RESUMEN
INTRODUCTION: Approximately, 25% of haemophilia A (HA) patients treated by factor VIII (FVIII), develop antibodies, known as inhibitors, neutralizing the activity of infused FVIII. This immune response involves B cells (BC), including FVIII-specific memory B cells (MBC). Production of anti-FVIII antibodies after stimulation of FVIII-specific MBC suggests a role of these cells in the immune response to FVIII. Animal models allowed the study of circulating FVIII-specific cells, however few data are available on HA patients. AIM AND METHODS: In the present study, we simultaneously detected, via ELISpot assay, different isotypes of MBC in the blood of HA patients, after polyclonal activation. Patients included: three with active inhibitors; three with a history of inhibitors; six without any past or active inhibitor. RESULTS: FVIII-specific MBC were detected in peripheral blood of HA patients: (i) patients with active inhibitors (IgG: 4-5.2/10(6) BC; IgA: 2.9-4/10(6) BC) (ii) patients with a past of inhibitors (no IgG BC; IgA: 5-7.5/10(6) BC) (iii) patients without inhibitors (no IgG BC or IgA BC except one patient had two FVIII-specific IgA BC/10(6) BC). CONCLUSION: FVIII-specific IgA MBC were detected in HA patients with past and current immune responses against FVIII and FVIII-specific IgG MBC were found only in those with positive inhibitors. This study shows the possibility to detect and characterize easily and simultaneously the MBC from patient blood and that MBC seem different according to anti-FVIII immune history. It could be a useful tool to study anti-FVIII response and Immune Tolerance Induction cellular mechanisms.
Asunto(s)
Linfocitos B/metabolismo , Ensayo de Immunospot Ligado a Enzimas , Factor VIII/inmunología , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Adolescente , Adulto , Anticuerpos Neutralizantes/sangre , Linfocitos B/citología , Estudios de Casos y Controles , Niño , Citometría de Flujo , Hemofilia A/patología , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Adulto JovenRESUMEN
OBJECTIVE: This study was performed to investigate the hyporeactivity of purified B lymphocytes from HIV-1-infected patients. DESIGN: Given the importance of the B-cell Ag receptor (BCR) and CD40 in B-lymphocyte activation, we assessed the capacity of purified peripheral blood B lymphocytes from HIV-1-infected patients to differentiate into Ig-secreting cells in a T-cell- and accessory-cell-independent system of BCR and CD40 costimulation. METHODS: B lymphocytes from 21 HIV-1-infected patients were purified by immunomagnetic cell separation and costimulated with immobilized anti-CD40 monoclonal antibodies and Staphylococcus aureus Cowan I particles in the presence of interleukin (IL)-2 and IL-10. Homotypic aggregate formation, apoptosis, cell cycle entrance, proliferation and Ig secretion of B cells were analysed. RESULTS: Costimulation by the BCR and CD40 induced proliferation and differentiation of B lymphocytes into Ig-secreting cells in 13 patients (group I) but not in eight patients (group II). For three patients in group II, the dual triggering induced apoptosis of B cells. The unexpected inability of these cells to differentiate was associated with a high CD38 expression and a weak spontaneous production of Ig or anti-HIV-1 antibodies in patients with a high viral load and a low CD4+ lymphocyte count. Despite this anomaly, the B cells from group II were able to progress through the cell cycle after stimulation with a combination of phorbol ester and ionomycin in complete medium, suggesting an impairment in BCR and CD40 early signal transduction. CONCLUSION: Intrinsic in vitro hyporeactivity of B lymphocytes to dual triggering of BCR and CD40 was observed in advanced HIV-1 disease and appeared to be related to in vivo hyperactivation of B cells.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/inmunología , Infecciones por VIH/inmunología , VIH-1 , Receptores de Antígenos de Linfocitos B/inmunología , Adulto , Anticuerpos Monoclonales , Apoptosis , Ciclo Celular , Diferenciación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Anticuerpos Anti-VIH/sangre , Humanos , Inmunoglobulina G/sangre , Separación Inmunomagnética , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
Up to 20% of health care workers are considered as non-responders to hepatitis B vaccination (anti-HBs<10 m UI/ml in serum). We have explored memory B cells differentiated in vitro into anti-HBs antibody-secreting cells (anti-HBs-SCs) by ELISPOT assay. Anti-HBs-SCs were detected in vaccinated responders (n = 11) and non-responders (n = 10) but IgG anti-HBs-SCs were significantly lower in the non-responder group (p<0.001). Low amounts of HBs antibodies were also quantified by ELISA in non-responders' sera. These results indicate that a suboptimal B cell response exists in non-responders to HBV vaccination. This B cell response may mediate a protection against clinically significant breakthrough hepatitis B infection.
