RESUMO
Cutaneous leishmaniasis (CL) is an important public health issue worldwide. The control of Leishmania infection depends on cellular immune mechanisms, and the inflammatory response may contribute to pathogenesis. A beneficial role of CD8(+) T lymphocytes has been proposed; nevertheless, other studies suggest a cytotoxic role of CD8(+) T lymphocytes involved in tissue damage, showing controversial role of these cells. The goal of the current study was to understand the immunopathology of CL and determine the profile of cytotoxic cells--such as CD4(+) T, natural killer and natural killer T cells--that might be involved in triggering immunological mechanisms, and may lead to cure or disease progression. The frequencies of cytotoxic cell populations in peripheral blood, obtained from patients with active disease, during treatment and after clinical healing, were assessed by flow cytometry. Cytotoxicity could not be related to a deleterious role in Leishmania braziliensis infection, as patients with active CL showed similar percentages of degranulation to healthy individuals (HI). Cured patients exhibited a lower percentage of degranulating cells, which may be due to a downregulation of the immune response. The understanding of the immunopathological mechanisms involved in CL and the commitment of cytotoxic cells enables improvements in therapeutic strategies.
Assuntos
Leishmaniose Cutânea/imunologia , Adulto , Antiprotozoários/uso terapêutico , Contagem de Linfócito CD4 , Degranulação Celular , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/parasitologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/tratamento farmacológico , Masculino , Meglumina/uso terapêutico , Antimoniato de Meglumina , Pessoa de Meia-Idade , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/parasitologia , Compostos Organometálicos/uso terapêutico , Adulto JovemRESUMO
The cutaneous leucocyte-associated antigen receptor (CLA) can direct Leishmania-specific T lymphocytes towards inflamed skin lesions. Homing receptors [CLA, lymphocyte-associated antigen 1 (LFA-1) or CD62L] were analysed in lymphocytes from blood and cutaneous leishmaniasis (CL) lesions. CL patients with active lesions (A-CL) presented lower levels of T lymphocytes expressing the CLA(+) phenotype (T CD4(+) = 10.4% +/- 7.5% and T CD8(+) = 5.8% +/- 3.4%) than did healthy subjects (HS) (T CD4(+) = 19.3% +/- 13.1% and T CD8(+) = 21.6% +/- 8.8%), notably in T CD8(+) (P < 0.001). In clinically cured patients these percentages returned to levels observed in HS. Leishmanial antigens up-regulated CLA in T cells (CLA(+) in T CD4(+) = 33.3% +/- 14.1%; CLA(+) in T CD8(+) = 22.4% +/- 9.4%) from A-CL but not from HS. An enrichment of CLA(+) cells was observed in lesions (CLA(+) in T CD4(+) = 45.9% +/- 22.5%; CLA(+) in T CD8(+) = 46.4% +/- 16.1%) in comparison with blood (CLA(+) in T CD4(+) = 10.4% +/- 7.5%; CLA(+) in T CD8(+) = 5.8% +/- 3.4%). Conversely, LFA-1 was highly expressed in CD8(+) T cells and augmented in CD4(+) T from peripheral blood of A-CL patients. In contrast, CD62L was not affected. These results suggest that Leishmania antigens can modulate molecules responsible for migration to skin lesions, potentially influencing the cell composition of inflammatory infiltrate of leishmaniasis or even the severity of the disease.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T/imunologia , Adulto , Animais , Antígenos de Diferenciação de Linfócitos T , Antígenos de Neoplasias/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Selectina L/análise , Ativação Linfocitária , Contagem de Linfócitos , Antígeno-1 Associado à Função Linfocitária/análise , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Receptores de Retorno de Linfócitos/metabolismo , Pele/imunologia , Estatísticas não Paramétricas , Linfócitos T/metabolismo , Adulto JovemRESUMO
Immunity to yellow fever (YF) is conferred by the interplay of humoral and cellular immune response. Despite the extensive literature on the humoral immune response to the YF vaccine virus, little is known about its cellular immune response to vaccination. The analysis of cytokine production by ex-vivo antigen-stimulated T cells has been considered as a valuable tool for understanding cellular immune response. Thus, we have analyzed two T(H)1/T(H)2 signature cytokines (IFN-gamma and IL-4) from 12 healthy first-time adults vaccinated with YF17DD virus. The cells, harvested on day 0 (before vaccination) and 7, 15 and 30 days after immunization were antigen-stimulated and analyzed by ELISpot. A significant increase in the number of spot-forming cells during the response to YF 17DD live virus stimulation by ELISpot assay was observed. IFN-gamma-and IL-4-producing cells were significantly increased on the 15th day after vaccination in all volunteers. These results presented herein are important for understanding the role of cytokines in the immune response to YF 17DD virus.
