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[This corrects the article DOI: 10.1099/acmi.0.000245.].
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The current tuberculosis (TB) vaccine Mycobacterium bovis BCG has been employed for some 70 years in Brazil and lessons from its use should be taken in account for the development or improvement of new TB vaccines. The vast majority of the current population has been vaccinated with BCG, with the possible requirement for a booster immunisation in adulthood for TB protection. BCG Moreau strain also protects against leprosy, meningitis and extrapulmonary forms of TB. Factors related to differences in strain, dosage and BCG administering protocol have been responsible for the variable efficacy of BCG. This vaccine is clearly affected by, as yet unclear, host and/or environmental variables. In this brief review, we describe some aspects of BCG immunisation observed in Brazil that may be of importance for improving or replacing BCG.
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Adyuvantes Inmunológicos/uso terapéutico , Vacuna BCG/uso terapéutico , Tuberculosis Pulmonar/prevención & control , Brasil , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Inmunización Secundaria , Salud Rural , Resultado del TratamientoRESUMEN
Tuberculosis (TB) remains among the world's leading cause of mortality. For its control, studies of TB vaccines are needed. Since live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently in use, studies on the protective role of BCG are required. In this study, we analyzed host cells purified directly from whole blood of human immunodeficiency virus (HIV)-negative volunteers, comprising adult healthy donors (HD) and neonates (umbilical cord bloods, UCB), with the aim to directly compare in vitro immune responses with distinct BCG strains in human mononuclear cells. The Moreau, Pasteur, and Danish BCG strains were used to infect mononuclear cells in vitro for 48 h; bacilli viability and cell-death were subsequently detected by flow cytometry. In addition, cell culture supernatants were used in cytokine detection assays. Overall, the Moreau BCG strain induced higher levels of apoptosis than the Pasteur and Danish BCG strains in both the HD and UCB groups (p-value < 0.05), and a human monocytic cell-line mirrored those cell-death patterns after BCG infection. The Moreau BCG strain, exclusively, induced Th1 cytokines at the highest levels in cells from adults (p-value < 0.05) when compared with both Pasteur and Danish BCG strains, whereas TGF-ß1 levels were reduced significantly (p-value < 0.01) in the HD group when cells were infected with the Moreau BCG vaccine. As expected, eight out of 22 pro-inflammatory cytokines were secreted at significant levels (p-value < 0.05) above the baseline rates in all BCG-infected cell cultures, in the HD group only. When analyzing these results, we excluded confounding factors related to storage and viability of the BCG strains used. These findings suggest that Moreau BCG is a more potent immunostimulating agent than the Pasteur and Danish BCG strains. Clinical trials will be needed to confirm these findings.
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Apoptosis/inmunología , Vacuna BCG/inmunología , Citocinas/inmunología , Leucocitos Mononucleares/inmunología , Tuberculosis/prevención & control , Adulto , Vacuna BCG/genética , Voluntarios Sanos , Humanos , Inmunogenicidad Vacunal , Recién Nacido , Mycobacterium bovis/genética , Mycobacterium bovis/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
This study compares the role of hepatic cytosolic glutathione transferases (cGST) isoforms of three different bivalve species to a Microcystis aeruginosa extract and purified MC-LR exposure (both at 150 µg MC-LR L-1) for 24 h. Characterization and alterations of the cytosolic GST activities in Mytilus galloprovincialis, Ruditapes philippinarum and Corbicula fluminea were measured using four class-specific substrates and changes in individual GST isoforms expression were achieved by a subsequent two-dimensional electrophoresis analysis. Evaluation of cGST activity basal levels using the four class-specific substrates denoted quantitative differences between the three bivalves. Purified MC-LR did not induce any significant response from bivalves. On the other hand, cell extracts caused significant alterations according to bivalves and substrates. Among the three bivalves, only R. philippinarum showed a significant induction of cGST activity using generic 1-chloro-2,4-dinitrobenzene (CDNB) substrate. However, no significant alterations were detected in these clams by cell extracts using the other specific substrates. In contrast, C. fluminea revealed significant induction of cGST activity when using 3,4-dichloronitrobenzene (DCNB) and ethacrynic acid (EA). In M. galloprovincialis, cell extracts promoted a significant decrease of cGST activity when using EA substrate. Altered protein expression was quantitatively detected upon exposure to cell extracts for one spot in R. philippinarum and another for C. fluminea, both upregulated (2.0 and 8.5-fold, respectively) and identified as a sigma1-class GST in the case of the first. The results showed that the three bivalves presented specific adaptive biotransformation responses to MCs and other cyanobacteria compounds supported by the modulation of distinct cGST classes.
