RESUMO
A 40-year-old Ghanaian woman presented with fever and exanthema. She had anemia, leukopenia, increased erythrocyte sedimentation rate (ESR), creatinin kinase, lactate dehydrogenase (LDH), and liver enzymes. She was diagnosed with schistosomiasis and was cured with praziquantel. During the following years, she developed polymyositis, chronic nephritis, and life-threatening perimyocarditis. High numbers of Epstein-Barr virus (EBV)-encoded RNA copies were demonstrated in CD8+ T-lymphocytes from endomyocardial biopsies. There was no evidence of any underlying immunosuppression or an EBV-related malignancy. Chronic active EBV infection was diagnosed, a clinical picture not described in an adult African previously. Interestingly, among all therapy attempts, only rituximab was effective at stabilizing the disease.
Assuntos
Infecções por Vírus Epstein-Barr/diagnóstico , Herpesvirus Humano 4/isolamento & purificação , RNA Viral/isolamento & purificação , Adulto , Anticorpos Monoclonais Murinos/uso terapêutico , Sedimentação Sanguínea , Linfócitos T CD8-Positivos/virologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Feminino , Humanos , Fígado/enzimologia , Fígado/patologia , Nefrite/patologia , Praziquantel/uso terapêutico , Rituximab , Esquistossomose/diagnóstico , Esquistossomose/tratamento farmacológicoRESUMO
In virtually all cells including hepatocytes cell volume regulation is accomplished during cell swelling by cellular ion release (activation of K (+) channels and/or anion channels, KCl-cotransport, parallel activation of K (+)/H (+) exchange and Cl (-)/HCO (3)(-) exchange) and following cell shrinkage by cellular ion uptake (activation of Na (+), K (+), 2Cl (-) cotransport, Na (+)/H (+) exchange in parallel to Cl (-)/HCO (3)(-) exchange and Na (+)-channels). Moreover, cell shrinkage triggers the cellular accumulation of organic osmolytes (e. g., myoinositol, betaine, phosphorylcholine, taurine). Cell volume is a powerful regulator of hepatic metabolism. Cell shrinkage stimulates and cell swelling inhibits proteolysis and glycogenolysis. Moreover, cell volume influences the generation of and sensitivity to oxidants. Cell volume regulatory mechanisms furthermore do play a role in fibrosing disease. Kinases stimulating cell volume regulatory mechanisms include the serum and glucocorticoid inducible kinase SGK1, which is expressed in the liver, is genomically up-regulated by cell shrinkage, stimulates a wide variety of channels and transporters including Na (+), K (+), 2Cl (-) cotransport and Na (+)/H (+) exchange and is known to participate in the stimulation of fibrosis. Accordingly, excessive SGK1 expression is observed in liver cirrhosis. The case is made that SGK1 participates in the regulation of liver cell volume and thus in the regulation of hepatic metabolism.
Assuntos
Tamanho Celular , Hepatócitos/citologia , Hepatócitos/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Fígado/citologia , Fígado/metabolismo , Modelos Biológicos , Proteínas Serina-Treonina Quinases/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Animais , HumanosRESUMO
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.
