Asunto(s)
Antígenos de Plantas/metabolismo , Toma de Decisiones Clínicas/métodos , Material Particulado/metabolismo , Polen/metabolismo , Rinitis Alérgica Estacional/diagnóstico , Adulto , Alérgenos/inmunología , Antígenos de Plantas/inmunología , Fagales , Femenino , Humanos , Masculino , Región Mediterránea/epidemiología , Material Particulado/inmunología , Pinales , Poaceae , Polen/inmunología , Rinitis Alérgica Estacional/epidemiología , Estaciones del AñoRESUMEN
Primary immunodeficiencies (PIDs) are rare diseases characterized by an increased susceptibility to infections. Early diagnosis and appropriate treatment are critical for reducing morbidity and mortality. Based on available data, the efficacy of antibiotic administration for the prophylaxis of infections remains uncertain, and recommendations supporting this practice are poor. The use of antimicrobial prophylaxis is mainly based on single institution-specific experience without controlled measurements of patient safety and quality health outcomes. To address this issue an Italian Network on Primary Immunodeficiencies (IPINet) has been set up in 1999 within the Italian Association of Pediatric Hematology and Oncology (AIEOP) to increase the awareness of these disorders among physicians. Further, diagnostic and treatment guideline recommendations have been established to standardize the best clinical assistance to all patients, including antibiotic prophylaxis, and for a national epidemiologic monitoring of PIDs. The aim of this review is not only to give a scientific update on the use of antimicrobial prophylaxis in selected congenital immunological disorders but also to draw a picture of this practice in the context of the Italian Primary Immunodeficiency Network (IPINet). Controlled multicenter studies are necessary to establish if, when and how you should start an efficacious antimicrobial prophylaxis.
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Profilaxis Antibiótica , Síndromes de Inmunodeficiencia/complicaciones , Inmunodeficiencia Variable Común/complicaciones , Síndrome de DiGeorge/complicaciones , Enfermedad Granulomatosa Crónica/complicaciones , Humanos , Deficiencia de IgA/complicaciones , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/complicacionesRESUMEN
Increased levels of serum IgE and eosinophilia have been described in human immunodeficiency virus (HIV) infection, almost exclusively in patients with CD4+ cell count < 200 cells/microliters. IgE production is regulated by CD4+ T helper type 2 (Th-2) lymphocytes, producing interleukin 4 (IL-4) and expressing a ligand for the B cell-specific CD40 molecule (CD40 ligand [L]). A shift to a Th-2-like pattern of cytokine secretion has been postulated to be associated with progression toward acquired immunodeficiency syndrome (AIDS). We studied three AIDS patients with very high levels of IgE and almost complete depletion of CD4+ lymphocytes, suggesting that IgE synthesis could not be driven by CD4+ cells. IgE in vitro synthesis by cells from such patients was, however, inhibited by anti-IL-4. We show that both CD8+ T cell lines and the majority of CD8+ T cells clones derived from these patients produce IL-4, IL-5, and IL-6 in half of the cases together with interferon gamma (IFN-gamma). 44% of CD8+ T cell clones expressed a CD40L, and the supernatants of the clones were capable of inducing IgE synthesis by normal B cells costimulated with anti-CD40. CD8+ T cells in these patients therefore functionally mimic Th-2 type cells and may account for hyper-IgE and eosinophilia in the absence of CD4+ cells. The presence of such CD8+ cells may also provide a source of IL-4 directing the development of predominant Th-2 responses in HIV infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Linfocitos T CD8-positivos/inmunología , Hipergammaglobulinemia/metabolismo , Inmunoglobulina E/biosíntesis , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Ligando de CD40 , Citocinas/genética , Expresión Génica , Humanos , Linfocinas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana EdadRESUMEN
OBJECTIVE: To investigate the contribution of inherited and acquired thrombophilic defects to the clinical manifestations of mixed cryoglobulinaemia vasculitis. METHODS: The following thrombophilic defects were investigated in 64 consecutive patients with HCV-associated mixed cryoglobulinaemia: aPLs, lupus anti-coagulant, homocysteinaemia, protein C and protein S concentrations, activated protein C resistance, plasminogen activator inhibitor-1 4G4G and 5G5G genotypes, and the presence of mutations of factor V (Leiden and H1299R), of prothrombin (G20210A) and of methyl tetrahydrofolate reductase (C677T and A1298C). Additional variables were demographic data, duration of the disease, cryocrit level and vascular risk factors (diabetes, hypertension, hypercholesterolaemia and smoking habit). The following clinical manifestations of mixed cryoglobulinaemia were analysed as dependent covariates: severity of purpura, presence of necrotic skin ulcers, presence of peripheral neuropathy and presence of kidney disease. RESULTS: Logistic regression analysis identified hyperhomocysteinaemia as a risk factor for severe purpura (P < 0.0001) and for the presence of skin ulcers (P < 0.0001), whereas none of the other thrombophilic defects influenced the clinical presentation of mixed cryoglobulinaemia. Purpura improved in two patients after lowering homocysteine with vitamin supplementation. CONCLUSIONS: Hyperhomocysteinaemia may be a risk factor for severe cutaneous manifestations in mixed cryoglobulinaemia.
