Identification of New, Functionally Relevant Mutations in the Coding Regions of the Human Fos and Jun Proto-Oncogenes in Rheumatoid Arthritis Synovial Tissue.

In rheumatoid arthritis (RA), the expression of many pro-destructive/pro-inflammatory proteins depends on the transcription factor AP-1. Therefore, our aim was to analyze the presence and functional relevance of mutations in the coding regions of the AP-1 subunits of the fos and jun family in peripheral blood (PB) and synovial membranes (SM) of RA and osteoarthritis patients (OA, disease control), as well as normal controls (NC). Using the non-isotopic RNAse cleavage assay, one known polymorphism (T252C: silent; rs1046117; present in RA, OA, and NC) and three novel germline mutations of the cfos gene were detected: (i) C361G/A367G: Gln121Glu/Ile123Val, denoted as "fos121/123"; present only in one OA sample; (ii) G374A: Arg125Lys, "fos125"; and (iii) C217A/G374A: Leu73Met/Arg125Lys, "fos73/125", the latter two exclusively present in RA. In addition, three novel somatic cjun mutations (604-606ΔCAG: ΔGln202, "jun202"; C706T: Pro236Ser, "jun236"; G750A: silent) were found exclusively in the RA SM. Tansgenic expression of fos125 and fos73/125 mutants in NIH-3T3 cells induced an activation of reporter constructs containing either the MMP-1 (matrix metalloproteinase) promoter (3- and 4-fold, respectively) or a pentameric AP-1 site (approximately 5-fold). Combined expression of these two cfos mutants with cjun wildtype or mutants (jun202, jun236) further enhanced reporter expression of the pentameric AP-1 construct. Finally, genotyping for the novel functionally relevant germline mutations in 298 RA, 288 OA, and 484 NC samples revealed no association with RA. Thus, functional cfos/cjun mutants may contribute to local joint inflammation/destruction in selected patients with RA by altering the transactivation capacity of AP-1 complexes.

Anti-coxsackievirus B3 activity of 2-amino-3-nitropyrazolo[1,5-a]pyrimidines and their analogs.

A novel class of 2-amino-4-nitropyrazolo[1,5-a]pyrimidines has been identified as potent inhibitors of coxsackievirus B3 replication. The synthesis of these compounds is based on the regioselective reaction of 3,5-diamino-5-nitropyrazole with unsymmetrical beta-diketones at catalysis by hydrochloric acid leading to 2-amino-4-nitropyrazolo[1,5-a]pyrimidines as key steps.

Co-expression of interleukin-2 to increase the efficacy of DNA vaccine-mediated protection in coxsackievirus B3-infected mice.

DNA immunizations with the major structural protein VP1 of coxsackievirus B3 (CVB3) have been previously found to protect mice from a lethal challenge with CVB3. The function of this vaccination procedure is mainly based on accelerated antibody induction with an early cytokine expression and increased virus-specific cytotoxic activity of spleen cells causing decreased myocyte destruction and reduced viral replication. Here, we report that the co-expression of the immune-stimulatory interleukin-2 (IL-2) can increase the efficacy of the inoculated DNA vaccine depending on the route of administration and the mouse strain used.

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers.

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.

Influenza infection of the embryonated hen's egg/an alternative model for in vivo evaluation of antiviral compounds.

The embryonated hen's egg can be infected with influenza A virus in laboratory experiments leading to death of chick embryos within 8 days post infection. This model can be used for rapid and reliable in vivo evaluation of potential anti-influenza inhibitors. It offers a realistic alternative to experiments with small laboratory rodents. By an example of treatment with the antiviral drugs amantadine (CAS 665-66-7), rimantadine (CAS 1501-84-4) and zanamivir (CAS 139110-80-8), it could be demonstrated that chick embryos survive a lethal influenza A virus infection when these drugs are administered immediately before or after the infective agent is inoculated. In conclusion, the use of influenza virus-infected chick embryos for evaluation of new antiviral substances can lead to a considerable reduction in the number of small laboratory rodents required.

