RESUMEN
Long-term survival of solid-organ transplants is limited as a result of chronic transplant dysfunction (CTD), which is characterized by occlusion of intragraft vascular tissue due to myointimal hyperplasia. Recent studies have shown a role for infections in vascular pathologies. For example, Chlamydia pneumoniae (Cpn) has been shown to aggravate atherosclerosis, and Cpn immunoglobulin (Ig)G titers correlate with severity of allograft atherosclerosis after cardiac transplantation. In this study, we evaluated the effect of Cpn infection on CTD using a rat aortic allograft model. Orthotopic abdominal aorta transplantations (Tx) were performed with Brown Norway rats as donors and Lewis rats as recipients. Rats were humanely killed at 1 or 8 weeks after surgery. The graft was processed for DNA isolation and histological examination. Influx of macrophages and T cells was assessed using immunohistochemistry. At 1 week after Tx, the perivascular influx of inflammatory cells in the graft was not affected by Cpn infection. Furthermore, only limited numbers of Cpn DNA copies were found in the graft at 1 week after Tx. In addition, Cpn did not alter the severity of myointimal hyperplasia in the rat aortic allograft model at 8 weeks after surgery. Our data suggested that, in the rat aortic allograft model, Cpn infection did not influence the influx of inflammatory cells or the severity of CTD.
Asunto(s)
Aorta/trasplante , Infecciones por Chlamydia/fisiopatología , Chlamydophila pneumoniae , Trasplante Homólogo , Animales , Masculino , Modelos Animales , Complicaciones Posoperatorias/fisiopatología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Trasplante IsogénicoRESUMEN
We have identified and characterized two antisense transcripts from the rat cytomegalovirus (RCMV) major immediate early (MIE) region. These transcripts, designated IE-AS1 and IE-AS2, are complementary to part of the sense IE1 transcript. The IE-AS transcripts were first detected in peripheral blood leukocytes (PBL) of RCMV-infected rats at 7 days post-infection (pi) in the absence of IE1 transcription. Nevertheless, both the IE1 and IE-AS transcripts were found at the same time in the salivary glands of RCMV-infected rats at 7 and 120 days pi as well as in RCMV-infected rat embryo fibroblasts (REFs) at 48 h pi.
Asunto(s)
Antígenos Virales/genética , Proteínas Inmediatas-Precoces/genética , Muromegalovirus/genética , ARN sin Sentido/análisis , ARN Mensajero/análisis , ARN Viral/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Fibroblastos/virología , Leucocitos/virología , Datos de Secuencia Molecular , ARN sin Sentido/genética , ARN Complementario/genética , ARN Mensajero/genética , ARN Viral/genética , Ratas , Glándulas Salivales/virologíaRESUMEN
OBJECTIVE: Atherosclerosis is an inflammatory process and is characterised by the presence of T-lymphocytes in the lesions. To study the role of Chlamydophila pneumoniae (C. pneumoniae) in this process and the effect of infection on T-cell influx, we infected Apo E3-Leiden mice with C. pneumoniae and investigated the effect on lesion development and T-cell influx in atherosclerotic lesions at different time points post infection (pi). METHODS: Nine week old mice, fed an atherogenic diet, were either mock-infected or infected with C. pneumoniae and sacrificed at 1, 6 and 9 months pi. Longitudinal sections of the aortic arches of the mice were stained with hematoxylin-eosin for atherosclerotic lesion type and lesion area analysis, or with rabbit-anti-CD3(+) to detect the presence of T-cells in the atherosclerotic lesions. T-cell influx was expressed as number of T-lymphocytes/lesion area. RESULTS: At 1 month pi, type 1, 2 and 3 lesions were present. At other time points pi, more complex lesion types 4, 5a and 5b were also present. Although infection did not influence the total lesion number or area, we observed an effect of C. pneumoniae infection on lesion type. Infection resulted in a significant shift in lesion formation from type 3 to type 4 (P=0.022) at 6 months pi, and from type 4 to type 5a (P=0.002) at 9 months pi. T-cells were observed at every time point pi. At 1 month pi, a significant increase in T-cell influx in the C. pneumoniae-infected atherosclerotic lesions was observed (P=0.0005). CONCLUSION: This study shows that C. pneumoniae infection enhances the inflammatory process by increasing T-lymphocytes in the plaque and accelerates the formation of complex lesions.
