RESUMEN
Human cytomegalovirus (HCMV), a betaherpesvirus, has developed several ways to evade the immune system, notably downregulation of cell surface expression of major histocompatibility complex class I heavy chains. Here we report that HCMV has devised another means to compromise immune surveillance mechanisms. Extracellular accumulation of both constitutively produced monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor-superinduced RANTES (regulated on activation, normal T cell expressed and secreted) was downregulated in HCMV-infected fibroblasts in the absence of transcriptional repression or the expression of polyadenylated RNA for the cellular chemokine receptors CCR-1, CCR-3, and CCR-5. Competitive binding experiments demonstrated that HCMV-infected cells bind RANTES, MCP-1, macrophage inflammatory protein (MIP)-1beta, and MCP-3, but not MCP-2, to the same receptor as does MIP-1alpha, which is not expressed in uninfected cells. HCMV encodes three proteins with homology to CC chemokine receptors: US27, US28, and UL33. Cells infected with HCMV mutants deleted of US28, or both US27 and US28 genes, failed to downregulate extracellular accumulation of either RANTES or MCP-1. In contrast, cells infected with a mutant deleted of US27 continues to bind and downregulate those chemokines. Depletion of chemokines from the culture medium was at least partially due to continuous internalization of extracellular chemokine, since exogenously added, biotinylated RANTES accumulated in HCMV-infected cells. Thus, HCMV can modify the chemokine environment of infected cells through intense sequestering of CC chemokines, mediated principally by expression of the US28-encoded chemokine receptor.
Asunto(s)
Quimiocinas CC/metabolismo , Citomegalovirus/fisiología , Receptores de Quimiocina/metabolismo , Receptores Virales/metabolismo , Replicación Viral/inmunología , Sitios de Unión , Células Cultivadas , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/metabolismo , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/metabolismo , Quimiocinas CC/genética , Medios de Cultivo/metabolismo , Citomegalovirus/genética , Fibroblastos/metabolismo , Fibroblastos/virología , Eliminación de Gen , Humanos , Líquido Intracelular/metabolismo , Receptores CCR2 , Receptores de Quimiocina/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Regulation of alternative splicing events is an essential step required for the expression of functional cytoskeleton and sarcomere proteins in cardiomyocytes. About 3% of idiopathic dilated cardiomyopathy cases present mutations in the RNA binding protein RBM20, a tissue specific regulator of alternative splicing. Transcripts expressed preferentially in skeletal and cardiac muscle, including TTN, CAMK2D, LDB3, LMO7, PDLIM3, RTN4, and RYR2, are RBM20-dependent splice variants. In the present study, we investigated the RBM20 involvement in post-transcriptional regulation of splicing variants expressed by Formin homology 2 domain containing 3 (FHOD3) gene. FHOD3 is a sarcomeric protein highly expressed in the cardiac tissue and required for the assembly of the contractile apparatus. Recently, FHOD3 mutations have been found associated with heart diseases. We identified novel FHOD3 splicing variants differentially expressed in human tissues and provided evidences that FHOD3 transcripts are specific RBM20 and PTBP1 targets. Furthermore, we demonstrated that the expression of RBM20 and PTBP1 promoted the alternative shift, from inclusion to exclusion, of selected FHOD3 exons. These results indicate that RBM20 and PTBP1 play a role in the actin filament functional organization mediated by FHOD3 isoforms and suggest their possible involvement in heart diseases.
Asunto(s)
Empalme Alternativo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Microfilamentos/biosíntesis , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteínas de Unión al ARN/metabolismo , Forminas , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Proteínas de Microfilamentos/genética , Proteína de Unión al Tracto de Polipirimidina/genética , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Eighty-two HIV-1-seropositive subjects were examined for the presence and quantification of human cytomegalovirus (HCMV) in peripheral blood polymorphonuclear leukocytes (PMNL) by polymerase chain reaction, culture and immunofluorescence in order to investigate the relationship between viraemia and immunosuppression. Patients were divided into three groups: (1) asymptomatic subjects with greater than 400 x 10(6)/l CD4 lymphocytes (n = 30); (2) asymptomatic subjects with less than 400 x 10(6)/l of CD4 lymphocytes and zidovudine (n = 20), and (3) AIDS-related complex (ARC)/AIDS patients on zidovudine (n = 32). Evidence of HCMV infection in circulating PMNL was found in 15 out of 29 ARC/AIDS patients examined (51.7%), whereas no infection was detected among the 50 asymptomatic HIV-1-seropositive subjects. HCMV-related symptoms were found only where the number of infected PMNL was greater than 50 per 2 x 10(5) cells.