Assuntos
Cardiomiopatias , Miocardite , Doença Aguda , Adulto , Idoso , Cardiomegalia/epidemiologia , Cardiomiopatias/diagnóstico , Cardiomiopatias/epidemiologia , Cardiomiopatias/terapia , Comorbidade , Comportamento Cooperativo , Feminino , Seguimentos , Doenças Genéticas Inatas/epidemiologia , Alemanha , Comportamentos Relacionados com a Saúde , Humanos , Infecções/epidemiologia , Masculino , Pessoa de Meia-Idade , Miocardite/diagnóstico , Miocardite/epidemiologia , Miocardite/terapia , Prognóstico , Qualidade de Vida , Sistema de Registros , Fatores de Risco , Fatores SocioeconômicosRESUMO
BACKGROUND: Although low (but increasing) rates of lung/lung-heart transplantations of scleroderma (systemic sclerosis [SSc]) patients have been reported, exclusive heart transplantation is a rare approach for treatment of heart failure due to SSc. CASES: We report on 2 cases of SSc patients receiving a heart transplantation (HTx) due to severe and progressive right heart failure without pulmonary artery hypertension. One patient received a hepatitis C virus (HCV)-positive donor heart and recovered excellently from viral transmission after administration of a direct-acting antiviral (DAA) regimen. This is the first published case of an SSc patient who underwent HTx using an HCV-positive donor heart. The clinical course of both patients was monitored by different serum SSc biomarkers. Only xylosyltransferase activity proved to be a promising biomarker for disease stage determination and therapeutic monitoring, precisely reflecting fibrotic remodeling and successful organ recovery. CONCLUSIONS: Successful implementation of the 2 cases described here demonstrates that HTx is a safe and effective therapeutic option for defined SSc sub-patient groups despite the progressive character of the underlying disease. In the future, xylosyltransferase activity might be conducive to simplify the identification of patients with low systemic involvement but a strong indication for single heart transplantation. Finally, we demonstrate that treatment of HCV viral transmission from HCV-positive donor to organ recipient using DAA gives us new opportunities to consider HCV-positive donor organs for HTx and might reveal new possibilities to ease the lack of donor organs.
Assuntos
Insuficiência Cardíaca/cirurgia , Transplante de Coração/métodos , Hepatite C Crônica/etiologia , Escleroderma Sistêmico/complicações , Doadores de Tecidos , Transplantes/virologia , Adulto , Antivirais/uso terapêutico , Feminino , Insuficiência Cardíaca/etiologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/transmissão , HumanosRESUMO
Molecular biological methods have confirmed the pathogenetic role of enteroviruses, primarily coxsackieviruses of group B (CVB), in the induction and maintenance of inflammatory cardiomyopathy. More recently, adenoviruses, various herpes viruses, and increasingly parvovirus B19 (B19) have been identified as potential cardiotropic agents. While cardiac myocytes are target cells for enterovirus and adenovirus infections with virus-induced cytolysis, B19-associated inflammatory cardiomyopathy is characterized by infection of intracardiac endothelial cells of small arterioles and veins, which may be associated with endothelial dysfunction, impairment of myocardial microcirculation, penetration of inflammatory cells, and secondary myocyte necrosis. Recent observations showed that B19 is involved in intracellular calcium regulation by the viral phospholipase. B19-induced caspase activation can lead to proinflammatory/proapoptotic processes through dysregulation of STAT signaling. These cellular interactions may contribute to mechanisms by which B19 establishes persistent infection in endothelial cells and play a critical role in viral pathogenesis of inflammatory cardiomyopathy.
Assuntos
Miocardite/patologia , Infecções por Parvoviridae/patologia , Viroses/patologia , Apoptose/fisiologia , Cálcio/metabolismo , Cardiomiopatias/patologia , Caspases/metabolismo , Vasos Coronários/patologia , Endotélio Vascular/patologia , Humanos , Microcirculação/fisiologia , Miócitos Cardíacos/patologia , Necrose , Parvovirus B19 Humano/patogenicidade , Fatores de Transcrição STAT/fisiologia , Transdução de Sinais/fisiologia , VirulênciaRESUMO
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.
Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Dilatada/virologia , Infecções por Coxsackievirus/fisiopatologia , Enterovirus Humano B/genética , Coração/fisiopatologia , Coração/virologia , Miocárdio/patologia , Animais , Cardiomiopatia Dilatada/patologia , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/isolamento & purificação , Enterovirus Humano B/fisiologia , Feminino , Genoma Viral , Ventrículos do Coração , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Regressão , Ensaio de Placa Viral , Replicação ViralRESUMO
BACKGROUND: The etiology of left ventricular (LV) isolated diastolic dysfunction often remains unclear. In the present study, we report a strong association between parvovirus B19 (PVB19) genomes and isolated LV diastolic dysfunction. METHODS AND RESULTS: In 70 patients (mean+/-SD age, 43+/-11 years) admitted with exertional dyspnea and/or reduced exercise tolerance despite preserved LV systolic contractility (ejection fraction=68%), isolated diastolic dysfunction was clinically suspected. Patients with classic risk factors for diastolic dysfunction such as hypertension, coronary heart disease, diabetes mellitus, or pulmonary disease had been excluded. Diastolic function was assessed by echocardiography and LV and RV catheterization. Endomyocardial biopsies (EMBs) were analyzed for the presence of storage or infiltrative diseases or myocarditis, including molecular screening for cardiotropic virus genomes. In a substudy of 24 patients who reported atypical angina, coronary endothelial function was additionally investigated with a coronary Doppler flow-wire technique. In 37 of 70 patients (53%), isolated diastolic dysfunction was confirmed as the cause of their clinical symptoms. No evidence for cardiac storage or infiltrative diseases was found in these cases, but in 35 of 37 of these patients (95%), cardiotropic virus genomes were detected in EMBs (P<0.001). PVB19 was the most frequent pathogen in 31 of 37 patients (84%). In a subgroup of 10 patients with diastolic dysfunction and coexisting endothelial dysfunction, all 10 (100%) were PVB19 positive. CONCLUSIONS: PVB19 genomes were predominant in patients with unexplained, isolated diastolic dysfunction. A strong association with the incidence of endothelial dysfunction was obvious, consistent with the hypothesis that PVB19-induced endothelial dysfunction may be a possible pathomechanism underlying diastolic dysfunction.
Assuntos
Diástole , Infecções por Parvoviridae/complicações , Parvovirus B19 Humano , Disfunção Ventricular Esquerda/virologia , Adulto , Biópsia , Angiografia Coronária , Endotélio/patologia , Endotélio/virologia , Feminino , Genoma Viral , Coração/fisiopatologia , Coração/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Parvovirus B19 Humano/genética , Prevalência , Disfunção Ventricular Esquerda/epidemiologia , Disfunção Ventricular Esquerda/etiologiaRESUMO
A panel of five quasimonomorphic mononucleotide repeats that dispenses with the need to analyse corresponding germline DNA was proposed by Suraweera et al for the detection of high-frequency microsatellite instability (MSI) in colorectal cancer. Using this panel, a simplified and a more sensitive (compared with the original) algorithm (p<0.05) was developed to define the instability of each repeat by assessing the morphological shape of its plot and not its absolute length. 103 cases of colorectal tumours were investigated and the results compared with those obtained by the analysis of five consensus microsatellites (Bethesda reference panel). By the proposed method, a higher specificity, but no loss of sensitivity, was found. Thus, the use of the five mononucleotide repeats in combination with the modified assessment technique simplifies the assessment of MSI, while retaining the sensitivity of the Bethesda panel for the detection of high-frequency MSI.
Assuntos
Neoplasias Colorretais/genética , Instabilidade Genômica , Repetições de Microssatélites/genética , Algoritmos , DNA de Neoplasias/genética , Humanos , Reação em Cadeia da Polimerase/métodos , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Infecções por Coxsackievirus/virologia , Enterovirus Humano B/patogenicidade , Interferon Tipo I/fisiologia , Animais , Infecções por Coxsackievirus/prevenção & controle , Modelos Animais de Doenças , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/fisiologia , Coração/virologia , Interferon Tipo I/genética , Interferon Tipo I/uso terapêutico , Interferon gama/fisiologia , Interferon gama/uso terapêutico , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Viral/metabolismo , Transdução de Sinais , Replicação Viral/efeitos dos fármacosRESUMO
We report on a 5-year-old girl who suddenly collapsed and died while dancing at a family party. Histological examination of the heart including the cardiac conduction system revealed lymphocytic infiltrations of the sinu-atrial node and perivascular infiltration in the atrio-ventricular region. Additionally, foci of mononuclear infiltrates were observed in the myocardium. Consequently, myocarditis was diagnosed as cause of death. The child also had lymphocytic conjunctivis, parotitis and tracheitis. Evaluation of infections by means of nested polymerase chain reaction revealed parvovirus B19 DNA (PVB19) in tissue samples of the trachea.
