RESUMEN
Recombinant Semliki Forest virus (SFV) is an attractive viral vector system owing to its ability to allow high efficiency of viral protein expression. To produce recombinant pseudotyped human immunodeficiency virus type 1 (HIV-1) virions, we designed a chimeric SFV/HIV vector system that contains both the HIV-1 cis- and trans-acting elements under the transcriptional control of the SFV replicase and investigated the ability of the hybrid SFV/HIV system to produce lentiviral particles capable of transducing target cells. Co-transfection of target cells with the two helper SFV packaging system RNAs along with each SFV/Gag-Pol, SFV/VSV(G) as well as SFV/HIV-1 vector unit replicon led to the generation of efficient transducing competent recombinant SFV/HIV particles. In contrast, co-transduction of target cells with the SFV/HIV chimeric virions produced recombinant particles with low transducing ability. Our data suggest that both the genomic and the subgenomic RNAs containing the HIV-1 vector unit were negatively selected for incorporation into recombinant particles, despite the fact that the SFV-driven HIV-1 vector replicon was the only one containing a lentiviral packaging sequence. The results of this study provide insights relevant to the design of chimeric lentiviral vectors.
Asunto(s)
VIH-1/genética , Virus de los Bosques Semliki/genética , Transactivadores/biosíntesis , Línea Celular , Vectores Genéticos/genética , Humanos , Recombinación Genética , Replicón/genética , Transactivadores/genética , Transducción Genética , Virión/genéticaRESUMEN
We investigated the incidence, risk factors and outcome of haemorrhagic cystitis (HC) in paediatric patients undergoing HSCT and the predictive value of BK viruria and viraemia for developing HC. Over a period of 54 months, 74 patients were recruited. The cumulative incidence of HC was 22%. Among 15 patients prospectively monitored for BK viruria and viraemia, four patients developed HC of grade > or =II. This group, which had two consecutive BK positive samples, showed a sensitivity of 100%, a specificity of 82%, a positive predictive value of 67%, and negative predictive value of 100% for developing HC. Analysed by a receiver-operator characteristic curve (ROC), a urine BK load >9 x 10(6) genomic copies/ml had a sensitivity of 95% and specificity of 90%; while a blood BK load >1 x 10(3) genomic copies/ml had a sensitivity of 40% and a specificity of 93% for HC, respectively. In univariate analysis, BK positivity was the only factor significantly associated with HC. After a median follow-up of 1.8 years, patients with HC showed a lower overall survival, 40 vs 65%, P 0.01, and a lower event-free survival, 42 vs 62%, P 0.03, compared to patients without HC. We conclude that BK detection in urine and/or plasma is a specific predictor for developing HC.
Asunto(s)
Virus BK/patogenicidad , Cistitis/virología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Infecciones por Polyomavirus/complicaciones , Infecciones Tumorales por Virus/complicaciones , Adolescente , Niño , Preescolar , Cistitis/epidemiología , Cistitis/fisiopatología , Supervivencia sin Enfermedad , Femenino , Humanos , Incidencia , Lactante , Italia/epidemiología , Masculino , Infecciones por Polyomavirus/epidemiología , Estudios Prospectivos , Trasplante Homólogo/efectos adversos , Infecciones Tumorales por Virus/epidemiología , Carga Viral , ViremiaRESUMEN
The Enterovirus may be the most common agent responsible for viral myocarditis and cardiomyopathy. Very little of the literature is available concerning the follow-up of patients who underwent transplantation with enteroviral positivity in native hearts. In the present study, 45 explanted hearts from patients who underwent orthotopic heart transplant at University of Padova were studied by reverse transcriptase (RT)-polymerase chain reaction (PCR): 27 patients had dilated cardiomyopathy (DC), 12 had ischemic cardiopathy (IC), 2 had valvular disease (VD), 2 had arrhythmogenic right ventricular cardiomyopathy (ARVC), 1 had giant cell myocarditis (GCM), and 1 had lymphocytic myocarditis (LM). Two sets of PCR primers from the highly conserved region of Enterovirus and Rhinovirus were used. Samples of both ventricles and septum were analyzed in every patients. The RT-PCR and nucleotide sequencing of amplicons were also performed on all post-transplantation follow-up biopsies in patients with Enterovirus positivity in the native heart. The viral genome was detectable in only 1 of 27 patients with DC (3%) and in 1 patient with LM. Nucleotide sequence analysis of the amplified product showed differences in nucleotide sequence of PCR samples compared with the sequence of the coxsackievirus B3 used in the current study. The patient with Enterovirus-positive DC showed a higher index of severe rejection (>3A) in the first 6 months, compared with the other patients tested. The patient with Enterovirus-positive LM died of disease recurrence 2 months after transplantation. The present study reveals a scarce presence of Enterovirus in the myocardium of patients with chronic myocardial disease. Because Enterovirus infection was predictive of a poor prognosis in these two patients, molecular studies are useful in excluding viral involvement in native hearts of transplanted patients.
