genetic disruption of IFN-gamma signaling in a mouse model of restenosis significantly reduced the vascular proliferative response. Our data suggest an important role of IFN-gamma in the control of neointima proliferation.

Cardioselective infection with coxsackievirus B3 requires intact type I interferon signaling: implications for mortality and early viral replication.

BACKGROUND: Interferons (IFNs) play an important role in antiviral defense and have therapeutic potential in coxsackievirus heart disease. However, little is known about the relative contributions of type I and type II IFN signaling in coxsackievirus B3 (CVB3) infection or their role in the cardioselective nature of CVB3 infection. METHODS AND RESULTS: Wild-type mice and mice deficient for either the type I or the type II IFN receptor (IFNR) were infected with CVB3. Infection of the type I IFNR-deficient mice with >10(3) plaque-forming units (pfu) of CVB3 resulted in 100% mortality within 2 to 4 days after infection. Death was rare in wild-type and type II IFNR-deficient mice after inoculation with as much as 10(8) pfu of CVB3. Surprisingly, the early mortality in the type I IFNR-deficient mice was not accompanied by higher virus titers in the heart. Unexpectedly, a dramatic increase of viral RNA in the liver was found to correlate with early mortality in type I IFNR-deficient mice. CONCLUSIONS: Type I but not type II IFN signaling is essential for the prevention of early death due to CVB3 infection. Interestingly, neither type I or type II IFN signaling has a dramatic effect on early viral replication in the heart. However, lethal viral replication in the liver is controlled by type I IFNs. These results demonstrate that the IFN system is capable of modulating both viral pathogenicity and tissue tropism.

Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation-contraction coupling and dilated cardiomyopathy.

Numerous studies have implicated Coxsackievirus in acute and chronic heart failure. Although enteroviral nucleic acids have been detected in selected patients with dilated cardiomyopathy, the significance of such persistent nucleic acids is unknown. To investigate the mechanisms by which restricted viral replication with low level expression of Coxsackieviral proteins may be able to induce cardiomyopathy, we generated transgenic mice which express a replication-restricted full-length Coxsackievirus B3 (CVB3) cDNA mutant (CVB3DeltaVP0) in the heart driven by the cardiac myocyte-specific myosin light chain-2v (MLC-2v) promoter. CVB3DeltaVP0 was generated by mutating infectious CVB3 cDNA at the VP4/VP2 autocatalytic cleavage site from Asn-Ser to Lys-Ala. Cardiac-specific expression of this cDNA leads to synthesis of positive- and negative-strand viral RNA in the heart without formation of infectious viral progeny. Histopathologic analysis of transgenic hearts revealed typical morphologic features of myocardial interstitial fibrosis and in some cases degeneration of myocytes, thus resembling dilated cardiomyopathy in humans. There was also an increase in ventricular atrial natriuretic factor mRNA levels, demonstrating activation of the embryonic program of gene expression typical of ventricular hypertrophy and failure. Echocardiographic analysis demonstrated the presence of left ventricular dilation and decreased systolic function in the transgenic mice compared with wild-type littermates, evidenced by increased ventricular end-diastolic and end-systolic dimensions and decreased fractional shortening. Analysis of isolated myocytes from transgenic mice demonstrate that there is defective excitation-contraction coupling and a decrease in the magnitude of isolated cell shortening. These data demonstrate that restricted replication of enteroviral genomes in the heart can induce dilated cardiomyopathy with excitation-contraction coupling abnormalities similar to pressure overload models of dilated cardiomyopathy.

Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: an approach to the study of enteroviral persistence in cardiac myocytes.

BACKGROUND: Enteroviral ribonucleic acids have been identified in heart muscle of a subset of patients with myocarditis and dilated cardiomyopathy as well as in a mouse model of persistent coxsackievirus B3 (CVB3) infection, suggesting that persistent viral infection along with activation of an immune response may contribute to the pathogenesis of ongoing cardiac disease and dilated cardiomyopathy in certain patients. It is still not known whether persistence of the viral genome contributes to the pathogenesis of dilated cardiomyopathy. METHODS AND RESULTS: To determine whether low-level enteroviral gene expression similar to that observed with viral persistence can induce myocytopathic effects without formation of infectious virus progeny, the full-length infectious cDNA copy of CVB3 was mutated at the VP0 maturation cleavage site. This prevented formation of infectious virus progeny. In myocytes transfected with this mutated cDNA copy of the viral genome, both positive- and negative-strand viral RNAs were detected, demonstrating that there was replication of the viral genome by the RNA-dependent RNA polymerase. The level of viral protein expression was found to be below limits of detection by conventional methods of protein detection, thus resembling restricted virus replication. Nonetheless, the CVB3 mutant was found to induce a cytopathic effect in transfected myocytes, which was demonstrated by inhibition of cotransfected MLC-2v luciferase reporter activity and an increase in release of lactate dehydrogenase from transfected cells. CONCLUSIONS: This study demonstrates that restricted replication of enteroviral genomes in myocytes in a pattern similar to that observed in hearts with persistent viral infection can induce myocytopathic effects without generation of infectious virus progeny.

NF-kappaB-mediated inhibition of apoptosis is required for encephalomyocarditis virus virulence: a mechanism of resistance in p50 knockout mice.

Apoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-kappaB suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-kappaB1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 -/-) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI -/-) mice, we find that this resistance is not mediated by IFN-beta as previously thought. While no IFNRI -/- mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals' ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 -/-, and p65 -/- embryos, we found that NF-kappaB is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 -/- and p65 -/- MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-kappaB-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-kappaB has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-kappaB1 is required for EMCV virulence in vivo. The attenuation in p50 -/- mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.

Comparison of systemic radiotherapy with I-131-labeled monoclonal antibody BW575/9 to external beam radiotherapy in human neuroblastoma xenografts.

The therapeutic effectiveness of external beam radiotherapy (XRT) and radioimmunotherapy (RIT) was investigated in a human neuroblastoma (SK-N-SH) xenotransplanted to nude mice. This tumor model seems especially suitable for comparison of the relative biological effectiveness of RIT vs. XRT, as--in contrast to most tumor models--it shows an unusually homogenous uptake of the labeled MAb, thus providing a homogenous intratumoral dose distribution. XRT was performed using single fractions of 800, 1600, 2000 and 2400 cGy and RIT was delivered by intravenous injection of 15, 19 and 26 MBq of the I-131-labeled monoclonal antibody (MAb) BW575/9. Therapeutic efficiency of the two radiation modalities was assessed in terms of tumor volume doubling time (VDT). Miniature thermoluminescent (mini-TLD) dosimetry and MIRD-based dose calculations were used to evaluate the absorbed doses delivered by RIT and to assess the degree of homogeneity of the dose distribution. RIT with 19 MBq of the I-131 BW575/9 delivered a tumor dose of 2820 cGy measured by TLD and resulted in a tumor VDT of 32 days (vs. one day in controls). An equivalent effect on VDT was achieved by a single fraction XRT of 1600 cGy. The relative efficiency of XRT compared with RIT (ratio of dose XRT/dose RIT required to give the same VDT) was 0.57. Application of 26 MBq of the MAb (= 3200 cGy) resulted in complete tumor regression after ten days as did XRT with 2400 cGy, corresponding to a relative efficiency of 0.75.