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Toxinas Bacterianas/toxicidad , Bivalvos/efectos de los fármacos , Glutatión Transferasa/antagonistas & inhibidores , Hígado/enzimología , Microcistinas/toxicidad , Animales , Biotransformación , Bivalvos/enzimología , Isoenzimas/antagonistas & inhibidores , Toxinas Marinas , Microcystis , Proteómica , Extractos de Tejidos/toxicidadRESUMEN
The Bacillus Calmette-Guerin (BCG) vaccine is not a single organism, but consists of substrains that vary in genotypes and phenotypes. Actually, BCG is the common name given to a family of vaccines created in 1921 by the in vitro attenuation of a virulent Mycobacterium bovis in France. Even nearly a century of use, the BCG vaccine lingers generating confusion and debate due to its diversity and failure to protect against tuberculosis (TB). That is probably owing to the enduring lack of standardization during production, distribution and administration procedures. Since the 1940s, substantial sequence modifications among the BCG substrains have been described. To increase the level of complexity, even though that the prolific generation of recombinant BCG vaccines has been promising, the relationships between those candidates used in current clinical trials and their parental substrains are either unsatisfactorily connected or have been never fully delineated. Consequently, the use of the most protective BCG substrain as the background or platform in the development of all recombinant BCG vaccine candidates has not been standardized. In order to schematize and to clarify the subject regarding substrains commonly used to generate those novel vaccines, a sequential emergence of the parental BCG vaccine substrains and their matching recombinant ones, if any, was built. Hence, for a total of 24 BCG substrains currently in circulation worldwide, 9 have been used to sustain one or more genetic modifications, resulting in around 21 novel recombinant BCG vaccines. Although this is a remarkable success, only 2 out of the 21 recombinant BCG substrains harbor a background representative of the most immunogenic group. Systematizing the novel BCG vaccines and their parental strains may facilitate our understanding of protection provided by BCG immunizations.
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Vacuna BCG/inmunología , Vacuna BCG/aislamiento & purificación , Evaluación de Medicamentos/normas , Mycobacterium bovis/genética , Vacunación/normas , Variación Genética , HumanosRESUMEN
Production of IFN-gamma guarantees helpful T cell-mediated immunity against Mycobacterium tuberculosis infection. We have evaluated the in vitro immune responses to M. tuberculosis antigens using IFN-gamma production among 43 Brazilian tuberculosis (TB) patients prior to and after specific treatment, and 18 community controls. Peripheral blood mononuclear cells (PBMC) were cultivated in the presence either of purified protein derivative, ferritin, 10 kDa, 38 kDa, MPT59, Ag85A or Ag85B. Also, the two M. tuberculosis and M. bovis heat-shock proteins (hsp) 65 and 70 kDa were compared, and 5 day supernatants were harvested for cytokine detection by ELISA. The results showed that the overall profile of primary PBMC in response to most M. tuberculosis antigens was well correlated, since high IFN-gamma levels were induced by Ag85A, Ag85B, 38 kDa, ferritin and 10 kDa, as well as M. tuberculosis hsp65 in TB patients. In addition, analysis was carried out of the in vitro expression of activation molecules on lymphocytes, as CD25 and CD69 expression assessed in 17 TB patients showed induction on CD4+ T cells by Ag85B. Overall, significantly low responses were found in untreated, in comparison with the treated TB patients. Furthermore, internal community but not healthy control individuals have higher immune responses than do TB patients.
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Antígenos Bacterianos/inmunología , Interferón gamma/biosíntesis , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunología , Adolescente , Adulto , Anciano , Brasil , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana EdadRESUMEN
Tuberculosis (TB) remains the world's leading cause of morbidity and mortality. Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine currently in use, its efficacy is highly variable. It has been suggested that early antigenic presentation is a pivotal event leading to a better immune response in TB vaccine models. To investigate this further, we compared in vitro cell-mediated immune responses in the context of early sensitization with TB (i.e. healthy adults vaccinated with BCG when they were young, HD; n = 25) to those in its absence (i.e., newborns with naïve immunity to TB, UV; n = 10) by challenging mononuclear cells with BCG Moreau. After 48 hours, CD4+ and CD8+ T cells were harvested from both groups and stained for PD-1/CD25/ FOXP3. In addition, supernatants were assayed for a broad range of cytokines using an array system. The HD group showed robust reactivity to Protein Purified Derivative and BCG while the naïve, UV group did not. Similarly, in terms of PD-1 expression and Treg cells (CD4+/CD25high(+)/FOXP3+), only the HD group showed higher levels in CD4 lymphocytes. Otherwise, only the UV group showed expression of CD25dim+ as an activation marker dependent on BCG infection. In terms of cytokines, the HD group showed higher levels of Th1 (IL-2/TNF-α/IFN-γ) and regulatory (IL-10) profiles, with monocytes, but not Tr1 cells, acting as the main source of IL-10. Taken together, our results highlight critical roles of early sensitization with TB in mounting cell-mediated immune responses.