Asunto(s)
Infecciones por Enterovirus/virología , Enterovirus/genética , Genoma Viral , Cardiopatías/virología , Trasplante de Corazón , Adolescente , Secuencia de Bases , Cartilla de ADN/química , Enterovirus/aislamiento & purificación , Infecciones por Enterovirus/genética , Estudios de Seguimiento , Rechazo de Injerto , Cardiopatías/genética , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Pronóstico , ARN Viral/análisis , Rhinovirus/genéticaRESUMEN
Different strategies proposed in the literature to attempt gene therapy of AIDS are based mainly on the intracellular production of RNA and protein therapeutics. This report describes the construction and the anti-human immunodeficiency virus type 1 (HIV-1) activity of a new type of antisense tRNA directed against a nucleotide region in the first coding exon of HIV-1 tat (nucleotides 5924 to 5943; Los Alamos data bank) which is conserved among many HIV-1 clones. The anti-tat antisense sequence was inserted into a tRNA(Pro) backbone by replacement of the anticodon loop, without altering the tRNA canonic tetraloop structure. The antisense tRNA was able to interact effectively with its target in vitro. Jurkat cells that constitutively expressed the anti-tat tRNA following retroviral vector transduction exhibited significant resistance to HIV-1 de novo infection. Resistance seemed to correlate with the level of antisense expression. This is the first time that such a tRNA antisense strategy has been shown to be effective as a genetic treatment of HIV-1 infection in tissue culture. The construct design proposed in this report has some intrinsic advantages: the transcript is driven by a polymerase III promoter, the short length of the RNA minimizes effects of intramolecular base pairing that may impair target recognition, and the antisense RNA has the stability and intracellular fate of a native tRNA molecule.
Asunto(s)
Productos del Gen tat/genética , Terapia Genética , VIH-1/genética , ARN sin Sentido/genética , ARN de Transferencia de Prolina/genética , Secuencia de Bases , Línea Celular , Técnicas de Transferencia de Gen , Vectores Genéticos , Infecciones por VIH/genética , Infecciones por VIH/terapia , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN sin Sentido/química , ARN de Transferencia de Prolina/química , Retroviridae/genética , Linfocitos T/virología , Replicación Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Endothelin-1 stimulates aldosterone secretion by interacting with specific receptors. Accordingly, we wished to investigate endothelin-1, endothelin-A (ETA) receptor, and endothelin-B (ETB) receptor gene expression, localization, and properties in aldosterone-producing adenomas and in the normal human adrenal cortex. We carried out 125I-endothelin-1 displacement studies with cold endothelin-1, endothelin-3, the specific ETA antagonist BQ-123, and the specific ETB weak agonist sarafotoxin 6 C and coanalyzed data with the nonlinear iterative curve-fitting program LIGAND. We also studied gene expression with reverse transcription-polymerase chain reaction with specific primers for endothelin-1, ETA, and ETB complementary DNA. Normal adrenal cortices from consenting kidney cancer patients (n = 2) and aldosterone-producing adenomas (n = 4) were studied; for the latter, surrounding normal cortex and kidney biopsy tissue served as controls. To further localize the receptor subtypes, tissue sections were studied by autoradiography in the presence and absence of 500 nmol/L BQ-123, 100 nmol/L sarafotoxin 6 C, and 1 mumol/L cold endothelin-1. In all tissues examined, endothelin-1, ETA, and ETB messenger RNAs were easily detected. However, in aldosterone-producing adenomas, both receptors' genes were expressed at a higher level than in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adenoma/metabolismo , Neoplasias de las Glándulas Suprarrenales/metabolismo , Aldosterona/biosíntesis , Endotelinas/metabolismo , Receptores de Endotelina/metabolismo , Autorradiografía , Unión Competitiva , Endotelinas/agonistas , Endotelinas/antagonistas & inhibidores , Humanos , Péptidos Cíclicos/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Endotelina/clasificación , Receptores de Endotelina/genética , Valores de Referencia , Transcripción Genética , Venenos de Víboras/farmacologíaRESUMEN
In this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa S3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K(+)-ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na+ and K+ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid peroxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K(+)-ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in non-phagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity.