Assuntos
Citocinas/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Adolescente , Adulto , Citocinas/análise , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Pessoa de Meia-Idade , Células Th1/química , Células Th1/imunologia , Células Th2/química , Células Th2/imunologia , Vacina contra Febre Amarela/administração & dosagemRESUMO
BACKGROUND: CD4+ and CD8+ T lymphocytes play different roles in the outcome of leishmaniasis. However, T-cell distribution in lesions shows significant variability in in situ immunocytochemical studies. OBJECTIVES: In this report flow cytometry was used to determine the predominant T-cell subsets in leishmaniasis lesions, and their relationship with Leishmania-responsive circulating T cells. PATIENTS AND METHODS: Mononuclear cells from lesions or peripheral blood (PBMC) of 34 cutaneous (CL), four mucosal (ML) and four disseminated leishmaniasis were phenotypically characterized by flow cytometry. Leishmania-responsive T cells were obtained after in vitro stimulation of PBMC with leishmanial antigens. RESULTS/CONCLUSIONS: Variable amounts of gammadelta lymphocytes were present in all lesions, with no association with duration of illness. The highest percentages of interleukin-2R- and interferon-gammaR-positive cells were observed in ML lesions and could render these T cells more susceptible to the effects of these cytokines. The distribution of intralesional T-lymphocyte subsets was quite variable (CD4+ > CD8+ = 18 cases, CD8+ > CD4+ = 12 cases and CD4+ congruent with CD8+ = 4 cases) without any association with clinical parameters, and could explain the controversy regarding proportions of these T-cell subsets in leishmaniasis lesions. Low percentages of Leishmania-reactive CD8+ T cells were observed in blood while an enrichment of CD8+ cells was shown in the inflammatory infiltrate, suggesting that local immunoregulatory factors could favour the recruitment and/or proliferation of local CD8+ lymphocytes. Increased percentages of CD8+ cells observed in older lesions are consistent with the hypothesis that they can mediate healing, although their involvement in tissue damage cannot be ruled out. It is possible that these mechanisms can influence the clinical outcome or even the response to therapy.
Assuntos
Leishmania braziliensis , Leishmaniose Mucocutânea/imunologia , Mucosa/imunologia , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.
Assuntos
Animais , Coelhos , Células Eritroides/citologia , Células Eritroides/metabolismo , Células Eritroides/ultraestrutura , Embrião de Mamíferos/citologia , Hemoglobinas/análise , Hemoglobinas/biossíntese , Hemoglobinas/imunologia , Citometria de Fluxo , Microscopia/métodosRESUMO
Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.
Assuntos
Antígenos Virais/análise , Vírus da Dengue/imunologia , Dengue/imunologia , Leucócitos Mononucleares/imunologia , Animais , Linhagem Celular/virologia , Separação Celular , Chlorocebus aethiops , Células Clonais/imunologia , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/isolamento & purificação , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/virologia , Receptores de Lipopolissacarídeos/análise , Células Vero/citologia , Células Vero/virologiaRESUMO
In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.
Assuntos
Apoptose/fisiologia , Citometria de Fluxo/métodos , Necrose , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , HumanosRESUMO
Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.
Assuntos
Citocinas/análise , Citoplasma/química , Citometria de Fluxo/métodos , Animais , Humanos , Leishmania braziliensis , Leishmaniose Cutânea/imunologiaRESUMO
Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation-reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.
Assuntos
Eritrócitos/fisiologia , Eritropoese/fisiologia , Heme/biossíntese , Mitocôndrias/metabolismo , Anemia Hemolítica , Animais , Anuros , Bufonidae , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Citometria de Fluxo/métodos , Isocitrato Desidrogenase/metabolismo , Cinética , Microscopia de Fluorescência/métodos , Mitocôndrias/fisiologia , Succinato Desidrogenase/metabolismoRESUMO
Callithrix jacchus is considered a reliable animal model for hepatitis A virus (HAV) infection. All three HAV orally inoculated marmosets developed hepatitis - the infection was monitored by continuous virus shedding, high levels of serum enzyme alanine aminotransferase, specific antibody and seroconversion 3-6 weeks after HAV inoculation. HAV antigen was detected in liver by immunofluorescence 4 days post inoculation (PI) and onwards. To gain insight into the biological role of inducible nitric oxide synthase (iNOS) during immune-related acute liver injury the enzyme was searched in frozen biopsies: immunofluorescent labeling was found in the cytoplasm of liver cells mainly Kupffer's cells and spleen macrophages (CD68+) starting 11 days PI with maximum intensity on the fifth to sixth week PI. Necroinflammatory liver lesions characteristic of viral hepatitis were also observed at 10 days PI with maximum severity at 4 to 6 weeks PI. Furthermore, T lymphocytes (CD2+) were raised at this time point. No difference was evident in the frequency of B lymphocytes (CD20+). Therefore, iNOS expression preceded necroinflammatory liver lesion and maximal immunofluorescence reaction was coincident with tissue injury, supporting the hypothesis that NO contributes to hepatic cytotoxic mechanism but also to virus clearance. The concomitant rise in T-lymphocyte population may suggest a role for these cells in this and/or other independent HAV-induced pathological changes.