Asunto(s)
Linfocitos B/inmunología , Personal de Salud/estadística & datos numéricos , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Vacunas contra Hepatitis B/inmunología , Adulto , Ensayo de Immunospot Ligado a Enzimas , Femenino , Hepatitis B/inmunología , Hepatitis B/prevención & control , Humanos , Memoria Inmunológica , Masculino , Persona de Mediana EdadRESUMEN
The enzyme-linked immunospot (ELISPOT) assay was adapted to detect and enumerate HIV-1-producing cells at the single cell level. With CEM cells or peripheral blood mononuclear cells (PBMC) infected in vitro with HIV-1, the ELISPOT assay detected cells that produced HIV-1 antigens and showed that between 5.4% and 9.5% of the p24 antigen-positive CEM cells and 11.1% to 23.6% of the p24 antigen-positive PBMC were productively infected. In HIV-1-infected patients in early stage of the disease and without antiretroviral therapy, up to 4.54 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in peripheral blood and up to 277.75 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in splenic lymphoid tissue. Our results indicate that the ELISPOT assay could represent a new tool to study HIV-1 replication in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por VIH/sangre , VIH-1 , Juego de Reactivos para Diagnóstico , Adulto , Humanos , Células Tumorales CultivadasRESUMEN
Two Brucella fractions, the murein-linked fraction PI and the murein-free fraction SF, behave as in vitro adjuvants for primary anti-sheep erythrocyte responses: added to Mishell and Dutton-type cultures of spleen cells from B6/DB F1 mice they significantly enhance the number of direct anti-sheep erythrocyte PFC observed on day 5. They exert both nonspecific, polyclonal activating effects and antigen-dependent specific adjuvanticity. These two functions, however, differ in their dose responses and in their cellular requirements and can therefore be dissociated. Thus, polyclonal activation requires high doses of the "adjuvant fraction," is enhanced by adherent-cell depletion, and is not impaired by T-cell depletion. Specific adjuvanticity, on the other hand, requires lower doses of the adjuvant fractions (very high doses are in fact suppressive) and is T-cell and adherent-cell dependent. Moreover, adjuvanticity can be transferred to unstimulated spleen cells (or restored in adherent-cell-depleted populations) by PI- or SF-stimulated adherent cells or by the filtered supernatants of such cultures; adjuvant-soluble factors are therefore involved in the phenomena of adherent, T- and B-cell cooperation required for the adjuvanticity of Brucella fractions.
Asunto(s)
Adyuvantes Inmunológicos , Formación de Anticuerpos , Brucella/inmunología , Animales , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Eritrocitos/inmunología , Femenino , Activación de Linfocitos , Macrófagos/inmunología , Ratones , Linfocitos T/inmunologíaRESUMEN
Two "protective" monoclonal anti-Brucella antibodies are described (SF311-IgG2a and S480-IgA) both of them accelerate the blood clearance of i.v. inoculated Brucella suis 1330 and decrease murine splenic infection on day 7.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Brucella/patogenicidad , Brucelosis/inmunología , Animales , Anticuerpos Monoclonales/análisis , Ensayo de Inmunoadsorción Enzimática , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Plasmacitoma/inmunologíaRESUMEN
Two lipid-A-free fractions which were extracted from Brucella melitensis and were designated PI and SF stimulated human unsensitized mononuclear cells to proliferate and to secrete immunoglobulins. Both of these effects were observed in cultures of peripheral blood, tonsils, and cord blood lymphocytes. Neither B cells nor T cells alone proliferated in the presence of these fractions, whereas the proliferative response of T cells plus B cells was largely independent of accessory cells. Polyclonal activation was estimated by counting the cells which secreted immunoglobulins of different isotypes into culture supernatants. This phenomenon was strongly T dependent.