Asunto(s)
Crioglobulinemia/complicaciones , Hiperhomocisteinemia/complicaciones , Úlcera Cutánea/etiología , Vasculitis/etiología , Resistencia a la Proteína C Activada , Adulto , Anciano , Estudios de Casos y Controles , Crioglobulinemia/genética , Factor V/genética , Femenino , Humanos , Hiperhomocisteinemia/genética , Modelos Logísticos , Inhibidor de Coagulación del Lupus/genética , Masculino , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Persona de Mediana Edad , Protrombina/genética , Factores de Riesgo , Piel/irrigación sanguínea , Úlcera Cutánea/genética , Vasculitis/genéticaRESUMEN
OBJECTIVE: Nine outbreaks of group A streptococcal (GAS) infections in nursing homes were reported to the Centers for Disease Control (Atlanta, Ga) during the past two winters. We conducted an intensive epidemiologic and laboratory investigation of one of these outbreaks to determine clinical characteristics, risk factors for transmission and infection, and methods of control and prevention. METHODS: Cases were detected using cultures and serologic tests. Matched case-control and retrospective cohort studies were performed to determine risk factors for infection. RESULTS: Between December 13, 1989, and January 31, 1990, 16 (20%) of 80 residents, and three (7%) of 45 staff, were infected with GAS. Eleven of the residents had invasive disease and four died. Isolates were available from four persons; all were serotype M-1, T-1. There was strong spatial clustering of cases within the nursing home; having a roommate with prior infection was the most important risk factor. Residents with preexisting decubiti had a reduced risk of infection, perhaps because of stricter infection control practices in their care. No evidence was found for common-source transmission of infection. No further cases occurred after improvement of infection control practices and administration of prophylactic antimicrobials to all residents and staff. CONCLUSIONS: Invasive GAS disease is increasing nationwide, and is a potentially serious problem in the growing and high-risk setting of nursing homes. These data suggest that, in this outbreak, a virulent GAS strain was introduced, with subsequent person-to-person transmission. Adherence to infection control practices can prevent or control GAS outbreaks. Prophylactic antimicrobials may be an effective adjunct to control severe or ongoing outbreaks.
Asunto(s)
Infección Hospitalaria/microbiología , Brotes de Enfermedades/estadística & datos numéricos , Hogares para Ancianos/estadística & datos numéricos , Casas de Salud/estadística & datos numéricos , Infecciones Estreptocócicas/epidemiología , Streptococcus pyogenes/aislamiento & purificación , Anciano , Estudios de Casos y Controles , Análisis por Conglomerados , Infección Hospitalaria/epidemiología , Femenino , Humanos , Masculino , North Carolina/epidemiología , Estudios Retrospectivos , Factores de Riesgo , Infecciones Estreptocócicas/prevención & controlRESUMEN
It has been reported that the period of latency between HIV-1 infection and the production of antibodies against the virus is sometimes prolonged for greater than 6 months. However, the data supporting this are still controversial and it is not known whether these individuals are actually infectious, especially through body fluids. We have performed a prospective study of 65 high-risk HIV-1-antibody-negative individuals who were followed-up for a period of at least 1 year. Twelve of these individuals were shown by polymerase chain reaction (PCR) to be carriers of HIV-1 proviral sequences. The virus was isolated from lymphocytes in five out of 10 PCR-positive subjects and from cell-free plasma in two. Our data indicate that in some cases delayed seroconversions may be associated with productive infection, suggesting that mechanism(s) other than viral latency may be responsible for the absence of antibody responses to HIV-1 proteins.