Assuntos
Hepatite A/enzimologia , Hepatovirus , Fígado/patologia , Óxido Nítrico Sintase/biossíntese , Linfócitos T/imunologia , Animais , Callithrix , Modelos Animais de Doenças , Indução Enzimática , Imunofluorescência , Hepatite A/patologia , Imunofenotipagem , Fígado/enzimologia , Fígado/virologia , Necrose , Óxido Nítrico Sintase Tipo II , Baço/virologia , Linfócitos T/virologiaRESUMO
Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 +/- 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 +/- 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 +/- 3.8 and 15.3 +/- 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Leishmaniose Cutânea/fisiopatologia , Adulto , Morte Celular , Corantes/administração & dosagem , Feminino , Citometria de Fluxo , Humanos , Leishmaniose Cutânea/imunologia , MasculinoRESUMO
Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 + or - 2.7 per cent) as compared with lesions undergoing spontaneous healing (mean = 17.8 + or - 2.2 per cent). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 + or - 3.8 and 15.3 + or - 3.0 per cent, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.
Assuntos
Humanos , Masculino , Feminino , Adulto , Apoptose , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Leishmaniose Cutânea/fisiopatologia , Morte Celular , Corantes/administração & dosagem , Dactinomicina/administração & dosagem , Citometria de Fluxo , Leishmaniose Cutânea/imunologiaRESUMO
This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.
Assuntos
Antígenos de Protozoários/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Adulto , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Humanos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Leishmania braziliensis/imunologia , Masculino , Fenótipo , Testes Cutâneos , EsterilizaçãoRESUMO
Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.
Assuntos
Leishmaniose Cutânea/imunologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Humanos , Interferon gama , Interleucina-4RESUMO
Clinical improvement has been described in AIDS patients submitted to zinc therapy, but the mechanisms involved are not well understood. In order to evaluate the effect of the zinc ions in the enhancement of the immune response, we tested its role in the lymphoproliferative response to a mitogen, as well as in the prevention of apoptosis. The mitogenic effect of zinc (10(-4)M ZnCl2) on the lymphocyte proliferative response was observed in healthy controls as well as in HIV-1+ asymptomatic individuals. Very low stimulation index could be observed in AIDS patients (CD4+<200/mm3). However, zinc treatment of phytohaemagglutinin (PHA; 5 microg/ml)-stimulated PBMC cultures significantly enhanced 3H-thymidine incorporation in both asymptomatic and symptomatic groups. A decreased percentage of apoptotic cells could be identified in cell cultures from HIV-1+ individuals submitted to zinc treatment compared with cells treated only with PHA, as detected by both flow cytometry and agarose gel electrophoresis. Further studies with zinc supplementation associated to anti-retroviral therapy would be of great interest to evaluate the in vivo role of this oligoelement in the improvement of the immunological functions of HIV-1-infected individuals and AIDS patients.
Assuntos
Adjuvantes Imunológicos/farmacologia , Apoptose/efeitos dos fármacos , Soropositividade para HIV/sangue , Soropositividade para HIV/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Zinco/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Feminino , Infecções por HIV/sangue , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/citologia , Ativação Linfocitária/imunologia , Masculino , Mitógenos/farmacologia , Fito-Hemaglutininas/farmacologiaRESUMO
Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of INF-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.
Assuntos
Humanos , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/fisiopatologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Interferon gama , Interleucina-4RESUMO
Acute Trypanosoma cruzi infection induces alterations in both lymphoid and microenvironmental compartments of the thymus. This prompted us to investigate whether the lymphoepithelial complex thymic nurse cell (TNC) was comprised in the thymic pathology occurring in experimental Chagas' disease. The isolation of TNCs from acutely T. cruzi-infected mice revealed a reduction in TNC numbers that paralleled thymic atrophy. This decrease does not seem to be stress-related since it was not seen following glucocorticoid hormone injection. Moreover, an increased intra-TNC cell death in complexes from infected animals was noticed. In addition, acute T. cruzi infection induced a decrease in size and granularity of TNC complexes, as well as several ultrastructural alterations indicating cell damage. The epithelial component of TNCs, independent of being infected in vitro or derived from infected animals, showed an enhancement of extracellular matrix proteins that is likely related to the enhanced thymocyte release observed in these complexes. Conjointly, these data show that TNCs are importantly affected in acute experimental T. cruzi infection, possibly contributing to the previously observed alterations in thymocyte differentiation.
Assuntos
Doença de Chagas/imunologia , Timo/patologia , Trypanosoma cruzi , Animais , Contagem de Células , Morte Celular , Tamanho Celular , Doença de Chagas/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Timo/imunologia , Timo/parasitologiaRESUMO
Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-gamma and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. approximately or = 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. approximately or = 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-gamma and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis.
Assuntos
Antígenos de Protozoários/imunologia , Citocinas/biossíntese , Leishmania braziliensis/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Linfócitos T/imunologia , Animais , Antimônio/uso terapêutico , Antiprotozoários/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Feminino , Humanos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Leishmaniose Cutânea/tratamento farmacológico , Ativação Linfocitária , Masculino , Meglumina/uso terapêutico , Antimoniato de Meglumina , Compostos Organometálicos/uso terapêuticoAssuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunocompetência , Hospedeiro Imunocomprometido , Leishmaniose Cutânea/imunologia , Animais , Humanos , Ativação Linfocitária , Linfocinas/biossíntese , CamundongosRESUMO
A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.