Asunto(s)
Linfocitos B/inmunología , Brucella/inmunología , Linfocitos T/inmunología , Adulto , Formación de Anticuerpos , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Sangre Fetal/citología , Humanos , Activación de Linfocitos , Cooperación Linfocítica , Mitógenos , Tonsila Palatina/citologíaRESUMEN
PI, a lipid A-free fraction (15) extracted from the cell wall of Brucella melitensis type I M.15 behaves as a non specific mitogen and a polyclonal B-cell activator (PBA) for human unsensitized lymphocytes from subjects with no previous history and no biological stigmata of brucellosis. Both effects may be evidenced, indifferently, in cultures of peripheral blood, tonsils or cord blood lymphocytes. Mitogenicity, estimated by the incorporation of tritiated thymidine is maximal on the 5th and 6th day of culture, for high cellular concentrations and for 10 micrograms/PI/well. PBA activity is estimated by the incidence of Prot. A reverse PFC and by a laser nephelometric measure of immunoglobulins in culture supernatants. The use of ET-rosetting for the preparation of purified and/or reconstituted populations demonstrates the T-dependence of the B cell activating effect of PI. On the other hand adherent cell depletion on Sephadex G 10 columns does not significantly impair these effects. Important individual differences are observed and suggest a genetic control of the level of B responses to this new bacterial PBA. These results are discussed in terms of a comparison of PI with other human PBA and of the interactions between non specific and specific responses of lymphocytes from vaccinated subjects or brucellosis patients.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Brucella/inmunología , Pared Celular/inmunología , Células Cultivadas , Sangre Fetal/inmunología , Humanos , Inmunidad Innata , Lípido A/inmunología , Mitógenos/inmunología , Tonsila Palatina/inmunología , Linfocitos T/inmunología , alfa-Macroglobulinas/inmunologíaRESUMEN
Immune sera have previously been shown to play a positive role in immune protection against murine experimental brucellosis. The protective properties of a panel of five anti-Brucella monoclonal antibodies (Mabs) were therefore assessed by estimation of the acceleration of the blood clearance of intravenously inoculated Brucella and of the reduction of splenic infection on Day 7 after infection. Three 'strongly protective', one 'weakly protective' and one 'non-protective' Mabs were identified. As a first step towards the study of the mechanism of this humoral protection, these Mabs were further compared for structural and functional properties such as immunoglobulin isotype, anti-Brucella specificity, anti-Brucella in vitro bacteriostasis, Brucella agglutination and complement fixation when complexed with tyndallized Brucella. No correlation was found between protection and either agglutination or direct bacteriostasis. On the other hand, the results observed suggest that isotypes (and especially the IgG2a isotype) could play an important role in in vivo immuno protection and that complement may be involved. However, the fact that one of the protective Mabs belongs to the IgA isotype, does not cross-react with the others in anti-Brucella epitopic specificity and does not fix complement underlines the probable diversity of the mechanisms involved.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Brucella/inmunología , Brucelosis/inmunología , Animales , Brucella/crecimiento & desarrollo , Brucelosis/microbiología , Pruebas de Fijación del Complemento , Ratones , Sepsis/inmunología , Bazo/microbiologíaRESUMEN
An attempt was made to test the hypothesis that immunological dysfunction occurs in primary polyenthesopathy (PP) by studying skin immunofluorescence, capillary microscopy, photoplethysmography and the lymphocyte populations in PP patients defined according to the usual criteria of Yunus. Skin immunofluorescence in 15 PP patients (14 women, 1 man, average age 50.9 years) failed to reveal any deposit of IgG, A, M, Clq, or C3c either at the dermoepidermal junction or in the vessels. 26 patients (24 women, 2 men, average age 50.5 years) were studied using capillary microscopy and 12 using digital photoplethysmography. The microvascularization abnormalities observed were mild and non specific. A study of the lymphocyte populations using Coulter flux cytometry in 35 PP patients (32 women, 3 men, average age 46 years) did not show any modification in the number of CD4 and CD8 lymphocytes by comparison with the controls. These results do not agree with the data given in the literature and this is probably due to the heterogeneity of the patients studied, with some studies showing a low concentration of antinuclear factors and an abnormal frequency of dry symptoms which, in the opinion of the authors, is considered as an exclusion criterion in the diagnosis of PP. In the author's view, polyenthesopathy is not an immune disease.
