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COVID-19/inmunología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Factor de Necrosis Tumoral alfa/análisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Genoma Viral , Humanos , Interleucina-6/análisis , Masculino , Persona de Mediana Edad , Sistema Respiratorio/química , Sistema Respiratorio/virología , Adulto JovenRESUMEN
INTRODUCTION: FVIII inhibitors consist of a polyclonal population of antibodies. Previous studies have demonstrated different distribution of IgG subclasses. IgG4 was associated to high level of FVIII inhibitors and failure of immune tolerance induction (ITI) treatment. This study monitored the relative distribution of IgG subclasses of anti-FVIII in patients with severe hemophilia A (SHA). METHODS: Anti-FVIII antibodies were measured employing an immunomethod, developed in our laboratory, that combines flow cytometry (FC) with microspheres coupled (FVIII-m) or not (Control-m) to FVIII. Seventy-five patients with SHA were studied, 17 without inhibitors (Group I); 58 with inhibitor history, 13 low responders: (LR: Group II), and 45 high responders (HR: Group III). Eight patients undergoing ITI were also included. RESULTS: We found anti-FVIII antibodies in 11 of 27 patients (40%) without inhibitors and in 45 of 48 with inhibitors at the moment of the study. IgG4 was predominant only in the Group III: P=0.02 in patients with low level of inhibitors and P=0.0001 with high titer of inhibitors. Longitudinal analysis performed on patients undergoing ITI showed a gradual decrease of IgG4 values that was associated to improvement of clinical parameters during treatment. CONCLUSION: We suggest the use of the FC method to supplement functional traditional assays and to help to improve the management of patients with SHA.
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Inhibidores de Factor de Coagulación Sanguínea , Factor VIII/antagonistas & inhibidores , Citometría de Flujo , Hemofilia A/sangre , Inmunoglobulina G , Inhibidores de Factor de Coagulación Sanguínea/sangre , Inhibidores de Factor de Coagulación Sanguínea/clasificación , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , MasculinoRESUMEN
Peripheral blood mononuclear cells (PBMC) from chronic hepatitis C virus-infected persons can harbour viral variants that are not detected in plasma samples. We explored the presence and persistence of HCV genotypes in plasma and PBMC cultures from 25 HCV-monoinfected and 25 HIV/HCV-coinfected patients with haemophilia. Cell cultures were performed at different time points between 1993 and 2010-2011, and the HCV genome was examined in culture supernatants. Sequential plasma samples were studied during the same time period. Analysing sequential plasma samples, 21% of patients had mixed-genotype infections, while 50% had mixed infections determined from PBMC culture supernatants. HIV coinfection was significantly associated with the presence of mixed infections (OR = 4.57, P = 0.02; 95% CI = 1.38-15.1). In our previous study, genotype 1 was found in 72% of 288 patients of this cohort. Similar results were obtained with the sequential plasma samples included in this study, 69% had genotype 1. However, when taking into account plasma samples and the results from PBMC supernatants, genotype 1 was identified in 94% of the population. The PBMC-associated variants persisted for 10 years in some subjects, emphasizing their role as long-term reservoirs. The presence of genotype 1 in PBMC may be associated with therapeutic failure and should not be disregarded when treating haemophilic patients who have been infected by contaminated factor concentrates. The clinical implications of persistent lymphotropic HCV variants deserve further examination among multiple exposed groups of HCV-infected patients.