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Células Sanguíneas/microbiología , Infecciones por VIH/microbiología , Seropositividad para VIH/microbiología , VIH-1/patogenicidad , Viremia , ADN Viral/sangre , Femenino , Infecciones por VIH/etiología , Infecciones por VIH/transmisión , Seropositividad para VIH/inmunología , VIH-1/aislamiento & purificación , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Factores de Riesgo , Conducta Sexual , Trastornos Relacionados con Sustancias/complicacionesRESUMEN
Little is known concerning the clinical features, the histological outcome, and the effects on the maturation of immune system of children with vertically-transmitted hepatitis C virus (HCV) infection. Specifically, no data are available on the peripheral distribution of T-cell subsets. The frequency of naive and memory cells, activated T cells, and cytokine-producing T cells was analyzed in nine HCV-infected children born to HCV-positive mothers. In HCV-infected children, the distribution of naive and memory cells was not significantly altered in the CD4 subset whereas within the CD8 subset, an increase of memory and a decrease of naive cells was observed. The frequency of HLA-DR-positive and Fas-positive T cells was increased in HCV-infected children in both CD4 and CD8 subsets. The distribution of Fas-expressing T cells was directly related to that of HLA-DR cells and inversely related to the frequency of naive T cells. In regard with cytokine production we found increased levels of both CD4 and CD8 interferon-gamma (IFN-gamma)-producing cells whereas no difference in the percentage of interleukin-2 (IL-2)-producing T cells was observed. No meaningful correlation was observed between individual T cell subsets and ALT levels or HCV viral load. In conclusion, our results indicate an increased T-cell activation and a shift to a T(H)1 pattern of cytokine production in children with vertically transmitted HCV infection. The cause of this kind of immune response could reside in the persistent antigenic stimulation by chronic HCV infection.
Asunto(s)
Hepatitis C/inmunología , Transmisión Vertical de Enfermedad Infecciosa , Linfocitos T/inmunología , Adolescente , Factores de Edad , Alanina Transaminasa/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Niño , Preescolar , Citocinas/biosíntesis , Femenino , Antígenos HLA-DR/análisis , Hepatitis C/transmisión , Humanos , Activación de Linfocitos , Masculino , Receptor fas/análisisRESUMEN
Lymphocytes from HIV-1-infected subjects undergo massive apoptosis when cultured in vitro, and this phenomenon might reflect pathogenetic mechanisms leading to immune dysfunction in vivo. However, (1) lymphocyte death is not restricted to CD4+ cells but seems to involve predominantly CD8+ cells, and (2) the same phenomenon occurs in other viral infections. Furthermore, it is not known whether a relationship exists between the HIV-1 burden and this type of cell death. In this work we sought to determine whether the HIV-1 provirus load correlates with the propensity to apoptosis of CD4+ and CD8+ cells. We studied 10 HIV-1-infected patients with CD4+ cell counts above 500/mm3 and free of concomitant infections. We correlated the frequency of HIV-1-infected CD4+ cells with the extent of culture-induced apoptosis as well as with the phenotype of the apoptotic lymphocytes. We found that the magnitude of apoptosis correlated with the frequency of HIV-1-infected CD4+ cells (p = 0.0007), and that increasing viral load and apoptosis were associated with a shift to the selective death of CD8+ cells. Our data support the view that, in addition to CD4+ cell killing, another immunopathogenic effect of HIV might be that of priming CD8+ cells to apoptosis. In vivo, this could eventually lead to the exhaustion of the cytotoxic T cell compartment.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1 , Linfocitos/inmunología , Linfocitos/virología , Adulto , Apoptosis , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Supervivencia Celular , Células Cultivadas , Femenino , Citometría de Flujo , Genoma Viral , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Antígenos HLA-DR/análisis , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Azelaic acid (C9- -dicarboxylic acid) is a competitive inhibitor of tyrosinase and some oxidoreductase in vitro, and in vivo has a beneficial effect on lentigo maligna and malignant melanoma. A definite cytotoxic effect in cultures of malignant melanocytes was also reported. In order to establish if the cytotoxic effect of the diacid is exerted equally in the absence of tyrosinase, lymphoma- and leukemia-derived cell lines were cultured for 72 hr with 10(-3) M, 10(-2) M and 5 X 10(-2) M C9 disodium salt. Normal resting lymphocytes, lymphocytes activated by phytohemoagglutinin, and mouse Balb/c 3T3 fibroblasts were also tested to study a possible effect of azelaic acid on DNA synthesis and cell duplication. At 10(-3) M C9 had no effect on the viability of all the cells tested; at 10(-2) M and 5 X 10(-2) M, C9 2Na had