Assuntos
Morte Súbita Cardíaca/etiologia , Eritema Infeccioso/diagnóstico , Parvovirus B19 Humano/isolamento & purificação , Pré-Escolar , Conjuntivite Viral/diagnóstico , Feminino , Humanos , Linfócitos/patologia , Miocardite/virologia , Miocárdio/patologia , Parotidite/virologia , Traqueíte/virologiaRESUMO
Lymphocytic myocarditis is thought to be a virus-induced disease. T cells expressing the alpha-beta T-cell receptor seem to play a central role in the pathogenesis and to mediate tissue injury in this disease. A case of active fulminant myocarditis is described, which was analyzed by immunohistochemical, molecular biologic, and serologic methods. Infiltration of the heart tissue predominantly by gamma-delta T cells was detected by immunohistochemistry. No evidence of viral disease could be obtained by in situ hybridization with different enterovirus-specific DNA probes; by reverse-transcriptase polymerase chain reaction using specific primers for enteroviruses, adenoviruses, herpes simplex viruses, influenza A and B viruses, and cytomegaloviruses; or by enzyme-linked immunosorbent assay and electron microscopy. Because gamma-delta T cells may have an autoimmune capacity, we propose that these cells may trigger autoimmune myocarditis. These findings may be important in order to identify subgroups of patients who may benefit from immunosuppressive therapy.
Assuntos
Doenças Autoimunes/patologia , Miocardite/patologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/química , Linfócitos T/patologia , Adulto , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , DNA/análise , DNA/genética , Ventrículos do Coração/química , Ventrículos do Coração/patologia , Ventrículos do Coração/ultraestrutura , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização In Situ , Masculino , Miocardite/imunologia , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/metabolismoRESUMO
The 5'-non-translated region (NTR) of enteroviruses contains secondary structures which do not only serve in the initiation of translation but also in the initiation of plus-strand RNA synthesis by binding of viral and cellular proteins. To investigate a very early step of enteroviral replication by cis- and trans-complementation, 220 nucleotides of the 5'-region of coxsackievirus B3 (CVB3) were exchanged with the corresponding region of poliovirus type 1 (PV1) to yield the chimeric virus CVB3[PV5']. The viability of this chimera demonstrates that the polioviral cloverleaf structure of the 5'-NTR is functional in the replication of a chimeric CVB3 RNA. The HeLa-generated chimera reveals a 4-nucleotide deletion (nt 232-235) within a short direct repeat. Besides clearly reduced growth characteristics in all permissive cell lines, the chimera exhibits a small-plaque phenotype. The host range is changed since the virus grows well in human HeLa cells, but does not replicate in murine YAC-1 and Ltk cells, although these cell lines are permissive for the replication of both parental viruses. Moreover, in simian Vero, COS-1, or FRhK-4 cells the HeLa-generated chimera CVB3[PV5'] exhibits a strict temperature sensitivity at 39 degrees C. After infection of simian cells with high m.o.i. in situ hybridization data reveal that the chimera replicates in single cells at almost normal rates indicating that only a small fraction of HeLa-generated virus is able to multiplicate in simian cell lines. After passaging the virus chimera in Vero cells two further mutations occur at nucleotide positions 185 and 227. Since this genome region is known to interact with viral proteins and several host cell factors during the initiation of replication and translation, interactions of such factors with either viral RNA or viral proteins may be disturbed but still functional at permissive temperatures in HeLa cells and simian cell lines, whereas murine cell lines are not permissive. These experiments suggest that phenomena like host range, tissue tropism and cell-type specificity may be explained as a complex interplay of cellular surface receptors and intracellular host factors. Such intracellular factors could be part of the enteroviral initiation complex during the plus-strand RNA synthesis or during translation initiation and could be expressed in a tissue-, organ- or species-specific way or might be regulated developmentally.