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Hemofilia A/complicaciones , Hepacivirus/aislamiento & purificación , Hepatitis C/complicaciones , Hepatitis C/virología , Leucocitos Mononucleares/virología , Adulto , Anciano , Coinfección/virología , Genotipo , Infecciones por VIH/complicaciones , Hepacivirus/clasificación , Hepacivirus/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The development of inhibitors is a complication of replacement treatment in Haemophilia. Loss of factor VIII-specific memory B cells in the spleen is associated with down regulation of antibodies in mice treated with high doses of FVIII, but changes in B cell memory have not been described in haemophilic patients. The aim of this study was to evaluate the phenotype of circulating lymphocytes in severe haemophilia A. Twenty patients with inhibitors (PI), 22 without inhibitors (P), nine patients during immune tolerance induction (ITI) treatment and 20 healthy donors (HD) were included. Peripheral blood lymphocytes were examined using flow cytometry. Anti-FVIII antibodies were measured using Bethesda and flow cytometry. Percentages of T subsets and B lymphocytes were similar in all groups. In contrast, memory B cells (CD27+) were decreased in PI and P compared with HD, but the level of significance was higher in PI (P = 0.001) than P (P = 0.01). PI with high level of anti-FVIII antibodies presented the lowest B memory values. CD70 expression was also lowest in PI. Non-switched CD27+ subpopulation (IgD+) was prevalent in PI, but did not show statistical significance. When ITI failed, the percentages of CD27+ B cells after 12 months of ITI were lowest. In a longitudinal study performed in four patients, an increased percentage of CD27+ and CD70+ B cells during ITI was found. This work suggests that different peripheral lymphocyte markers, such as CD27 and CD70 on B cells, may be helpful to evaluate anti-FVIII response and to monitor the success of ITI.
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Linfocitos B/inmunología , Factor VIII/inmunología , Hemofilia A/inmunología , Memoria Inmunológica/inmunología , Adolescente , Anticuerpos/análisis , Linfocitos B/metabolismo , Inhibidores de Factor de Coagulación Sanguínea/metabolismo , Ligando CD27/metabolismo , Niño , Preescolar , Citometría de Flujo , Hemofilia A/metabolismo , Humanos , Masculino , Fenotipo , Adulto JovenRESUMEN
In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme-linked immunosorbent assay (ELISA) that detects both inhibitory (I-Ab) and non-inhibitory (NI-Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I-Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII-m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control-m). Captured anti-FVIII antibodies were detected using anti-human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I-Ab, <0.5 BU mL(-1)); 13 PI (patients with I-Ab, 1.1-8200 BU mL(-1)). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII-m and control-m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r = 0.8; P = 0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI-Ab by FC, and two of them developed high levels of I-Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200 µL of plasma or serum is required especially making it useful for paediatric patients.
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Autoanticuerpos/inmunología , Factor VIII/inmunología , Citometría de Flujo/métodos , Hemofilia A/inmunología , Autoanticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Hemofilia A/sangre , Humanos , Microesferas , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
Microsatellite markers have become one of the most popular tools for germplasm characterization, population genetics and evolutionary studies. To investigate the mutational mechanisms of maize microsatellites, nucleotide sequence information was obtained for ten loci. In addition, Single-Strand Conformation Polymorphism (SSCP) analysis was conducted to assess the occurrence of size homoplasy. Sequence analysis of 54 alleles revealed a complex pattern of mutation at 8/10 loci, with only 2 loci showing allele variation strictly consistent with stepwise mutations. The overall allelic diversity resulted from changes in the number of repeat units, base substitutions, and indels within repetitive and non-repetitive segments. Thirty-one electromorphs sampled from six maize landraces were considered for SSCP analysis. The number of conformers per electromorph ranged from 1 to 7, with 74.2% of the electromorphs showing more than one conformer. Size homoplasy was apparent within landraces and populations. Variation in the amount of size homoplasy was observed within and between loci, although no differences were detected among populations. The results of the present study provide useful information on the interpretation of genetic data derived from microsatellite markers. Further efforts are still needed to determine the impact of these findings on the estimation of population parameters and on the inference of phylogenetic relationships in maize investigations.