a 50-80% cytotoxic effect on lymphoma- and leukemia-derived cell lines, while at the same concentrations it was not toxic to normal lymphocytes, either resting or stimulated, or to 3T3 fibroblasts. The experiments on cellular incorporation of (1-9 14C) azelaic acid showed that the radiocarbon uptake was two to three times higher for lymphoma- and leukemia-derived cell lines than for lymphocytes, either resting or stimulated, or 3T3 fibroblasts. Biochemical analysis revealed that the diacid underwent beta-oxidation in all the cell cultures. Fractionated centrifugations of 3T3 fibroblasts cultured in the presence of radiolabelled azelaic acid (2 X 10(-4) M) plus cold C9 2Na (10(-2) M), showed that the radioactivity was mainly concentrated in the cytoplasm. The results, being similar to those obtained by adding azelaic acid to cultures of melanoma cells, suggest that the cytotoxic effect of azelaic acid may be due to interference with mitochondrial oxido-reductase enzymes, rather than with tyrosinase. The difference in reaction between lymphoma- and leukemia-derived cell lines and normal or stimulated lymphocytes, and 3T3 fibroblasts, could be explained on the basis of a different degree of permeability of the cell membrane, and/or to a possible different sensitivity of reaction of mitochondrial functions. A similar argument could be used to explain the absence of an effect of dicarboxylic acids upon normal as compared with hyperactive or malignant melanocytes in vivo.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Dicarboxílicos/farmacología , Leucemia/metabolismo , Linfocitos/efectos de los fármacos , Linfoma/metabolismo , Animales , Radioisótopos de Carbono , Línea Celular , Células Cultivadas , Ácidos Dicarboxílicos/metabolismo , Fibroblastos/efectos de los fármacos , Humanos , Activación de Linfocitos , Ratones , Mitocondrias/efectos de los fármacosRESUMEN
We identified a subgroup of ataxia-telangiectasia (AT) patients (2 sibs and 1 unrelated case) characterized by typical clinical manifestations of the disease and cellular radiosensitivity intermediate between classical AT and normal subjects. Our data and a literature review of the intermediate radiosensitivity AT cases show that radioresistant DNA synthesis, cellular radiosensitivity (measured in terms of survival and chromosome breakage), and the clinical hallmarks behave independently. This raises a number of interesting questions about the correlation between radiobiological and clinical features, and about the nature of the AT gene(s).
Asunto(s)
Ataxia Telangiectasia/genética , Tolerancia a Radiación/genética , Adolescente , Línea Celular Transformada , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Niño , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , ADN/biosíntesis , Femenino , Rayos gamma , Humanos , Masculino , FenotipoRESUMEN
We describe 2 brothers with a malformation syndrome consisting of agenesis of the corpus callosum, cutaneous hypopigmentation, bilateral cataract, cleft lip and palate, and combined immunodeficiency. The clinical history of both patients was characterized by severe psychomotor retardation, seizures, recurrent severe respiratory infections, and chronic mucocutaneous candidiasis. The children died of bronchopneumonia at age 2 and 3 years, respectively. Immunological investigations showed, in one sib studied, skin anergy to recall antigens, profound depletion of T4+ lymphocytes, and serum IgG2 deficiency. Necropsy showed agenesis of the corpus callosum, hypoplasia of the cerebellar vermis, and profound hypoplasia of the thymus and of the peripheral lymphoid tissue. The distinctive features of these sibs appear to define a previously undescribed hereditary MCA/MR syndrome. The clinical and pathological findings seem to indicate, as a pathogenetic mechanism, a defect involving the embryonic organization of the central nervous system and of the immune system.
Asunto(s)
Anomalías Múltiples/genética , Agenesia del Cuerpo Calloso , Catarata/genética , Síndromes de Inmunodeficiencia/genética , Trastornos de la Pigmentación/genética , Preescolar , Labio Leporino/genética , Fisura del Paladar/genética , Humanos , Masculino , SíndromeRESUMEN
The aim of the study was to evaluate the latent structure of DSM-IV schizotypal personality disorder (SPD) diagnostic criteria. The sample consisted of 564 consecutively admitted inpatients and outpatients. Exploratory latent class analysis identified a four-class model as the best fitting model for DSM-IV SPD criteria. The first of the SPD latent classes was mainly characterized by odd thinking, inappropriate affect, and interpersonal features; the second class by cognitive/perceptual difficulties; the third class by paranoid features; and the fourth class by absence of SPD features. The conditional probability pattern of the fourclass solution could be safely replicated across confounder strata. Unlike previous findings, oddness, aloofness, and social withdrawal, rather than positive symptoms, best characterized SPD even in clinical samples.