Asunto(s)
Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/aislamiento & purificación , Infecciones por VIH/complicaciones , Neutrófilos/microbiología , Infecciones Oportunistas/complicaciones , Complejo Relacionado con el SIDA/complicaciones , Complejo Relacionado con el SIDA/microbiología , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/microbiología , Adulto , Secuencia de Bases , Infecciones por Citomegalovirus/diagnóstico , Infecciones por VIH/microbiología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Viremia/complicaciones , Viremia/diagnósticoRESUMEN
PURPOSE: Human retinal pigment epithelial (RPE) cells and endothelial cells (HUVECs) are targets of human cytomegalovirus (HCMV) infection in vivo with significantly protracted replication in vitro compared with that in fibroblasts. This study analyzes the kinetics and mechanisms of HCMV entry into both cell types. METHODS: RPE cells were obtained from donor eyes. HUVECs were isolated from human umbilical cords. HCMV entrance was followed by electron microscopy and immunofluorescence in the presence of lysosomotropic agents and cytochalasin B. RESULTS: Human cytomegalovirus entered into RPE cells and HUVECs as early as 5 minutes after virus- cell contact. Entry was mediated by endocytosis, whereas HCMV enters fibroblasts through fusion. Most internalized viral particles and dense bodies appeared to be degraded within vacuoles. Viral entry, transport of viral proteins to the nucleus, and onset of viral transcription (immediate early [IE] protein expression) were significantly blocked by cytochalasin B. Lysosomotropic agents did not significantly reduce IE expression in RPE cells or HUVECs. CONCLUSIONS: This study shows that HCMV penetrates these highly specialized relevant cells via endocytosis. The low level of infection and the delay in the onset of HCMV expression seen in these cells compared with fibroblasts may be related to the sequestration and degradation of incoming viral particles in endocytic vacuoles.
Asunto(s)
Citomegalovirus/fisiología , Endocitosis/fisiología , Endotelio Vascular/virología , Epitelio Pigmentado Ocular/virología , Cloruro de Amonio/farmacología , Membrana Celular/ultraestructura , Membrana Celular/virología , Células Cultivadas , Cloroquina/farmacología , Citocalasina B/farmacología , Citomegalovirus/química , Citomegalovirus/ultraestructura , Endocitosis/efectos de los fármacos , Endotelio Vascular/química , Endotelio Vascular/ultraestructura , Fibroblastos/virología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Fusión de Membrana , Epitelio Pigmentado Ocular/química , Epitelio Pigmentado Ocular/ultraestructura , Proteínas Virales/análisis , Replicación Viral/fisiologíaRESUMEN
To test the hypothesis that human cytomegalovirus (CMV) gB genotype may differ with geographic origin or patient demographics, CMV DNA was amplified for gB typing from immunocompromised patients in Italy and Africa and compared with previously reported frequencies in California. Increased gB2 frequency occurred in Italian homosexual AIDS patients, as compared with both Italian heterosexual injection drug users with AIDS and heterosexual Zimbabwe AIDS patients. Occurrence of gB3 in Italy was higher in injection drug users than in homosexual AIDS patients. The incidence of gB4 was higher overall in the Italian as compared with the California patients. Therefore geographic and demographic differences in patients affect gB distribution and should be considered before associations of gB genotypes and virulence are made.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Citomegalovirus/genética , Huésped Inmunocomprometido , Genotipo , Humanos , Italia , Estados Unidos , ZimbabweRESUMEN
Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.