Asunto(s)
Membrana Celular/fisiología , Herpesvirus Humano 1/fisiología , Membrana Celular/metabolismo , Membrana Celular/virología , Permeabilidad de la Membrana Celular , Efecto Citopatogénico Viral , Difenilhexatrieno/análogos & derivados , Colorantes Fluorescentes , Radicales Libres , Células HeLa , Humanos , Peroxidación de Lípido , Malondialdehído/metabolismo , Fluidez de la Membrana , Potenciales de la Membrana , Potasio/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Replicación ViralRESUMEN
Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.
Asunto(s)
Corteza Suprarrenal/metabolismo , Expresión Génica , Receptores de Endotelina/biosíntesis , Autorradiografía , Secuencia de Bases , Unión Competitiva , Cartilla de ADN , ADN Complementario/aislamiento & purificación , ADN Complementario/metabolismo , Endotelinas/metabolismo , Humanos , Radioisótopos de Yodo , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/análisis , Receptores de Endotelina/metabolismoRESUMEN
A ribozyme was constructed that specifically cleaves RNA that contains the first coding exon of the tat gene of HIV-1. This anti-tat ribozyme was incorporated into a Moloney murine leukemia virus vector. A sequence containing only the 48-nucleotide antisense region of the ribozyme was also inserted into the retroviral vector. Human T-cell lines constitutively producing the tat-antisense and the anti-tat ribozyme RNA were created by transduction into Jurkat cells. When challenged with HIV-1, both the tat-antisense and anti-tat ribozyme-producing cells inhibited the replication of HIV-1. The antisense vector conferred a greater resistance to HIV-1 replication than did the anti-tat ribozyme vector.
Asunto(s)
Productos del Gen tat/genética , VIH-1/genética , ARN sin Sentido/genética , ARN Catalítico/genética , Replicación Viral/genética , Secuencia de Bases , Northern Blotting , Línea Celular , ADN Viral , Células Gigantes/citología , VIH-1/fisiología , Humanos , Cinética , Datos de Secuencia Molecular , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Linfocitos T/microbiología , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The synthesis of thymidines selectively dansylated in the 3'- and 5'-positions is reported. The biological investigation showed that these fluorescent nucleosides behave as competitive inhibitors of thymidine kinase (TK) from herpes simplex virus (HSV) and are endowed with a certain degree of antiviral activity against HSV-2 and HSV-1.
Asunto(s)
Antivirales/síntesis química , Compuestos de Dansilo/síntesis química , Timidina/análogos & derivados , Células Cultivadas , Fenómenos Químicos , Química , Compuestos de Dansilo/farmacología , Simplexvirus/efectos de los fármacos , Espectrofotometría Ultravioleta , Timidina/síntesis química , Timidina/farmacología , Timidina Quinasa/antagonistas & inhibidores , Virus Vaccinia/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacosRESUMEN
The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants.
Asunto(s)
Aciclovir/farmacología , Simplexvirus/efectos de los fármacos , Timidina Quinasa/metabolismo , Aciclovir/análogos & derivados , Aciclovir/metabolismo , Sitios de Unión , Farmacorresistencia Microbiana/genética , Cinética , Mutación , Simplexvirus/enzimología , Simplexvirus/genética , Timidina Quinasa/genéticaRESUMEN
The nucleotide and deduced amino acid sequence of the thymidine kinase gene of a new strain of herpes simplex virus type 1 is reported in comparison with the published sequences for three other strains. The primary gene structure is shown to be highly conserved. Changes normally occur at the same positions in the protein molecule and are not distinctive of any specific strain since they can be found in each one of them. However, a unique substitution takes place in our strain at amino acid position 240 where a glutamic acid replaces a glycine. This modification apparently does not alter the enzyme activity and, therefore, must be located outside the catalytic site.