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Vacuna BCG/administración & dosificación , Vacuna BCG/inmunología , Leucocitos Mononucleares/inmunología , Subgrupos de Linfocitos T/inmunología , Adolescente , Adulto , Brasil , Células Cultivadas , Medios de Cultivo/química , Citocinas/análisis , Factores de Transcripción Forkhead/análisis , Voluntarios Sanos , Humanos , Subunidad alfa del Receptor de Interleucina-2/análisis , Leucocitos Mononucleares/química , Receptor de Muerte Celular Programada 1/análisis , Subgrupos de Linfocitos T/química , Adulto JovenRESUMEN
The acute phase of Chagas' disease was classified as early, intermediate, and late based on the levels of anti-Galalpha, 3Gal IgG (Gal) and specific IgM (M) and IgG (G) anti-T. cruzi reactivity. While the early phase was M+G-Gal-, the intermediate phase was M+G-Gal+, M+G+Gal-, or M+G+Gal+, and the late phase was M-G+Gal+. This sequence of stages was consistent with our previous studies on acute-phase proteins. Analysis by the polymerase chain reaction (PCR) of parasite DNA in 65 blood samples of children living in Cochabamba, Bolivia showed a significant correlation (90.8%) between ELISA and PCR positivity. A lower correlation was observed between indirect hemagglutination, PCR (58%), and ELISA. Electrocardiographic analysis of 43 children studied by the PCR did not show any alteration typical of acute chagasic myocarditis. The PCR positivity was observed in eight samples where only Gal was increased, suggesting a very early T. cruzi infection, when specific antibodies were not yet present. By associating anti-Gal IgG with specific serology, early T. cruzi infection can be detected with greater precision. We suggest the use of anti-Gal antibody reactivity as an aid for the detection of recent T. cruzi infections, at least in endemic areas where diseases caused by other trypanosomatids do not overlap.
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Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/clasificación , Enfermedad de Chagas/inmunología , Trypanosoma cruzi/inmunología , Adolescente , Animales , Bolivia/epidemiología , Enfermedad de Chagas/sangre , Enfermedad de Chagas/epidemiología , Niño , Preescolar , ADN Protozoario/sangre , Electrocardiografía , Ensayo de Inmunoadsorción Enzimática , Femenino , Galactosa/inmunología , Pruebas de Hemaglutinación , Humanos , Inmunoglobulina G/sangre , Masculino , Reacción en Cadena de la Polimerasa , Pruebas SerológicasRESUMEN
Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/biosíntesis , Lepra/inmunología , Mycobacterium leprae/inmunología , Tuberculosis Pulmonar/inmunología , Citoplasma/inmunología , Citometría de Flujo , Humanos , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-4/biosíntesis , Tuberculina/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
SETTING: Randomised trial comparing 9 months of isoniazid with 4 months of rifampicin for the treatment of high-risk tuberculin skin test positive subjects in Rio de Janeiro, Brazil. OBJECTIVES: To compare QuantiFERON®-TB Gold In-Tube (QFT-GIT) responses before and 1, 4 and 9 months after starting treatment for latent tuberculous infection (LTBI) according to adherence to one of the two regimens. DESIGN: Participants in the trial were invited to undergo serial QFT-GIT. Within-subject differences at different time points were analysed as quantitative responses and categorised as positive or negative using different cut-off points. RESULTS: Of 215 participants, 118 completed treatment, of whom 58 underwent all three tests; and 97 did not complete treatment, of whom 10 underwent all tests. After 1 month of treatment, there was no significant difference in QFT-GIT response between the groups. After 4 and 9 months, reversions were more frequent in non-adherent subjects. Marked within-subject fluctuations were observed. No cut-off point could be established at which QFT-GIT responses were consistently positive or associated with adherence or type of treatment. CONCLUSION: Frequent within-subject variability in QFT-GIT responses, not associated with LTBI treatment, makes it difficult for clinicians to interpret QFT-GIT conversions and reversions.