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Alelos , Repeticiones de Microsatélite , Mutación , Zea mays/genética , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Hepatitis C viraemia, in 38 human immunodeficiency virus positive (HIV+)/hepatitis C virus positive (HCV+) patients, was determined in haemophilic patients during the 4 years since initiation of highly active antiretroviral therapy (HAART). Six of 38 patients had persistently HCV-negative viraemia for more than 2 years. No correlation between HCV-negative viraemia and CD4+ T-cell counts, HIV viral load, age, type or severity of haemophilia could be established. Reduced levels of HIV viral load and the immune reconstitution that follows the initiation of HAART were not enough to explain the disappearance of HCV from plasma. Individuals who cleared plasma HCV had significantly higher CD8+ T-cell counts (P=0.0013) (mean +/- SE: 1153 +/- 117.8 cells microL(-1)) than those with HCV-positive viraemia (819.1 +/- 40.72 cells microL(-1)). Because HCV could maintain a low replication level in peripheral blood mononuclear cells (PBMC), we cultured PBMC of five of six patients with undetectable HCV viraemia. We found four of five HCV RNA-positive cultures. The presence of HCV RNA in our cultures proved that these cells may be an important viral reservoir that could contribute to HCV recurrence in plasma even after long periods of negative viraemia. In summary, our results indicate that in spite of prolonged HCV-negative plasma viraemia, HCV patients that are co-infected with HIV may harbour replication-competent HCV in their PBMC. Therefore, true clearance of HCV infection is difficult to achieve in these patients.
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Infecciones por VIH/complicaciones , Hemofilia A/complicaciones , Hepacivirus/aislamiento & purificación , Hepatitis C/complicaciones , Leucocitos Mononucleares/virología , Adulto , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Células Cultivadas , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Hemofilia B/complicaciones , Hepacivirus/fisiología , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Masculino , ARN Viral/análisis , Carga Viral , Viremia/complicaciones , Viremia/virología , Latencia del VirusRESUMEN
Sustained reduction of viral replication can be achieved in HIV infected patients after treatment with combinations of drugs (HAART) that inhibit the viral reverse transcriptase, and protease enzymes. However, replication competent virus can still be recovered from latently infected resting memory CD4+ T-cell lymphocytes. Moreover, "covert" virus replication has been demonstrated in patients who experienced reductions in plasma viremia to levels below the limit of detection of the most sensitive PCR assays. In most studies, preferential attention has been given to latent resting CD4+ T-lymphocytes as a source of HIV persistence. However, insufficient suppression of HIV replication could also lead to viral re-emergence after HAART interruption. In addition to CD4+ T- lymphocytes, other host cells such as long-lived resident macrophages or recently infected blood monocytes could also contribute to maintain persistent HIV replication after HAART. Establishing the origin of re-emerging HIV in patients under HAART upon treatment interruption is important to design optimal treatment schemes. Therapeutic strategies aimed at reducing the number of latently infected cells involve immune activation with IL-2, or other stimulatory factors, in the presence of antiretroviral drugs. Elimination of replication-competent virus would require intensification of HAART, or the use of antiretroviral drugs achieving an effective concentration at the site of HIV replication. In this review the mechanisms of HIV persistence and the methods that can be used to distinguish latent from covert HIV replication in different cell types will be discussed.