Asunto(s)
Fibromialgia/inmunología , Enfermedades del Sistema Inmune/complicaciones , Enfermedades Reumáticas/inmunología , Adulto , Anciano , Femenino , Humanos , Masculino , Microcirculación , Persona de Mediana Edad , Linfocitos TRESUMEN
Two lipid A-free fractions extracted from Brucella melitensis (fractions PI and SF) were shown to behave as B-cell non-specific mitogens for murine lymphocytes: they stimulated the uptake of tritiated thymidine by normal unsensitized murine splenic lymphocytes, by spleen cells from nude mice and by T-cell depleted but not by T-cell enriched and B-cell-deprived splenic populations. Since depletion of adherent cells leads to a two- to three-fold depression of PI- or SF-induced mitogenic responses these two fractions were shown to require accessory adherent cell participation for an optimal mitogenicity. Moreover they behaved as polyclonal activators for murine spleen cells. These results are discussed in terms of a possible classification of PI and SF amongst other B-cell mitogens and of the respective role of the peptidoglycan and lipoprotein moieties in B-cell activation by Brucella fractions.
Asunto(s)
Linfocitos B/inmunología , Vacuna contra la Brucelosis/farmacología , Animales , Adhesión Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Cinética , Activación de Linfocitos , Masculino , Ratones , Mitosis/efectos de los fármacosRESUMEN
Cytomegalovirus-specific in vitro antibody production (CMV-IVAP) by peripheral blood lymphocytes (PBL) was investigated in 12 renal transplant recipients. CMV-IVAP, CMV-serology, and viral cultures were carried out weekly during at least 8 weeks after transplantation. Nine episodes of CMV infection occurred in eight patients. CMV-IVAP was positive in eight episodes; IgG and/or IgA and/or IgM antibodies reacting with several CMV polypeptides were secreted by PBL. In two patients with primary CMV infection, only IgA antibodies were detected. Cytomegalovirus cultures were positive in eight episodes and serological evidence of CMV infection was obtained in five episodes. In one case, CMV-IVAP was observed before CMV isolation and in another case, before serological evidence of CMV infection. Our study indicates that CMV-IVAP could represent an additional tool for the diagnosis of CMV infection. Our study indicates that CMV-IVAP could represent an additional tool for the diagnosis of CMV infection in renal transplant recipients.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Trasplante de Riñón/inmunología , Adulto , Anciano , Especificidad de Anticuerpos , Biomarcadores , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/etiología , Femenino , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Técnicas In Vitro , Trasplante de Riñón/efectos adversos , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
25 HIV-infected antiretroviral-naive adults were included in a 24-week study to evaluate the efficacy and the tolerability of a zidovudine/didanosine combination therapy in which didanosine was administered once daily (200 mg if weight < 60 kg, 300 mg if weight > 60 kg) and zidovudine twice daily (500 mg/day if weight < 90 kg, 600 mg/day if weight > 90 kg). 5 patients discontinued their treatment early: 3 had poor compliance and 2 presented adverse events. Evaluation of treatment efficacy was based on CD4+ T cell enumeration and HIV RNA level quantitation in plasma (NASBA). Baseline values were 278 CD4+/mm3 and 5.42 log RNA copies/ml. Mean changes from baseline were +102 CD4+/mm3 and -2.14 log RNA copies/ml at week 8 and +156 CD4+/mm3 and -2.07 log RNA copies/ml at week 24. HIV RNA in plasma was lower than the detection limit (2.60 log RNA copies/ml) in 55% of patients at week 8 and in 30% at week 24. No major adverse events such as neuropathy or pancreatitis were observed. Once-daily administration of didanosine in combination with twice-daily administration of zidovudine is a well tolerated regimen that appears to be as effective ad the conventional zidovudine/didanosine combination regimen.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Didanosina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Zidovudina/uso terapéutico , Adulto , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/efectos de los fármacos , Quimioterapia Combinada , Femenino , VIH , Infecciones por VIH/inmunología , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/sangre , ARN Viral/efectos de los fármacosRESUMEN