Asunto(s)
Arteriosclerosis/microbiología , Infecciones por Chlamydia/complicaciones , Chlamydophila pneumoniae , Animales , Aortitis/inmunología , Aortitis/microbiología , Aortitis/patología , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Colesterol/sangre , Dieta Aterogénica , Progresión de la Enfermedad , Femenino , Hipercolesterolemia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de Riesgo , Linfocitos T/patología , Triglicéridos/sangreRESUMEN
We previously generated an RCMV strain in which the r144 gene, encoding a major histocompatibility complex class I homolog, had been deleted (RCMVdelta r144). To investigate the role of r144 during acute infection of neonatal rats, we infected three days-old neonatal rats with either RCMVdelta r144 or wild type (wt) RCMV and the presence of infectious virus as well as viral DNA in various organs was determined at either 3, 5 or 21 days p.i.. In addition, we assessed both type and number of inflammatory cells in these organs. Interestingly, a significantly lower concentration of infectious virus as well as viral DNA was found in spleens of RCMVdelta r144-infected rats than in those of wt RCMV-infected animals at 3 days p.i.. At the same time point, a significantly lower amount of infiltrating NK cells and monocytes/macrophages was seen in the spleens of RCMVdelta r144-infected rats than in spleens of rats infected with wt RCMV. At 21 days p.i., RCMVdelta r144-infected rats were found to have lower virus titers in the salivary glands than wt RCMV-infected animals. Significant differences between RCMVdelta r144- and wt RCMV-infected rats were detected neither at other time points nor at other sites. We conclude that after infection of neonatal rats, the replication of RCMVdelta r144 is severely restricted compared to wt RCMV.
Asunto(s)
Genes MHC Clase I/genética , Infecciones por Herpesviridae/virología , Muromegalovirus/genética , Proteínas Virales/genética , Enfermedad Aguda , Animales , Animales Recién Nacidos , Recuento de Células , ADN Viral/análisis , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Infecciones por Herpesviridae/inmunología , Leucocitos/virología , Macrófagos/virología , Masculino , Muromegalovirus/química , Ratas , Glándulas Salivales/virología , Bazo/inmunología , Bazo/virología , Factores de TiempoRESUMEN
Rat cytomegalovirus (RCMV) open reading frame R44 is the homolog of human cytomegalovirus gene UL44, which encodes the DNA polymerase accessory protein. Here, we show that R44 is transcribed as a 3.6-kb mRNA within the early and late phases of infection in vitro. In order to find potential monoclonal antibodies (MoAbs) directed against the R44-encoded protein (pR44), a panel of anti-RCMV MoAbs was screened for binding to pR44 recombinant proteins. Thus, an anti-pR44 MoAb, termed RCMV8, was identified. By using this MoAb, pR44 could be detected as early as 8 h after RCMV infection in vitro. The pR44 protein was determined to have a molecular mass of approximately 55 kDa and was found to be localized to the nucleus of RCMV-infected cells.
Asunto(s)
Citomegalovirus/genética , Genes Virales , Proteínas Nucleares/genética , Sistemas de Lectura Abierta , Animales , Peso Molecular , Proteínas Nucleares/análisis , RatasRESUMEN
The rat cytomegalovirus (RCMV) r144 gene encodes a polypeptide homologous to major histocompatibility complex class I heavy chains. To study the role of r144 in virus replication, an RCMV r144 null mutant strain (RCMVDeltar144) was generated. This strain replicated with efficiency similar to that of wild-type (WT) RCMV in vitro. Additionally, WT RCMV and RCMVDeltar144 were found not to differ in their replication characteristics in vivo. First, the survival rate was similar among groups of immunosuppressed rats infected with either RCMVDeltar144 or WT RCMV. Second, the dissemination of virus did not differ in either RCMVDeltar144- or WT RCMV-infected, immunosuppressed rats, either in the acute phase of infection or approximately 1 year after infection. These data indicate that the RCMV r144 gene is essential neither for virus replication in the acute phase of infection nor for long-term infection in immunocompromised rats. Interestingly, in a local infection model in which footpads of immunosuppressed rats were inoculated with virus, a significantly higher number of infiltrating macrophage cells as well as of CD8(+) T cells was observed in WT RCMV-infected paws than in RCMVDeltar144-infected paws. This suggests that r144 might function in the interaction with these leukocytes in vivo.
Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/inmunología , ADN Viral , Genes MHC Clase I , Genes Virales , Humanos , Huésped Inmunocomprometido , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Datos de Secuencia Molecular , Mutagénesis , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Proteínas Virales/fisiología , Replicación Viral/fisiologíaRESUMEN
Cytomegalovirus (CMV) infection is a major problem in the immunosuppressed patient. It is thought that besides direct CMV induced cell lysis, immunological damage is part of CMV pathogenesis. New antiviral drugs, which combine immunomodulating and antiviral qualities, could be beneficial. Recently, it has been described that desferrioxamine (DFO) and calcium trinatrium diethylenetriaminepentaacetic acid (DTPA) exhibit both properties. In this report the antiviral effects of both compounds against rat CMV (RCMV) are described in vitro and in vivo using a generalised and local infection model. In vitro, both compounds exhibited a significant antiviral effect, DTPA being more potent than DFO. However, in the generalised infection model no effect was seen on mortality, morbidity or presence of virus in internal organs. In rats infected subcutaneously in the hind paw, no effect was seen locally on paw thickness, presence of viral antigens and inflammatory response. In addition, these rats suffered from a generalised infection of low magnitude at 15 days post infection, although both DFO and DTPA were able to lower the level of viral replication. In conclusion, our data indicate that despite in vitro activity, in vivo usage of DFO or DTPA for acute CMV infection is not warranted.
Asunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/tratamiento farmacológico , Citomegalovirus/efectos de los fármacos , Deferoxamina/farmacología , Ácido Pentético/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antígenos Virales/análisis , Células Cultivadas , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Hígado/virología , Ratas , Ratas Endogámicas Lew , Glándulas Salivales/virología , Bazo/virología , Ensayo de Placa ViralRESUMEN
OBJECTIVE: Recently, we have found that rat CMV (RCMV) infected smooth muscle cells (SMCs) in rat carotid arteries when administered 14 days after balloon injury. In the present study we investigated (1) the long term effects of CMV infection on neointimal cross-sectional area, and (2) whether the phenotype of the intimal SMCs influences their susceptibility to active CMV infection. METHODS: In the first part of the study, rats received RCMV intravenously, two weeks after balloon catheterisation of the left carotid artery and were sacrificed twenty weeks after catheterisation. Continuous BrdU infusion was performed by subcutaneously implanted osmotic pumps during the last two weeks of life. In the second part RCMV was administered eight weeks after catheterisation and rats were sacrificed two weeks later. Immunohistochemistry was used to detect viral antigens, to determine BrdU incorporation as well as the contents of alpha-actin, desmin and vimentin in the carotid arteries. Intima and media cross-sectional areas were determined using computerized morphometry. RESULTS AND CONCLUSIONS: RCMV infection did not induce any differences in intima or media cross-sectional areas of the injured carotid artery, nor in the extent of SMC proliferation as shown by BrdU incorporation, 20 weeks after balloon catheterisation. Eight weeks after balloon catheterisation, RCMV no longer infected neointimal SMCs. This non-responsiveness to RCMV was associated with "re-differentiation" of the eight weeks old neointima, compared with two weeks after catheterization, as shown by the contents of alpha-actin, desmin and vimentin. Our data suggest that intimal SMC phenotype determines its susceptibility to active RCMV infection in vivo. Since de-differentiation of neointimal SMCs is associated with enhanced proliferation of these cells it is stated that de-differentiation or proliferation is prerequisite for infection.
Asunto(s)
Traumatismos de las Arterias Carótidas , Cateterismo , Infecciones por Citomegalovirus/transmisión , Músculo Liso Vascular/virología , Túnica Íntima/virología , Análisis de Varianza , Animales , Antígenos Virales/análisis , Biomarcadores/análisis , Arteria Carótida Común/patología , Arteria Carótida Común/virología , Diferenciación Celular , División Celular , Infecciones por Citomegalovirus/patología , Desmina/análisis , Susceptibilidad a Enfermedades , Músculo Liso Vascular/patología , Distribución Aleatoria , Ratas , Ratas Endogámicas WKY , Factores de Tiempo , Túnica Íntima/patologíaRESUMEN
In 8 of 10 wild rats trapped in The Netherlands, an infectious viruslike agent was isolated predominantly from the salivary glands and could be serially passed in laboratory rats. In rat embryo cells a typical cytomegalo-like cytopathic effect was produced. The morphologic and cultural characteristics of the isolated agent were comparable with those of the mouse cytomegalovirus (MCMV). The virus-nucleocapsid had a size of 92 nm and was not ether-resistant. The extracellular nucleocapsids were often enclosed by an outer layer of very variable shape and size. The formation of Fc receptors on cells infected with the rat virus could be demonstrated. The wild rats possessed neutralizing antibodies to the isolated agent. The rat agent grew only in rat embryo fibroblast cells while MCMV grew in rat and mouse embryo cells. The rat agent gave plaques in REF monolayers. Electron microscope studies showed the presence of nucleocapsids in the nucleus.