Asunto(s)
Infecciones por Citomegalovirus/microbiología , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Huésped Inmunocomprometido , Neutrófilos/microbiología , Reacción en Cadena de la Polimerasa , Viremia/microbiología , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Antígenos Virales/sangre , Secuencia de Bases , Citomegalovirus/genética , Infecciones por Citomegalovirus/complicaciones , Cartilla de ADN , ADN Recombinante/genética , Estudios de Seguimiento , Trasplante de Corazón/inmunología , Humanos , Datos de Secuencia Molecular , Complicaciones Posoperatorias/microbiologíaRESUMEN
Fifty AIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, while 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMV-related clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10(5) cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCR-positive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Síndrome de Inmunodeficiencia Adquirida/microbiología , Antígenos Virales/análisis , Antivirales/farmacología , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , ADN Viral/sangre , Foscarnet , Ganciclovir/farmacología , Humanos , Neutrófilos/microbiología , Ácido Fosfonoacético/análogos & derivados , Ácido Fosfonoacético/farmacología , Retinitis/microbiología , Proteínas Virales/sangreRESUMEN
Following amplification by PCR of a portion of the matrix phosphoprotein pp150 gene, electrophoretic analysis revealed the simultaneous presence of two viral variants of human cytomegalovirus in the blood of a heart transplant recipient. Repeated denaturation-annealing cycles during the amplification reaction led to the formation of heteroduplex molecules with altered electrophoretic mobility. Sequence analysis of the amplification products showed the presence of a viral variant carrying an in-frame three nucleotide deletion, which caused the absence of an aspartic acid in the corresponding protein. Attempts to plaque-purify the deletion mutant were unsuccessful, suggesting that the variant was growth-defective.
Asunto(s)
Citomegalovirus/genética , Genes Virales/genética , Mutación/genética , Fosfoproteínas/genética , Proteínas de la Matriz Viral/genética , Proteínas Virales/genética , Secuencia de Bases , Citomegalovirus/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , FenotipoRESUMEN
Cytomegalovirus (CMV) retinitis in patients infected with human immunodeficiency virus (HIV) is a significant clinical problem. Seventy-five patients with CD4 T cell counts <100/mm3 were monitored prospectively every 2 months for CMV DNA burden. The target for DNA amplification was a 162-bp fragment from the CMV immediate early gene. CMV DNA burden, at levels of > or =320 in white blood cells or > or =32 in plasma (P = .001), particularly when sustained (P = .005 and .008, respectively), distinguished patients who developed retinitis from those who remained free of disease. Progression to retinitis was not consistently accompanied by increases in CMV burden, indicating that quantitation of CMV burden beyond threshold levels is not necessary to predict risk for development of retinitis. Virus isolation from WBC, but not urine, was also significantly associated with risk for retinitis (P = .001).
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Retinitis/etiología , Viremia/complicaciones , Adulto , Recuento de Linfocito CD4 , Citomegalovirus/genética , Femenino , Dosificación de Gen , Humanos , Masculino , Persona de Mediana EdadRESUMEN
In permissive cells, human cytomegalovirus encodes the protein US28, a functional CC chemokine receptor. US28 polyadenylated mRNA could be detected by RT-PCR as early as 2 h post-infection. US28 mRNA appeared after major IE1 transcripts (UL123), but before transcripts of the early genes pp65 (UL83) and gB (UL55), and the late gene pp150 (UL32). This temporal appearance indicates that US28 is transcribed earlier than previously reported. Furthermore, US28 mRNA could be detected in semi- and non-permissive cells.
Asunto(s)
Citomegalovirus/genética , Receptores de Quimiocina/genética , Transcripción Genética/genética , Animales , Línea Celular , Células Cultivadas , Cicloheximida/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/crecimiento & desarrollo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/genética , Humanos , Sistemas de Lectura Abierta/genética , ARN Mensajero/metabolismo , Receptores CCR2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Proteínas Virales/genética , Replicación ViralRESUMEN
An in vitro model of human CMV infection of primary retinal pigment epithelial (RPE) cells was used to study the effects of cytokines on CMV replication in these cells, which are targets of CMV infection in vivo. IFN-gamma and IFN-beta were potent inhibitors of CMV replication in RPE cells, while TNF-alpha, IL-1beta, or TGF-beta2 did not affect viral replication. Inhibition by IFN-gamma, and to a lesser extent IFN-beta, was almost completely reversed by addition of L-tryptophan to the culture medium, strongly implicating the indoleamine 2,3 dioxygenase (IDO) pathway. Polyadenylated IDO mRNA accumulation was detected as early as 2 h after IFN stimulation. Furthermore, CMV blocked the production of nitric oxide by the inducible form of nitric oxide synthase. This inhibition depended on a functional viral genome. However, exogenous nitric oxide significantly inhibited viral protein expression in RPE cells. Thus, CMV infection blocks the inducible nitric oxide synthase pathway activated by IFN-gamma and IL-1beta, but cannot counteract the IFN-induced IDO pathway, which ultimately controls its replication in primary human RPE cells.