Asunto(s)
Trastorno de la Personalidad Esquizotípica/diagnóstico , Conducta Social , Adulto , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Modelos Psicológicos , Escalas de Valoración Psiquiátrica , Valores de Referencia , Trastorno de la Personalidad Esquizotípica/clasificaciónRESUMEN
The effect of hydrogen peroxide on the rate of semi-conservative DNA synthesis in ataxia telangiectasia (AT) and normal human lymphoblastoid cells was investigated. The rate of DNA synthesis in AT cells was not depressed to a lesser extent than in normal cells, as might have been expected since H2O2 is a radiomimetic agent. On the contrary, 4 AT cell lines displayed a higher sensitivity to the inhibitory effect of H2O2 on DNA synthesis than 2 normal cell lines. Comparable levels of cytotoxicity were detected in cell viability studies. Furthermore, neither the level of DNA breakage produced by H2O2, nor the rate of repair of these lesions was significantly different in normal and AT cells. Together, these results indicate that the AT cell lines utilized in this study are not hypersensitive to the oxidant. It is suggested that H2O2 may not induce lethality via the direct action of the hydroxyl radical (OH.).
Asunto(s)
Supervivencia Celular/efectos de la radiación , Replicación del ADN/efectos de la radiación , Peróxido de Hidrógeno/farmacología , Ataxia Telangiectasia , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN/biosíntesis , ADN/efectos de los fármacos , ADN/efectos de la radiación , Daño del ADN , Replicación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Humanos , CinéticaRESUMEN
The passive-aggressive (negativistic) personality disorder (PAPD) is one of the most controversial personality disorders. In order to assess DSM-IV PAPD psychometric properties and comorbidity pattern in a mixed psychiatric sample, 379 consecutively admitted in- and outpatients were administered SCID-II, Version 2.0. Confirmatory factor analysis showed that DSM-IV PAPD is a unidimensional construct with adequate internal consistency (K-R 20 = .85). A strong, specific association (odds ratio = 10.38, 95% CI = 4.83-22.30) was observed between DSM-IV PAPD and narcissistic personality disorder (NPD). Confirmatory factor analysis showed that DSM-IV PAPD should be considered as a subtype of a broader narcissistic disorder.
Asunto(s)
Trastorno de Personalidad Pasiva Agresiva/diagnóstico , Escalas de Valoración Psiquiátrica , Adulto , Femenino , Humanos , Masculino , Psicometría , Reproducibilidad de los ResultadosRESUMEN
Three hundred consecutively admitted in- and outpatients were administered the Personality Diagnostic Questionnaire-4+ (PDQ-4+). The Structured Clinical Interview for DSM-IV Axis II Personality Disorders, Version 2.0 (SCID-II) was used as the external diagnostic standard for personality disorder (PD) assessment. SCID-II was administered blind to PDQ-4+ scores. Low agreement between PDQ-4+ and SCID-II was observed for both dimensional and categorical PD evaluations. Receiver operating characteristic (ROC) analysis showed a definitively satisfactory discriminatory capability only for two PDQ-4+ PD scales (dependent, and antisocial). In agreement with previous studies, these results showed that PDQ-4+ was not a substitute for a structured diagnostic interview.
Asunto(s)
Trastornos de la Personalidad/diagnóstico , Pruebas de Personalidad/normas , Psicometría/normas , Encuestas y Cuestionarios/normas , Adulto , Intervalos de Confianza , Femenino , Humanos , Entrevista Psicológica/normas , Italia , Masculino , Trastornos Mentales/complicaciones , Análisis Multivariante , Curva ROC , Reproducibilidad de los Resultados , Método Simple CiegoRESUMEN
Hospitals use various methods to establish performance benchmarks. This may include cooperative data shared between organizations to allow broad, general comparisons. These can, however, be misinterpreted as representing standards of patient care. In the authors' institution, a more complete examination was made of one of these quality indicators when it appeared quality indicator standards were in conflict with standards of patient care. The authors conclude that quality indicators are valuable when screening a hospital, just as we utilize screening tests to identify patients at potential risk. Neither should we apply broad quality indicators as standards of care without a full understanding of their strengths and weaknesses and the foundation on which they are built.