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Antituberculosos/uso terapéutico , Isoniazida/uso terapéutico , Tuberculosis Latente/tratamiento farmacológico , Rifampin/uso terapéutico , Adulto , Antituberculosos/administración & dosificación , Brasil , Femenino , Humanos , Ensayos de Liberación de Interferón gamma , Isoniazida/administración & dosificación , Tuberculosis Latente/microbiología , Masculino , Cumplimiento de la Medicación , Persona de Mediana Edad , Rifampin/administración & dosificación , Factores de Tiempo , Resultado del Tratamiento , Prueba de Tuberculina , Adulto JovenRESUMEN
Mononuclear cells have been implicated in the primary inflammatory response against mycobacteria. Yet, little is known about the interaction of Mycobacterium bovis bacillus Calmette-Guerin (BCG) with human monocytes. Here, we investigated the potential of BCG Moreau strain to induce in vitro specific cell-death utilizing a flow cytometry approach that revealed an increase in apoptosis events in BCG-stimulated monocytes from healthy adults. We also detected a concomitant release of interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), but not metalloproteinase (MMP)-9. In addition, annexin V-propidium iodide double staining demonstrated an enhancement of monocytes necrosis, but not apoptosis, following BCG Moreau strain stimulation of umbilical vein cells from naïve, neonate. This pattern was paralleled by different pro-inflammatory cytokine levels, as well as MMP-9 induction when compared to the adults. Our findings support the hypothesis that BCG induces distinct cell-death patterns during the maturation of the immune system and that this pattern might set the stage for a subsequent antimycobacterial immune response that might have profound effects during vaccination.
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Vacuna BCG/inmunología , Muerte Celular , Interleucina-1beta/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Monocitos/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Embarazo , Adulto JovenRESUMEN
Multidrug-resistant tuberculosis (MDR-TB) is known as having a poor prognosis with a weak response to therapy and very high death rates. The aim of this work was to assess the immune response to the RD1-encoded antigen ESAT-6 of Mycobacterium tuberculosis in MDR-TB patients and compare to non-resistant (NR) TB patients and healthy controls (HC). Evaluation of interferon (IFN)-gamma production showed that, although 55% of the MDR patients were responsive to ESAT-6, they produced lower IFN-gamma levels (553 +/- 11 pg/ml) when compared to NR-TB (1179 +/- 163 pg/ml; P < 0.05) but not to controls (412 +/- 65.7 pg/ml). Differences in the response to ESAT-6 and to its overlapping peptides mixture were also significant between MDR versus treated pulmonary NR-TB. Furthermore, a very low rate of response to PPD (23.5%) and to Ag85B (33.3%) was noted in MDR-TB patients as compared to the other groups. To determine the inflammatory response in patients' groups, detection of tumour necrosis factor (TNF)-alpha was assessed in their sera before and during chemotherapy. Mean TNF-alpha levels in MDR-TB (43.8 +/- 9 pg/ml) paralleled those found in treated pulmonary, and it was significantly different (P < 0.05) from the values found in untreated NR and HC. Interestingly, secretion of IFN-gamma and TNF-alpha were predominant in MDR patients who presented with bilateral pulmonary lesions and lung cavitation. The present data indicate that the overall immune response to mycobacterial antigens is decreased in resistant TB and the major role inflammatory cytokines may play in perpetuating pulmonary tissue damage.
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Interferón gamma/análisis , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Factor de Necrosis Tumoral alfa/análisis , Adulto , Antígenos Bacterianos/inmunología , Antituberculosos/uso terapéutico , Proteínas Bacterianas , Estudios de Casos y Controles , Células Cultivadas , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Tuberculosis Resistente a Múltiples Medicamentos/sangre , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
We performed a cross-sectional flow cytometric analysis of peripheral blood mononuclear cells to evaluate human immunologic status during early stages of Trypanosoma cruzi infection in children. We identified major immunological features corresponding to three proposed phases of disease: early acute (EA) phase, late acute (LA) phase and recent chronic (RC) phase. EA phase was accompanied by expansion of conventional B cells, up-regulation of CD54 on monocytes and down-regulation of CD54 on T cells and not associated with monocyte-activation phenotypes or changes of natural killer (NK) population. LA phase was characterized by a selective increase in a distinct lineage of NK cells (CD16+CD56-), as well as a persistent expansion of B cells and down-regulation of CD54 on T cells. RC phase showed persistent low levels of CD54 molecule on T cells and an increase of B cells, mainly triggered by expansion of the B1-cell subset, as well as increased expression of human leucocyte antigen (HLA-DR) by monocytes. These findings reinforce the hypothesis that T. cruzi-derived antigens are able to activate NK cells before the development of T-cell-mediated immunity. Moreover, our data support previous findings of increased levels of B1 lymphocytes during human Chagas' disease and show that this event is already present during initial stages of chronic infection.