Frequent detection of parvovirus B19 genome in the myocardium of adult patients with idiopathic dilated cardiomyopathy.

Aside from enteroviruses and other viruses, e.g., adenoviruses, which are known to be associated with idiopathic dilated cardiomyopathy (IDC), a cardiac tropism is also attributed to parvovirus B19 (PVB19). The purpose of the present study was to determine the prevalence of enterovirus, adenovirus and PVB19 genomes in the myocardium of adult patients with IDC and to analyze the significance of PVB19 with regard to the course of the disease, as compared to the other cardiotropic viruses. In 52 adult patients with IDC and 10 control patients with normal left ventricular ejection fraction (> or =55%) undergoing coronary artery bypass surgery, myocardial tissue samples were investigated for enteroviral RNA using polymerase chain reaction (PCR) and Southern blot hybridization of the PCR product. Specific nested PCR was used to assess the prevalence of adenovirus and PVB19 DNA, in addition to sequencing of the latter. The clinical and echocardiographic course of the disease was followed for a mean (+/- SD) period of 21.1+/-9.5 months. Fourteen of the 52 patients (27%) were enterovirus-positive, 2/52 (4%) patients were adenovirus-positive, 14/52 (27%) patients were PVB19-positive, 8/52 (15%) patients were enterovirus plus PVB19-positive, and in 14/52 (27%) patients no viral genomes were found. Six patients died during the follow-up period, without any significant difference between the patient groups: 1/14 (7%) in the enterovirus-positive, 0/2 (0%) in the adenovirus-positive, 2/14 (14%) in the PVB19-positive, 1/8 (12.5%) in the enterovirus plus PVB19-positive, and 2/14 (14%) in the virus-negative group. PVB19 genome was found in 4 of the 10 (40%) control patients, but no enterovirus or adenovirus genomes were detected in these patients. In conclusion, in the myocardium of patients with IDC, PVB19 is detectable as frequently as enteroviral genome. PVB19-positive patients with IDC have a rather favorable prognosis and do not differ significantly from the other virus-positive or virus-negative patient groups with respect to survival. Finally, the pathogenetic and prognostic significance of PVB19 in IDC still remains unclear.

Coxsackievirus B3-induced myocarditis: differences in the immune response of C57BL/6 and Balb/c mice.

Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.

Nitric oxide donors inhibit the coxsackievirus B3 proteinases 2A and 3C in vitro, virus production in cells, and signs of myocarditis in virus-infected mice.

The antiviral effect of nitric oxide (NO)-releasing compounds was investigated. Using bacterially expressed and purified proteinases 2A and 3C of coxsackievirus B3, in vitro assays demonstrated the inhibition of the 2A proteinase activity in the presence of S-nitroso- N-acetyl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), 4-phenyl-3-furoxancarbonitrile (PFC), glyceryl trinitrate (GTN), and isosorbide dinitrate (ISDN). Sodium nitroprusside (SNP), which releases NO after metabolization, had no effect. The 3C proteinase was inactivated by SNAP, GTN, and ISDN. The vasodilators GTN and ISDN, widely used in the treatment of angina pectoris, exhibited antiviral activity in CVB3-infected GMK cells. CVB3-infected NMRI outbred mice showed significantly reduced signs of myocarditis after treatment with GTN or ISDN. Inhibitors of the cellular inducible NO synthase (iNOS) such as N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NNA), and S-methyl-isothiourea (SMT), had no deleterious effect on CVB3-infected NMRI mice, indicating that endogenous NO synthesis is unlikely to be a major defense mechanism after enterovirus infection of outbred mice.

Direct interferon-gamma-mediated protection caused by a recombinant coxsackievirus B3.