Asunto(s)
Benchmarking , Administración Hospitalaria/normas , Atención al Paciente/normas , Indicadores de Calidad de la Atención de Salud , Acreditación , Profilaxis Antibiótica/normas , Humanos , Joint Commission on Accreditation of Healthcare Organizations , North Carolina , Cuidados Preoperatorios/normas , Estándares de Referencia , Estados UnidosRESUMEN
The clinical and pathologic features of a Burkitt-like lymphoma in a bisexual HIV-sero-positive man are reported. Emphasis is placed on some histologic characteristics present in a hyperplastic lymph node removed 2 years before the development of the B-cell lymphoma.
Asunto(s)
Bisexualidad , Linfoma de Burkitt/patología , Seropositividad para VIH , Adulto , Linfoma de Burkitt/etiología , Humanos , Hiperplasia , Ganglios Linfáticos/patología , MasculinoRESUMEN
Evidence for a direct cell-to-cell virus transfer could be provided by an agent that inhibits plaque formation without interfering with the processes that determine plaque growth in the exit and reinfection pathway of virus transfer. We studied the process of Vero cell infection by herpes simplex virus type 1 laboratory strain F [HSV-1 (F)] in the presence of monoclonal antibody (mAb) F10, an anti gD mAb that inhibits plaque development but does not neutralize virus infectivity in the absence of complement. In virus growth curves, cell associated virus was inhibited at low (0.01) but not at high (10) MOI when all cells are simultaneously infected, showing that the target of the mAb is the process of progressive cells recruitment and not the rate of virus replication. The mAb slightly inhibited virus exit and delayed virus entry. However these two additional inhibitory activities were not responsible for inhibition of virus spread, at least at early time of infection. In fact inhibition of virus spread, as measured by reduction of infectious centers (IC) from infected monolayers, could be appreciated before the appearance of extracellular virus in control cultures. We obtained electron microscope evidence that, both in the absence and in the presence of mAb, extracellular virus was initially concentrated at the interspaces between adjacent cell membranes, with little or no virus present at free cell surfaces. At more advanced stages of infection, only virus at free cell surfaces was found. The results of the study of virus replication in the presence of the mAb confirmed the hypothesis of the existence of a pathway of virus transfer between adjacent cells independent from extracellular virus. However, no electron microscope evidence for a direct cell-to-cell virus passage or for a modification of virus transfer brought about by the plaque inhibiting mAb was obtained. Interestingly, electron microscope studies suggested a targeting of the virions to different extracellular spaces, intercellular spaces and free cell surfaces, in intact and damaged cells respectively.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 1/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Membrana Celular/ultraestructura , Membrana Celular/virología , Chlorocebus aethiops , Herpesvirus Humano 1/ultraestructura , Microscopía Electrónica , Células Vero , Ensayo de Placa Viral , Virión/ultraestructuraRESUMEN
OBJECTIVES: Terminal restriction fragment (TRF) analysis of human telomeres was used to calibrate flow-fluorescence in situ hybridization (FF) measures of telomere lengths to expand the range of measures and increase power of resolution of our previously published protocol. TRF data used as the gold standard should be obtained by electrophoresis with suitable resolution applied to appropriately isolated genomic DNA. When we considered TRF attained by correct methods, we found our method to be insufficiently accurate, thus we have reviewed our previously published FF protocol to obtain the best coefficient of determination (r(2)) between our experimental results and valid TRF lengths. MATERIALS AND METHODS: Using human telomere-specific PNA probe, Cy5-OO-(CCCTAA)3 , we measured telomere lengths of continuous cell line and of peripheral blood lymphocytes by FF. We modified hybridization, stringency, negative control handling, stoichiometric DNA staining and telomere fluorescence assessment of the protocol. RESULTS: We realized a procedure with increased power of resolution, improved TRF versus FF r(2) values that allowed simultaneous analysis of DNA and telomere duplication. Notwithstanding multiple steps in formamide sampling, recovery was satisfactory. DISCUSSION: The reviewed FF protocol appeared at least as suitable as the TRF method. Measures obtained by TRF can be affected by chromosome end variability, DNA fragmentation, incomplete digestion and unsuitable electrophoresis. In contrast, the FF technique analyses telomeric sequences confined to preserved nuclei thus overcome most previous limitations. As yet, however, the FF telomere measure cannot be performed together with immunophenotyping and/or generation study by the dye dilution method.