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Enfermedad de Chagas/inmunología , Leucocitos/inmunología , Adolescente , Antígenos CD19/análisis , Complejo CD3/análisis , Antígeno CD56/análisis , Niño , Preescolar , Antígenos HLA-DR/análisis , Humanos , Inmunofenotipificación , Molécula 1 de Adhesión Intercelular/análisis , Receptores de IgE/análisisRESUMEN
A rapid, sensitive, specific, and reliable enzyme-linked immunosorbent assay (ELISA) is proposed for determination of the levels of anti-Trypanosoma cruzi IgM in acute chagasic sera (ACD). The efficiency of this ELISA as a diagnostic method was compared with that of parasite DNA detection by polymerase chain reaction (PCR) and that of indirect immunofluorescence (iIF) anti-T. cruzi IgM detection. We tested whether this ELISA using fixed epimastigotes (epi) could detect anti-T. cruzi IgM in serum samples from two groups of children with acute Chagas' disease from a hyperendemic area in Bolivia. In a comparison of the ELISA method with other techniques, 95% and 71% of the results correlated with PCR and iIF findings, respectively. At the serum dilution applied (1:250), rheumatoid factor (RF) did not influence the results, and samples from patients carrying leishmaniasis or mixed Leishmania and T. cruzi infection could also be excluded from ACD. Highly specific and reliable results were obtained, a great number of the sera could be tested in only one assay, and a quantitative index of reactivity (IR) could be calculated without serial titration. Using test samples in triplicate, the method provides a useful tool for the detection of early acute-phase T. cruzi infection in humans.
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Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina M/sangre , Enfermedad Aguda , Adolescente , Bolivia , Enfermedad de Chagas/epidemiología , Niño , Preescolar , Reacciones Cruzadas , Enfermedades Endémicas , Ensayo de Inmunoadsorción Enzimática/normas , Fijadores , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Formaldehído , Humanos , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa/métodos , Factor Reumatoide/inmunología , Sensibilidad y EspecificidadRESUMEN
Here, we analysed the use of Vbeta-TCR regions by CD4+ and CD8+ T cells from acute and chronic chagasic patients using flow cytometry. We determined the Vbeta expression in cells freshly isolated from patients, as well as after in vitro stimulation with antigens derived from epimastigote (EPI) or trypomastigote (TRYPO) forms of Trypanosoma cruzi. Analysis of Vbeta-TCR expression of T cells freshly isolated from patients showed a decrease in Vbeta5 expression in the CD4+ T-cell population from acutely infected individuals, whereas CD4+Vbeta5+ T cells were found to be increased in chronic patients with the cardiac, but not indeterminate, clinical form. After culturing peripheral blood mononuclear cells (PBMC) from chronic patients with EPI or TRYPO, we found that both antigenic preparations led to a preferential expansion of CD4+Vbeta5+ T cells. EPI stimulation also led to the expansion of CD8+Vbeta5+ T cells, whereas TRYPO led to the expansion of this cell population only if PBMC were from cardiac and not indeterminate patients. We observed that TRYPO stimulation led to an increase in the frequency of CD4+Vbeta17+ T cells in cultures of PBMC from indeterminate patients, whereas an increase in the frequency of CD8+Vbeta17+ T cells was found upon TRYPO stimulation of PBMC from cardiac patients. Despite this increase in the frequency of Vbeta17+ T-cell populations upon TRYPO stimulation, the same antigenic preparation led to a much higher expansion of Vbeta5+ T cells. These results show a differential expression of Vbeta5-TCR in cells freshly isolated from chagasic patients in different stages of the disease and that parasite-specific antigens stimulate a portion of the T-cell repertoire with preferential usage of Vbeta5-TCR.
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Enfermedad de Chagas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Enfermedad Aguda , Animales , Antígenos de Protozoos/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Enfermedad Crónica , Cardiopatías/inmunología , Humanos , Trypanosoma cruzi/inmunologíaRESUMEN
Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.