Assuntos
Enterovirus Humano B/crescimento & desenvolvimento , Poliovirus/crescimento & desenvolvimento , Vírus Reordenados/crescimento & desenvolvimento , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA , Enterovirus/genética , Enterovirus/crescimento & desenvolvimento , Enterovirus Humano B/genética , Células HeLa , Humanos , Hibridização In Situ , Macaca mulatta , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Poliovirus/genética , RNA Viral/química , Vírus Reordenados/genética , Especificidade da Espécie , Células Tumorais Cultivadas , Células VeroRESUMO
Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.
Assuntos
Infecções por Coxsackievirus/etiologia , Enterovirus Humano B/patogenicidade , Enterovirus/patogenicidade , Animais , Animais Recém-Nascidos , Infecções por Coxsackievirus/microbiologia , Modelos Animais de Doenças , Enterovirus/classificação , Enterovirus/genética , Enterovirus Humano B/classificação , Enterovirus Humano B/genética , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , RNA Viral/genética , RNA Viral/metabolismo , Sorotipagem , Especificidade da EspécieRESUMO
Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the presentation of foreign antigenic epitopes was further characterized. Insertion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimeras containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Analysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site 1 (N-Ag1) of CVB4 which is located in the BC loop of the structural protein VP1. A significant neutralization of a viable chimera with the deletion of CVB4-specific amino acid Ser-83 at the amino terminus of the VP1 BC loop was obtained with CVB4 serotype-specific polyclonal antisera. This neutralization was reduced after further deletion of the adjacent Ala-84, suggesting that this amino acid either constitutes the beginning of N-Ag1 of CVB4 or is essential for the conformation of the adjacent epitope. In contrast, exchange of amino acid Ser-86 to alanine, in the middle of the BC loop, led to complete loss of reactivity with CVB4-specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutralizing antisera. Furthermore, we observed that manipulations of the VP1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar.
Assuntos
Antígenos Virais/imunologia , Enterovirus Humano B/fisiologia , Proteínas Estruturais Virais/imunologia , Sequência de Aminoácidos , Antígenos Virais/química , Sequência de Bases , Primers do DNA , Enterovirus Humano B/genética , Enterovirus Humano B/imunologia , Epitopos/análise , Variação Genética , Genoma Viral , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Testes de Neutralização , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Deleção de Sequência , Transfecção , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/química , Replicação ViralRESUMO
The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the P1 region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the P1 region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the P1 region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The P1 region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the P1 region. Furthermore, a chimera which only contained the 5' part of the P1 region derived from CB3NRD and the remaining P1 sequence from CB3N was able to replicate in RD cells, suggesting that the VP2 polypeptide contains at least one determinant for the RD phenotype.
Assuntos
Enterovirus Humano B/genética , Genoma Viral , Mapeamento de Nucleotídeos , Rabdomiossarcoma/microbiologia , Sequência de Bases , Capsídeo/genética , Proteínas do Capsídeo , Linhagem Celular , Quimera , Enterovirus Humano B/crescimento & desenvolvimento , Variação Genética , Dados de Sequência Molecular , Mapeamento de Nucleotídeos/métodos , Fenótipo , Células Tumorais Cultivadas , Replicação ViralRESUMO
Viral infection is a complex process which includes binding and interaction of the virus with specific cell surface receptors, uptake and uncoating of the virus, and finally replication. Chinese hamster ovary (CHO) cells are non-permissive towards infection with coxsackieviruses of group B (CVB), although they do express a putative CVB-specific receptor protein. In order to localize the block of infection in CHO cells, these cells were tested for binding of radiolabelled CVB3, receptor-mediated transformation of virions into A-particles, and replication of the viral genome. Binding of CVB3 to CHO cells was found to be comparable to the binding of this virus to permissive cell lines. Detergent-solubilized membrane proteins of CHO cells were tested in virus overlay protein-binding assays (VOPBAs) and shown to express a 100 kDa CVB-binding membrane protein similar to the CVB receptor protein which we recently described for permissive HeLa cells. Incubation of CVB3 with intact CHO cells resulted in transformation of cell-bound virions into non-infectious A-particles (deprived of capsid protein VP4), demonstrating the functional activity of the CVB receptor protein on CHO hamster cells. Transfection of recombinant CVB3 cDNA or viral RNA into CHO cells resulted in the production of infectious CVB3 virions, implying that the failure of CVB to infect CHO cells is not caused by a defect in virus replication but results from a block in the uptake of virus particles into the cell after the initial steps of virus-receptor interactions.