Asunto(s)
Citomegalovirus/fisiología , Interferón gamma/fisiología , Óxido Nítrico Sintasa/fisiología , Epitelio Pigmentado Ocular/enzimología , Epitelio Pigmentado Ocular/virología , Triptófano Oxigenasa/fisiología , Replicación Viral/inmunología , Antivirales/antagonistas & inhibidores , Antivirales/fisiología , Citocinas/fisiología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Triptófano/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
In an AIDS patient with a disseminated human cytomegalovirus (HCMV) infection, presence of HCMV in blood was repeatedly excluded by the shell vial culture method with the HCMV immediate-early (IE) antigen-specific monoclonal antibody (MAb) 5D2 currently employed for rapid HCMV identification, whereas it was repeatedly confirmed by all other assays (conventional virus isolation from blood, antigenemia, and DNAemia). Sequence analysis of the HCMV strain revealed a point mutation in exon 2 of the IE gene, which led to a serine-to-proline substitution at position 20 of the corresponding protein. Cloning and expression of a region of the IE gene containing the mutation showed that this was responsible for the lack of reactivity of MAb 5D2. A pool of IE antigen-reactive MAbs instead of a single MAb must be used for rapid HCMV identification to detect all viral strains.
Asunto(s)
Antígenos Virales/genética , Citomegalovirus/genética , Citomegalovirus/inmunología , Genes Virales , Proteínas Inmediatas-Precoces/genética , Mutación Puntual , Infecciones Oportunistas Relacionadas con el SIDA/complicaciones , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Citomegalovirus/clasificación , Infecciones por Citomegalovirus/complicaciones , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/microbiología , ADN Complementario/genética , ADN Viral/genética , Humanos , Datos de Secuencia Molecular , Viremia/complicaciones , Viremia/diagnóstico , Viremia/microbiologíaRESUMEN
Fourteen immunocompromised patients were examined for viremia, pp65 and p72 antigenemia, and presence of viral DNA in leukocyte fractions of polymorphonuclear leukocytes (PMNL), monocytes/macrophages (M/M), and B and T lymphocytes after purification by fluorescence-activated cell sorting. Nearly all PMNL and M/M fractions were positive for DNA and pp65 antigenemia, while p72 antigenemia was detected in 73% and 62%, respectively. The virus isolation rate was 45% from PMNL and 17% from M/M. T lymphocytes were positive for DNA in 50% of cases and for pp65 and p72 antigenemia in only 11%, while B lymphocytes were DNA-positive in 43% of samples and consistently negative for antigenemia; neither T nor B lymphocytes had virus isolated. Immediate-early (IE)1 RNA was present in 23 (85.2%) of 27 dextran-enriched DNA-positive p72-positive PMNL samples and, in sequential PMNL samples from two heart-transplanted patients, was detected during peak infection in association with p72. Thus, PMNL and M/M are the subpopulations primarily involved in HCMV infection; PMNL may undergo IE replicative events and are not merely passive carriers of phagocytized viral material.
Asunto(s)
Infecciones por Citomegalovirus/microbiología , Citomegalovirus/fisiología , Proteínas Inmediatas-Precoces , Neutrófilos/microbiología , Viremia/microbiología , Adulto , Antígenos Virales/sangre , Antígenos Virales/química , Antígenos Virales/genética , Secuencia de Bases , Separación Celular , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/sangre , ADN Viral/sangre , ADN Viral/química , Estudios de Seguimiento , Trasplante de Corazón , Humanos , Huésped Inmunocomprometido , Persona de Mediana Edad , Datos de Secuencia Molecular , Fosfoproteínas/sangre , Reacción en Cadena de la Polimerasa , ARN Viral/sangre , ARN Viral/química , Proteínas de la Matriz Viral/sangre , Viremia/sangre , Replicación ViralRESUMEN
Fifty human cytomegalovirus (HCMV) isolates were recovered from different clinical specimens (buffy coat, throat washing and urine) obtained from fifty patients (23 AIDS patients, 20 heart transplant recipients, 1 bone marrow transplant recipient, 2 newborns with congenital HCMV infection and 4 immunocompetent individuals with acute HCMV infection). The isolates were previously identified by immunological methods and then examined for identification by the polymerase chain reaction. In parallel, reference HCMV strains as well as other human members of the Herpesviridae family (reference and wild strains) were examined as controls. Two pairs of primers relevant to the immediate-early and late regions of HCMV DNA, respectively, were used. The DNA amplification product was highly specific; in addition, all fifty HCMV isolates were amplified by both pairs of primers and thus identified as HCMV. These preliminary results show that selected pairs of primers are able to amplify DNA regions conserved enough to allow virus identification among a large number of HCMV strains. In addition, they are highly promising in view of the use of PCR for direct detection of HCMV DNA in clinical samples.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
The presence of human cytomegalovirus (HCMV) DNA in blood was investigated by the polymerase chain reaction (PCR) in 293 blood samples from 86 immunocompromised patients. Of the 86 patients, 23 underwent clinical and virologic follow-up for HCMV infection. In parallel, blood samples were examined for viremia and antigenemia. Concordant results between PCR and assays for viremia and antigenemia were obtained on 124 positive and 110 negative samples, with an overall concordance of 79.8%, while 59 samples (most from patients with HCMV infection) were positive by PCR alone. PCR is a new powerful tool for detection of HCMV infections in blood samples from immunocompromised patients. However, its clinical significance appears to be restricted to the indication of a risk of reactivation of HCMV infection.