The in vitro secretion of cytomegalovirus (CMV)-specific antibodies by peripheral blood mononuclear cells (PBMC) was analyzed in patients with acute CMV infection and CMV-seropositive patients infected with human immunodeficiency virus. This active and spontaneous in vitro secretion was not affected by the depletion of T cells or adherent cells. The number of anti-CMV antibody-secreting cells was estimated to be 28-176/10(6) PBMC; 30%-65% of the total in vitro antibody production was CMV-specific. This secretion seemed to be independent of in vitro polyclonal B cell activation, in vitro antigen-specific lymphocyte stimulation, or in vivo Epstein-Barr virus-induced B cell transformation. In vitro anti-CMV antibody production may therefore result from an in vivo stimulation of the immune system by CMV antigens and might indicate CMV replication in the host.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Inmunoglobulina G/biosíntesis , Leucocitos Mononucleares/inmunología , Enfermedad Aguda , Antígenos Virales/inmunología , Western Blotting , Células Cultivadas , Infecciones por Citomegalovirus/complicaciones , Ensayo de Inmunoadsorción Enzimática , Infecciones por VIH/complicaciones , Humanos , ImmunoblottingRESUMEN
We followed the course of cytomegalovirus (CMV) viremia in 65 patients treated with zidovudine for symptomatic HIV-1 infection. Blood samples were tested for the presence of CMV before initiation of treatment and every 3 months thereafter. 13 patients (20%) showed a positive CMV viremia at initiation of treatment. After 3 months of therapy, only 2 patients (3%) remained viremic. However, the frequency of CMV viremia increased from the 6th month of treatment and 28 (43%) of our patients showed a persistence of, or conversion to, positive viremia during the course of treatment. CMV viremia was associated with a decline in the patients' clinical state in 79% of the cases. In contrast, among the 37 patients, who remained negative for CMV viremia, 73% did not show a progression of the HIV-associated disease. The results suggest that CMV viremia could be considered as a useful marker for HIV-associated disease and its progression as well as for the efficacy of therapy.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Infecciones por Citomegalovirus/tratamiento farmacológico , VIH-1 , Viremia/tratamiento farmacológico , Zidovudina/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/etiología , Adolescente , Adulto , Anciano , Citomegalovirus/aislamiento & purificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
We studied the spontaneous in vitro secretion of anti-HIV-1 antibodies by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Specific antibody production was detected in supernatants of PBMC cultures using an ELISA; HIV-1 specificity was confirmed by antigen adsorption and Western blotting. This antibody secretion was found to be an active phenomenon and was not due to a release of plasma antibodies passively adsorbed onto the cell membranes. In all positive supernatants, anti-HIV-1-secreted antibodies were directed against env-encoded antigens and many supernatants also contained antibodies to pol- and gag-encoded antigens. PBMC from all HIV-1-infected patients tested (140 adults and 18 infants) secreted anti-HIV-1 antibodies. This production was found during all the clinical stages of HIV-1 infection. Our results suggest that this spontaneous HIV-1-specific antibody secretion represents a marker of HIV-1 infection. Detection of these antibodies could be a valuable tool for early confirmation of HIV-1 infection in neonates born to HIV-1-seropositive mothers.