Assuntos
Células CHO/virologia , Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B/patogenicidade , Receptores Virais/metabolismo , Animais , Células CHO/metabolismo , Linhagem Celular , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/virologia , Cricetinae , Suscetibilidade a Doenças , Enterovirus Humano B/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Genoma Viral , Células HeLa/metabolismo , Células HeLa/virologia , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Peso Molecular , RNA Viral/biossíntese , Receptores Virais/isolamento & purificação , Vírion/metabolismo , Vírion/patogenicidade , Replicação ViralRESUMO
In humans and experimental murine models enteroviruses, and in particular coxsackieviruses of group B (CVB), may induce chronic myocarditis associated with a persistent type of heart muscle infection. Persistent myocardial infection has been characterized by restricted viral replication and gene expression, which is capable of sustaining chronic inflammation. Altered replication and transcription of the virus, in addition to an immune response insufficient to recognize and clear infected cells entirely, are essential mechanisms for initiation and maintenance of persistent heart muscle infection. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection, indicating that enterovirus myocarditis is a virus-induced rather than an immune-mediated disease. Notably, resistance to the development of persistent heart muscle infection is not linked to the H-2 haplotype of the host. In addition to persistently infected myocytes, detection of the replicative minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen, predominantly in splenic B lymphocytes, during the course of the disease. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac1+ macrophages, no enteroviral genomes were identified in CD8+ T cells. Detection of infected activated B lymphocytes both in heart tissue of CVB3-infected immunocompetent mice and syngenic SCID mice receiving splenocytes from CVB3-infected donors support the concept that B cell traffic may contribute to maintenance of chronic disease. Dissection of the diversity of viral and host-specific determinants in susceptible and resistant hosts will allow us to define the protective mechanisms that mediate resistance to the development of life-threatening acute and chronic enterovirus myocarditis.
Assuntos
Infecções por Enterovirus/virologia , Enterovirus , Coração/virologia , Sistema Imunitário/virologia , Animais , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B , Infecções por Enterovirus/patologia , Humanos , Sistema Imunitário/patologia , Camundongos , Miocardite/patologia , Miocardite/virologia , Baço/imunologia , Baço/virologiaRESUMO
In animal studies, smooth muscle cell phenotype conversion has been suggested to be an essential prerequisite for subsequent migratory and proliferative events leading to (neo)intima formation. To determine ultrastructural characteristics of individual smooth muscle cells and to relate them to specific lesion types and intimal cell density, we compared atherectomy samples from 17 restenotic and 32 primary coronary and peripheral lesions using transmission electron microscopy and histology. Ultrastructural analysis of cell-rich tissue, predominantly of restenotic origin, revealed smooth muscle cells full of synthetic organelles. Moreover, these cells were frequently found to be surrounded by loose extracellular matrix and partially fragmented basement membrane components. In contrast, plaques exhibiting low cell density, as exclusively seen with primary lesions, displayed an extensive buildup of extracellular matrix containing sparse numbers of microfilament-rich smooth muscle cells. The central finding of our study is a morphometrically quantitated, twofold greater (p <0.001) volume fraction of synthetic organelles (VS) within smooth muscle cells in restenotic versus primary plaques, indicating a more dedifferentiated cellular phenotype as a typical feature of restenotic lesions. Equally enhanced VS values were seen for restenotic coronary and peripheral plaques. No VS decrease was observed during time after angioplasty (2.2 to 30 months) regardless of previous revascularization procedures (balloon angioplasty or atherectomy). Despite intra- and interlesional variability, VS and intimal cell density were strongly correlated (r = 0.74; p <0.001). This correlation was observed more often with clinical restenoses and, importantly, in a portion (10% to 15%) of primary lesions. Data from restenotic lesions indicate that a dedifferentiated smooth muscle cell phenotype, pericellular matrix disintegration, and intimal hypercellularity are long-lasting biologic responses to previous smooth muscle cell injury. Similar tissue characteristics expressed in several primary lesions suggest that comparable pathogenic mechanisms are related to the progression and/or acuity of chronic lesions.