Asunto(s)
Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Reacción en Cadena de la Polimerasa/métodos , Antígenos Virales/sangre , Citomegalovirus/genética , Infecciones por Citomegalovirus/diagnóstico , Estudios de Evaluación como Asunto , Humanos , Huésped Inmunocomprometido , Viremia/diagnóstico , Virología/métodosRESUMEN
The aim of this study was to investigate peripheral blood polymorphonuclear leukocytes and, whenever possible, aqueous humor from 65 AIDS patients with ophthalmoscopically diagnosed human cytomegalovirus (HCMV) retinitis to determine (i) whether patients consistently carry viral DNA and (ii) to what extent foscarnet induction treatment decreases viral DNA levels. HCMV DNA was quantified by PCR using densitometric analysis of hybridization products obtained from external standards and a standard curve from which the number of genome equivalents of test samples, normalized by using an internal amplification control, was interpolated. Results showed that 56 of 65 patients (86.1%) were positive for HCMV DNA prior to induction treatment. Of 41 of the 56 patients (73.2%) whose blood had become DNA negative after induction, only 5 had a high viral load (> 5,000 genome equivalents per 2 x 10(5) polymorphonuclear leukocytes) prior to induction, whereas as many as 13 of the 15 (26.8%) patients remaining DNA positive after induction had a high viral load prior to induction. Finally, of the nine patients (13.8%) with DNA-negative blood prior to induction treatment, three were shifted to foscarnet from ganciclovir, while six were erroneously enrolled in the study. Pre- and postinduction aqueous humor samples were obtained from 12 patients; all of these were DNA positive prior to induction, whereas after induction, 4 became negative, 6 showed a marked decrease in viral DNA, and 2 had nearly stable low DNA levels. In conclusion, PCR is a valuable tool for etiologic diagnosis and monitoring of HCMV retinitis treatment in AIDS patients. HCMV DNA is consistently present in the blood and aqueous humor of all patients with HCMV retinitis. Foscarnet induction treatment is highly effective in suppressing or reducing DNA levels in both blood leukocytes and aqueous humor.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/microbiología , Humor Acuoso/metabolismo , Retinitis por Citomegalovirus/microbiología , Citomegalovirus/metabolismo , ADN Viral/biosíntesis , Foscarnet/farmacología , Neutrófilos/microbiología , Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/metabolismo , Adulto , Humor Acuoso/efectos de los fármacos , Citomegalovirus/efectos de los fármacos , Retinitis por Citomegalovirus/tratamiento farmacológico , Retinitis por Citomegalovirus/metabolismo , ADN Viral/análisis , Foscarnet/efectos adversos , Foscarnet/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismoRESUMEN
A quantitative DNA amplification assay for human cytomegalovirus (CMV) DNA has been used to evaluate the relationship between quantities of CMV DNA in plasma and those in infected leukocytes (WBC) from human immunodeficiency virus-infected patients. The target sequence for DNA amplification was a region of the immediate-early 1 gene of CMV. The quantitation assay uses an internal control that is coamplified with each patient sample DNA and contains a sequence for detection by colorimetric hybridization with the same bases, but in different order than in the CMV immediate-early 1 region used for hybridization of amplified patient sample DNA. Results showed that patients with CMV disease had more CMV DNA in both WBC and plasma than those without disease. However, in this study, copy numbers of CMV DNA in WBC were higher than those in plasma. The gB and gH variants were the same in plasma and WBC.