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Terapia Antirretroviral Altamente Activa/métodos , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Terapia Antirretroviral Altamente Activa/estadística & datos numéricos , Portador Sano/tratamiento farmacológico , Portador Sano/inmunología , Portador Sano/virología , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Inmunidad Celular/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
OBJECTIVES: To study the factors that determine malignant B cell growth in human immunodeficiency virus type 1 (HIV-1)-infected patients. STUDY DESIGN: B-cell lines (lymphocyte cell lines [LCL]) were developed after nonstimulated culture of peripheral blood mononuclear cells (PBMC) from HIV-1-positive (HIV-1(+)) patients. Human immunodeficiency virus type 1 replication in culture, Epstein-Barr virus (EBV) latent oncogene expression, and cell-to-cell interaction were studied after nonstimulated culture of HIV-1(+) PBMC, analyzing their contribution to LCL appearance. METHODS: Nonstimulated PBMC cultures of HIV-1(+) PBMC and controls (N-PBMC) were established. Lymphocyte cell lines were characterized. Epstein-Barr virus latent membrane protein 1 (LMP-1) and Epstein-Barr nuclear antigen 2 were detected by polymerase chain reaction (PCR). Clonality of LCL was determined by light chain restriction (flow cytometry) and immunoglobulin H chain rearrangement (semi-nested PCR). Peripheral blood mononuclear cell phenotypes were studied at different intervals of culture. RESULTS: Lymphocyte cell lines were obtained in 73% of HIV-1(+) PBMC cultures, compared with 6% in N-PBMC. All LCL were EBV-positive (EBV(+)). B-cell lineage was established, and up to 12 different B-cell clones were expanded from the same individual. Occurrence of LCL was more frequent in cultures with HIV-1 replication, high LMP-1 expression in viable B cells, and high CD4:CD8 ratio. Human immunodeficiency virus type 1 replication persisted in 53% of the LCL. CONCLUSIONS: In vitro HIV-1 replication and persistence of viable EBV(+) lymphoblasts favor spontaneous in vitro outgrowth of LCL in HIV-1(+) patients.
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Linfocitos B/fisiología , Linfocitos B/virología , Línea Celular Transformada , Infecciones por VIH/virología , VIH-1/fisiología , Herpesvirus Humano 4/fisiología , Leucocitos Mononucleares/virología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Células Cultivadas , Citometría de Flujo , Hemofilia A/complicaciones , Humanos , Leucocitos Mononucleares/fisiologíaRESUMEN
Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.
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Técnicas de Cultivo de Célula/métodos , Infecciones por VIH/virología , VIH/aislamiento & purificación , Monocitos/virología , Terapia Antirretroviral Altamente Activa , Diferenciación Celular , División Celular , Células Cultivadas , Medios de Cultivo Condicionados/química , Femenino , VIH/crecimiento & desarrollo , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Antígeno Ki-67/análisis , Cinética , Macrófagos/química , Macrófagos/citología , Macrófagos/virología , Masculino , Monocitos/química , Monocitos/citología , Replicación ViralRESUMEN
A new method of culture of peripheral blood leukocytes (PBMC) from HIV+ patients, in the absence of exogenous stimuli (allogeneic cells or cytokines) (PBMC w/s) was used for the detection of persistent viral infection in HIV patients who had undergone successful highly active antiretroviral therapy (HAART) lowering their viral burden to undetectable levels (< 50 RNA copies/ml). Infected cells were always of the monocyte/macrophage lineage (M). No infection could be detected in these patients using the classical system (co-culture with HIV-CMP activated with PHA and IL-2). Differences in the class of target cells (higher proportion of proliferating M and CCR5 expressing cells in the PBMC w/s system than in PBMC-PHA cultures) may determine the relative sensitivity of each technique to achieve successful isolation of HIV from different patients.
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Terapia Antirretroviral Altamente Activa/métodos , Infecciones por VIH/tratamiento farmacológico , VIH/aislamiento & purificación , Macrófagos/virología , Carga Viral , VIH/fisiología , Humanos , Replicación ViralRESUMEN
Variations of the expression of CXCR4 and CCR5 HIV co-receptors after non stimulated culture of peripheral blood mononuclear cells (PBMC) from HIV+ patients were studied. Expression of CCR5 on both CD4+ and CD8+ T lymphocytes was reduced after 7 days and remained low throughout the culture. CXCR4 levels remained stable in both lymphocyte subpopulations. No significant changes were observed in control HIV- PBMC cultures. In order to ascertain if the CCR5 changes were associated to in vitro HIV replication, 6 days pre-cultured HIV- PBMC were infected with HIV+ culture supernatants. After 3 days CCR5 expression was reduced both in CD4+ and in CD8+ T lymphocytes, while CXCR4 expression was not, coincident with initiation of HIV replication in culture. These results suggest that CCR5 down modulation in CD4+ or CD8+ T lymphocytes is a consequence of HIV replication.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , VIH/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Replicación ViralRESUMEN
A new method of culture of peripheral blood leukocytes (PBMC) from HIV+ patients, in the absence of exogenous stimuli (allogeneic cells or cytokines) (PBMC w/s) was used for the detection of persistent viral infection in HIV patients who had undergone successful highly active antiretroviral therapy (HAART) lowering their viral burden to undetectable levels (< 50 RNA copies/ml). Infected cells were always of the monocyte/macrophage lineage (M). No infection could be detected in these patients using the classical system (co-culture with HIV-CMP activated with PHA and IL-2). Differences in the class of target cells (higher proportion of proliferating M and CCR5 expressing cells in the PBMC w/s system than in PBMC-PHA cultures) may determine the relative sensitivity of each technique to achieve successful isolation of HIV from different patients.