Assuntos
Arteriosclerose/patologia , Arteriosclerose/cirurgia , Aterectomia , Doença das Coronárias/patologia , Doença das Coronárias/cirurgia , Perna (Membro)/irrigação sanguínea , Músculo Liso Vascular/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Recidiva , Túnica Íntima/patologiaRESUMO
The limited efficacy of current therapeutic approaches for a number of socially relevant human diseases such as cancer and cardiovascular pathologies, has required the exploration of alternative and more effective therapeutic strategies. In the last two decades, nucleic acid based drugs have emerged as an attractive and novel alternative with great therapeutic potential. Among these molecules, hammerhead ribozymes were the first to be extensively studied and predicted to be of potential practical utility. Hammerhead ribozymes are catalytic RNA molecules capable of inducing the site-specific cleavage of a phosphodiester bond within an RNA molecule. Thus, they can be used to reduce the intracellular level of a specific mRNA coding for a protein which affects cellular metabolism or environment, causing disease. As hammerhead ribozymes can be engineered to reduce the level of virtually any mRNA, they have a very broad applicability. Among the several pathological conditions amenable for a hammerhead ribozyme based therapeutic approach, we focused our attention on pathologies sustained by a dis-regulated and excessive cellular proliferation, being sure to properly demonstrate their usefulness. Trying to be as objective as possible in regard to the feasibility of hammerhead ribozyme employment as therapeutics, a technical section, describing some of the unresolved problems in this field, has been also included. Although some aspects of hammerhead ribozymes as therapeutics can and should be optimized, the encouraging results displayed so far fully justifies further efforts, economic and scientific, to bring them closer to the clinical practice.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Reestenose Coronária/tratamento farmacológico , Terapia Genética/métodos , Glomerulonefrite/tratamento farmacológico , Neoplasias/tratamento farmacológico , RNA Catalítico/uso terapêutico , Vitreorretinopatia Proliferativa/tratamento farmacológico , Animais , Artrite Reumatoide/genética , Reestenose Coronária/genética , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Inativação Gênica , Terapia Genética/tendências , Glomerulonefrite/genética , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/tratamento farmacológico , Índice de Gravidade de Doença , Vitreorretinopatia Proliferativa/genéticaRESUMO
As enterovirus infections of the heart cause myocarditis and eventually congestive heart failure, the antiviral activity of ribavirin was studied in coxsackie virus B3 (CVB3)-infected carrier cultures of human myocardial fibroblasts. Cultures were infected 7 days before application of ribavirin and effects were evaluated over a period of 16 days by plaque assays and in situ hybridization. Compared to the low antiviral activity in HeLa cells, ribavirin was highly active in reducing infectious virus yields in human myocardial fibroblasts, for example, to 2.0 x 10(3) pfu/ml with 25 microg/ml and to 1.3 x 10(2) pfu/ml with 50 microg/ml (4.3 x 10(4) pfu/ml in infected controls). Moreover, 100 microg ribavirin/ml completely suppressed infectious virus progeny in two of three cultures, and reduced the number of infected cells from 14.3 to 0.3% as determined by in situ hybridization, whereas up to 3200 microg ribavirin/ml did not result in a significant cytotoxic effect. Interaction with interferon-alpha (IFN-alpha) was additive to slightly synergistic in reducing the number of infected cells and virus yields. In conclusion, our results suggest a cell-specific high activity of ribavirin in human myocardial fibroblasts and indicate the importance of using organ-specific cells for testing antiviral agents in myocarditis. Furthermore, the usefulness of in situ hybridization for determining the long term effects of antivirals in carrier state cell cultures was demonstrated.