Asunto(s)
Infecciones por Citomegalovirus/sangre , ADN Viral/aislamiento & purificación , Infecciones por VIH/complicaciones , Leucocitos/virología , Secuencia de Bases , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/patología , ADN Viral/sangre , Genotipo , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Semen/citología , Semen/virologíaRESUMEN
Human cytomegalovirus (CMV) DNA copy number in white blood cells from both human immunodeficiency virus (HIV)-seronegative and HIV-seropositive patients was amplified from the immediate-early region of CMV DNA and quantified by colorimetric detection of the hybridization of the amplification product to a detector oligonucleotide probe in microtiter wells. By Mann-Whitney U test, significantly higher (P < .05, two-tailed) copy numbers of CMV DNA were detected in HIV-seropositive patients with retinitis than in either patients with < 100 CD4 cells/mm3 and no symptomatic CMV disease or HIV-seropositive patients with > 100 CD4 cells/mm3. By prospective monitoring for increases in CMV DNA copy number, it may be possible to identify HIV-seropositive patients who are at imminent risk for development of symptomatic CMV retinitis.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Retinitis por Citomegalovirus/diagnóstico , Citomegalovirus/genética , ADN Viral/sangre , Infecciones por VIH/virología , Leucocitos/virología , Secuencia de Bases , Biomarcadores/sangre , Recuento de Linfocito CD4 , Citomegalovirus/aislamiento & purificación , Seronegatividad para VIH , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Factores de Riesgo , Sensibilidad y EspecificidadRESUMEN
Fourteen heart transplant recipients were monitored for human cytomegalovirus (HCMV) infection based on determination of antigenemia, viremia, and DNAemia (by polymerase chain reaction [PCR]) in peripheral blood polymorphonuclear leukocytes (PMNL). Three patients had symptomatic primary, 10 had recurrent (3 asymptomatic), and 1 (seronegative) had no HCMV infection. Severe clinical symptoms appeared when levels of viremia/antigenemia were greater than 50 infected PMNL/2 x 10(5) cells examined. Of 200 blood samples examined, 93 (46.5%) were positive for viremia/antigenemia and DNAemia, whereas 48 (24.0%) were positive for DNAemia only; 59 (29.5%) were negative in all assays. Follow-up of HCMV infections in heart transplant recipients showed that PCR can detect viral appearance in blood 7-10 days earlier than assays for antigenemia/viremia. On the other hand, viral disappearance from blood, as assessed by PCR, occurred weeks or months later than revealed by other assays. Detection of virus by PCR only was never associated with overt HCMV-related clinical symptoms. Of the 8 symptomatic patients treated with ganiclovir, 2 became PCR-negative at the end of treatment and 1 cleared virus from blood in the following weeks, whereas 5 showed persistent or recurrent infection.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , Ganciclovir/uso terapéutico , Trasplante de Corazón , Complicaciones Posoperatorias/diagnóstico , Antígenos Virales/sangre , Secuencia de Bases , Citomegalovirus/genética , Citomegalovirus/inmunología , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/etiología , ADN Viral/sangre , Estudios de Seguimiento , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Chemokines play a major role in inflammatory responses and affect hematopoiesis both negatively and positively. We show that fresh isolates and laboratory strains (Towne and Ad-169) of human cytomegalovirus (HCMV) induce production of the CC chemokine RANTES in fibroblasts. Induction of extracellular RANTES production occurred as early as 8 h after infection, peaked around 24 h after infection, and was almost undetectable by 48 and 72 h. Upregulation occurred in the absence of viral DNA synthesis, suggesting that it was due to immediate-early-early HCMV gene expression. CMV infection stimulated RANTES transcription, since reverse transcription-PCR detected a sharp increase in RANTES RNA which persisted even when extracellular RANTES was no longer detected. Induction of RANTES in fibroblasts was not due to prior induction of tumor necrosis factor alpha or interleukin 1 beta. Down-regulation required an active viral genome. Decrease of RANTES in culture supernatants may be associated with the appearance of the HCMV CC chemokine receptor US28, since we show that this gene is transcribed as early as 8 h after infection. Modulation of CC chemokine production early during CMV infection might have a regulatory effect on viral replication, as well as affect immune surveillance.