Coxsackievirus B3 (CVB3) is one of the most important causes of viral myocarditis. Cytokines are involved in the control of CVB3 replication and pathogenesis. Local expression of specific cytokines by recombinant CVB3 confers prevention of virus-caused myocarditis. Expression of IFN-gamma by CVB3(IFN-gamma) protected BALB/c and C57BL/6 mice when the lethal infection with the highly pathogenic CVB3H3 variant was given directly after or prior to CVB3(IFN-gamma) inoculation by decreasing the viral load and spread as well as tissue destruction. This direct effect was not restricted to the homologous virus. In vitro, cocultivation of CVB3(IFN-gamma)-infected cells induced a reduction of CVB3H3 replication and virus-induced cytopathogenicity.

Sequencing of porcine enterovirus groups II and III reveals unique features of both virus groups.

The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5'-cloverleaf structure.

Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C.

The initiation of enteroviral positive-strand RNA synthesis requires the presence of a functional ribonucleoprotein complex containing a cloverleaf-like RNA secondary structure at the 5' end of the viral genome. Other components of the ribonucleoprotein complex are the viral 3CD proteinase (the precursor protein of the 3C proteinase and the 3D polymerase), the viral 3AB protein and the cellular poly(rC)-binding protein 2. For a molecular characterization of the RNA-binding properties of the enteroviral proteinase, the 3C proteinase of coxsackievirus B3 (CVB3) was bacterially expressed and purified. The recombinant protein is proteolytically active and forms a stable complex with in vitro-transcribed cloverleaf RNA of CVB3. The formation of stable complexes is also demonstrated with cloverleaf RNA of poliovirus (PV) 1, the first cloverleaf of bovine enterovirus (BEV) 1, and human rhinovirus (HRV) 2 but not with cloverleaf RNA of HRV14 and the second cloverleaf of BEV1. The apparent dissociation constants of the protein:RNA complexes range from approx. 1.7 to 4.6 microM. An electrophoretic mobility shift assay with subdomain D of the CVB3 cloverleaf demonstrates that this RNA is sufficient to bind the CVB3 3C proteinase. Binding assays using mutated versions of CVB3 and HRV14 cloverleaf RNAs suggest that the presence of structural features rather than a defined sequence motif of loop D are important for 3C proteinase-RNA interaction.

Functional features of the bovine enterovirus 5'-non-translated region.

The bovine enterovirus (BEV) serotypes exhibit unique features of the non-translated regions (NTRs) which separate them from the other enteroviruses. Their most remarkable property is an additional genome region of 110 nt located between the 5'-cloverleaf and the internal ribosome entry site (IRES). This genome region has the potential to form an additional cloverleaf structure (domain I*) separated from the 5'-cloverleaf (domain I) by a small stem-loop (domain I**). Other characteristics involve the putative IRES domains III and VI. In order to investigate the features of the 5'-NTR, several full-length coxsackievirus B3 (CVB3) cDNA plasmids with hybrid 5'-NTRs were engineered. After exchange of the CVB3 cloverleaf with the BEV1 genome region representing both cloverleafs, a viable virus chimera was generated. Deletion of domain I** within the exchanged region also yielded viable virus albeit with reduced growth capacity. Deletion of sequences encoding either the first or the second BEV cloverleaf resulted in non-infectious constructs. Hybrid plasmids with exchanges of the IRES-encoding sequence or the complete 5'-NTR were non-infectious. Transfection experiments with SP6 transcripts containing 5'-NTRs fused to the luciferase message indicated that IRES-driven translation is enhanced by the presence of the CVB3 cloverleaf and both BEV1 cloverleaf structures, respectively. Deletion of either the first or the second BEV cloverleaf domain reduced but did not abolish enhanced luciferase expression. These results suggest that the substitution of two putative BEV cloverleaf structures for the putative coxsackieviral cloverleaf yields viable virus, while BEV sequences encoding the IRES fail to functionally replace CVB3 IRES-encoding sequences.