RESUMEN
Variations of the expression of CXCR4 and CCR5 HIV co-receptors after non stimulated culture of peripheral blood mononuclear cells (PBMC) from HIV+ patients were studied. Expression of CCR5 on both CD4+ and CD8+ T lymphocytes was reduced after 7 days and remained low throughout the culture. CXCR4 levels remained stable in both lymphocyte subpopulations. No significant changes were observed in control HIV- PBMC cultures. In order to ascertain if the CCR5 changes were associated to in vitro HIV replication, 6 days pre-cultured HIV- PBMC were infected with HIV+ culture supernatants. After 3 days CCR5 expression was reduced both in CD4+ and in CD8+ T lymphocytes, while CXCR4 expression was not, coincident with initiation of HIV replication in culture. These results suggest that CCR5 down modulation in CD4+ or CD8+ T lymphocytes is a consequence of HIV replication.
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We report here a case of nonhepatosplenic gammadelta T-cell lymphoma with undescribed initial localization in testis, without hepatosplenomegaly or adenopathies, and subsequent development in the maxillary sinus. The maxillar mass biopsy revealed a T-cell infiltration, and its immunologic characterization by flow cytometry showed a gammadelta T-cell phenotype (CD45+, CD3+, CD2+, TCR gammadelta+), without expression of CD7, CD5, CD1a, TdT, CD4, CD8, TCR alphabeta, or NK antigens (CD16, CD56, and CD57). Clonal gamma-chain gene rearrangement by polymerase chain reaction (PCR) was detected in testicular and maxillar biopsies. Epstein-Barr virus type 1 (EBV) sequences were detected by molecular biology in the biopsy material, suggesting that this oncogenic virus may play a role in the genesis of the clonal expansion of gammadelta T-cells. The patient was initially treated with standard chemotherapeutic protocols, with poor response and aggressive course.
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Neoplasias Hepáticas/patología , Linfoma de Células T/patología , Neoplasias del Bazo/patología , Neoplasias Testiculares/patología , Humanos , Masculino , Neoplasias del Seno Maxilar/patología , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T gamma-delta/análisisRESUMEN
Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.
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Apoptosis , Linfocitos T CD4-Positivos/fisiología , Infecciones por VIH/virología , VIH/fisiología , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Humanos , Activación de Linfocitos , Fagocitos/fisiología , Fagocitosis/fisiología , Carga Viral , Replicación ViralRESUMEN
Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.
RESUMEN
Generalized activation of the immune system after HIV infection leads to an exacerbation of all apoptotic mechanisms. CD4+, CD8+ T lymphocytes and B lymphocytes are sensitized to apoptotic stimuli. Macrophages are important in the removal of apoptotic cells, they prevent apoptotic cell accumulation during in vitro culture and they may lead to enhanced CD4+ T lymphocyte cell death through indirect mechanisms. A simple procedure for prolonged culture of peripheral blood mononuclear cells from HIV+ patients is discussed, in relation to its convenience to evaluate apoptosis, cell to cell interaction and HIV replication in the absence of exogenously added stimuli or co-culture of allogeneic cells. In this system, HIV replication takes place primarily in cells of macrophage lineage that may be activated into differentiation through removal of apoptotic debris during the culture.