Asunto(s)
Anticuerpos Anti-VIH/análisis , Infecciones por VIH/inmunología , VIH-1/inmunología , Leucocitos Mononucleares/inmunología , Adolescente , Adulto , Anciano , Células Cultivadas , Productos del Gen gag/análisis , Proteína p24 del Núcleo del VIH , Seropositividad para VIH/inmunología , Humanos , Persona de Mediana Edad , Proteínas del Núcleo Viral/análisisRESUMEN
The spontaneous secretion in vitro of anti-Toxoplasma gondii antibodies by peripheral blood mononuclear cells was assessed in 69 patients with AIDS-related brain lesions. The sensitivity and specificity of this assay in the diagnosis of toxoplasmic encephalitis (TE) were found to be 85.4% and 92.8%, respectively. Twenty-four patients with TE were observed during 1-year follow-up after initiation of anti-Toxoplasma treatment and classified on the basis of their clinical and radiologic responses as sustained responders (SR; n = 11), incomplete responders (IR; n = 7) or transient responders (TR; n = 6). In vitro anti-T. gondii antibody secretion decreased as early as the first month after initiation of treatment and disappeared within the year in SRs, persisted in IRs, and decreased but rebounded at relapse in the TR patients. In vitro anti-T. gondii antibody, which reflects immune system activation by parasitic antigens, could be a surrogate marker of TE in AIDS patients.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Anticuerpos Antiprotozoarios/biosíntesis , Encefalitis/inmunología , Leucocitos Mononucleares/inmunología , Toxoplasmosis Cerebral/inmunología , Infecciones Oportunistas Relacionadas con el SIDA/diagnóstico , Adulto , Anciano , Antiprotozoarios/uso terapéutico , Encefalitis/diagnóstico , Francia , Humanos , Masculino , Persona de Mediana Edad , Pirimetamina/uso terapéutico , Estudios Retrospectivos , Toxoplasmosis Cerebral/diagnóstico , Combinación Trimetoprim y Sulfametoxazol/uso terapéuticoRESUMEN
Reverse transcriptase-coupled polymerase chain reaction (Amplicor HIV-1 Monitor), the branched DNA (bDNA) method (Quantiplex HIV-1 RNA) and the nucleic acid sequence-based assay (NASBA HIV-1 RNA QT) were comparatively evaluated for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma. Among 60 plasma specimens from HIV-1 infected patients, HIV-1 RNA was detected in 56 by Amplicor (sensitivity, 93.3%), in 41 by bDNA (sensitivity, 68.3%), and in 60 by NASBA (sensitivity, 100%). HIV-1 RNA was not detected by any of these methods in 34/34 plasma specimens from HIV-1-seronegative blood donors (specificity, 100%). The HIV-1 RNA levels as determined by the different methods were correlated significantly. The frequency of concordant results (log difference < 0.50) was 80.4% between Amplicor and NASBA, 77.5% between Amplicor and bDNA, and 58.6% between bDNA and NASBA. After initiation of antiviral therapy, HIV-1 RNA level variations observed with the three methods were similar. HIV-1 RNA levels were inversely correlated with the CD4+ T cell counts, whereas no correlation was found with HIV-1 p24-antigen levels. When the methods were evaluated for reproducibility, coefficients of variation ranged from 11% to 40% for Amplicor, from 6% to 35% for bDNA, and from 13% to 62% for NASBA. Quantitation of HIV-1 RNA in culture supernatants from HIV-1 subtype A to H strains showed that bDNA can be used to quantitate RNA from all HIV-1 subtypes, whereas Amplicor failed to detect RNA from subtype A strains and NASBA subtype G strains.
Asunto(s)
Técnicas Genéticas , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , ARN Viral/sangre , Virología/métodos , Recuento de Linfocito CD4 , Estudios de Evaluación como Asunto , Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/clasificación , VIH-1/genética , Humanos , Técnicas de Amplificación de Ácido Nucleico , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Viremia/virologíaRESUMEN
In the present study, we show that IL-2, IL-4, IL-7, and IL-15 are able to induce functional CXCR4 surface expression on resting in vitro-generated CD4+ CXCR4- CCR7+ memory T cells. Cytokine-mediated induction of CXCR4 expression was associated with an increase in CXCR4 transcription, enhanced stromal-derived factor-1-induced T cell migration in vitro, and increased susceptibility of these cells to infection with X4 strains of HIV-1. CXCR4 expression could also be induced through an alternative pathway, following coculture of these cells with CD40-activated, autologous, CD34+ progenitor-derived dendritic cells. Although these dendritic cells express transcripts for IL-7 and IL-15, addition of neutralizing anti-IL-7R and IL-15 mAbs did not block induction of CXCR4 expression. Indeed, dendritic cell-mediated up-regulation of CXCR4 expression was found to depend on CD40/CD154 and CD134/CD134L interactions. Whereas activated autologous dendritic cells induced the expression of both CXCR4 and CD25 on a portion of CCR7+ memory T cells, concomitant CD3-mediated activation of these cells further enhanced CD25 expression, but, in contrast, prevented induction of CXCR4 expression. This observation suggests that triggering of the CD134 and CD154 molecules, in contrast to TCR/CD3 complex-mediated stimulation, results in simultaneous T cell activation and CXCR4 expression. Taken together, these results show that common gamma-chain-interacting cytokines as well as signals mediated via noncognate interactions between activated dendritic cells and memory T cells are involved in the up-regulation of CXCR4 expression.