RESUMO
There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Citocinas/biossíntese , Ativação Linfocitária/imunologia , Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/imunologia , Febre de Chikungunya/patologia , Febre de Chikungunya/virologia , Citocinas/imunologia , Citotoxicidade Imunológica/imunologia , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/imunologia , Granzimas/biossíntese , Granzimas/imunologia , Humanos , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-17/biossíntese , Interleucina-17/imunologia , Lectinas Tipo C/biossíntese , Lectinas Tipo C/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/biossíntese , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Perforina/biossíntese , Perforina/imunologia , Receptor fas/biossíntese , Receptor fas/imunologiaRESUMO
Myocarditis is a common cardiovascular disease and frequently occurs in children and teenagers. It is believed to be caused by both endogenous and exogenous factors, among which FAS/FASL gene pair-induced cell apoptosis is a major mechanism of myocardial cell injury. A previous study has detected low expression of microRNA (miR)-98 in myocarditis patients. Therefore, in this study we investigated the functional implications of miR-98 with respect to the disease. We carried out a case-control study including 50 myocarditis patients and 50 healthy individuals. Total RNA was extracted from peripheral blood plasma. Expression levels of miR-98 and the FAS/FASL gene pair were determined by real-time fluorescent quantitative polymerase chain reaction. The interaction between miR-98 and the FAS/FASL pair was visualized by dual-luciferase reporter assay. The expression of the FAS/FASL gene pair was further detected by transfecting with an miR-98 mimic or an miR-98 inhibitor. The content of miR-98 in the peripheral blood of the myocarditis patients was significantly lower than in the healthy individuals. However, the FAS/FASL genes were upregulated by 1.68-fold in the myocarditis patients. miR-98 was shown to interact with the 3'-untranslated region of the FAS/FASL gene pair. The inhibition/facilitation of miR-98 expression in myocardial cells can modulate apoptosis. miR-98 was downregulated in the peripheral blood of myocarditis patients. It may interact with the FAS/FASL gene pair to further modulate cell apoptosis.
Assuntos
Proteína Ligante Fas/biossíntese , MicroRNAs/biossíntese , Miocardite/genética , Receptor fas/biossíntese , Apoptose/genética , Estudos de Casos e Controles , Proteína Ligante Fas/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Miocardite/patologia , Miocárdio/patologia , Receptor fas/genéticaRESUMO
Sevoflurane, the most widely used anesthetic in clinical practice, has been shown to induce apoptosis, inhibit neurogenesis, and cause learning and memory impairment in young mice. However, the underlying mechanism is still unknown. In this study, wild-type and the FAS- or FAS ligand (FASL)-knockout mice (age 7 days) were exposed to sevoflurane or pure oxygen. Western blotting was used to examine the expression of FAS protein. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and bromodeoxyuridine (BrdU) staining were employed to quantify the apoptotic cells and newborn cells in the hippocampus and Morris water maze (MWM) in order to evaluate learning and memory status. Sevoflurane significantly increased the expression of FAS protein in wild-type mice. Compared to FAS- and FASL-knockout mice treated with sevoflurane, sevoflurane-treated wild-type mice exhibited more TUNEL-positive hippocampal cells and less BrdU-positive hippocampal cells. The MWM showed that compared with FAS- and FASL-knockout mice treated with sevoflurane, sevoflurane treatment of wild-type mice significantly prolonged the escape latency and reduced platform crossing times. These data suggest that sevoflurane induces neurotoxicity in young mice through FAS-FASL signaling.
Assuntos
Proteína Ligante Fas/genética , Éteres Metílicos/efeitos adversos , Síndromes Neurotóxicas/genética , Receptor fas/genética , Anestésicos/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Proteína Ligante Fas/biossíntese , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Sevoflurano , Receptor fas/biossínteseRESUMO
The aim of this study was to investigate the roles of Fas/FasL, Bcl-2/Bax, and Caspase-8 mRNA expressions in nonalcoholic fatty liver disease (NAFLD). The apoptosis percentage was measured by flow cytometry, the immunohistochemical assay was performed for the determination of Fas, FasL, Bcl-2, and Bax expressions, and a real-time polymerase chain reaction (PCR) assay was performed to detect Caspase-8 mRNA expression. Flow cytometry showed that the apoptosis percentage of the rat liver in the experimental group increased, which increased more obviously with the extension of modeling time. Immunohistochemistry showed that with increasing hepatic steatosis, Fas and FasL protein staining intensified and the number of positive cells increased; the number of positive cells for Bcl-2 and Bax gradually increased on the 4th, 8th, and 12th weeks in the experimental group, whereas the Bcl-2/Bax ratio decreased. The real-time PCR assay showed that Caspase-8 mRNA expression increased with increasing hepatic steatosis and inflammation, exhibiting a progressively rising trend. Hepatocyte apoptosis could promote NAFLD progression; Fas, FasL, and Caspase-8 mRNA activation were important contributing factors to NAFLD. The upregulation of Bax and Bcl-2 expression might be one important mechanism of the apoptosis in NAFLD.
Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Caspase 8/biossíntese , Proteína Ligante Fas/biossíntese , Hepatopatia Gordurosa não Alcoólica/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2/biossíntese , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Caspase 8/genética , Proteína Ligante Fas/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , Ratos , Proteína X Associada a bcl-2/genéticaRESUMO
Fas/FasL protein expression of bone marrow hematopoietic cells was investigated in severe aplastic anemia (SAA) patients. Fas expression was evaluated in CD34(+), GlycoA(+), CD33(+), and CD14(+) cells labeled with monoclonal antibodies in newly diagnosed and remission SAA patients along with normal controls. FasL expression was evaluated in CD8(+) cells in the same manner. In CD34(+) cells, Fas expression was significantly higher in the newly diagnosed SAA group (46.59 ± 27.60%) than the remission (6.12 ± 3.35%; P < 0.01) and control (8.89 ± 7.28%; P < 0.01) groups. In CD14(+), CD33(+), and GlycoA(+) cells, Fas levels were significantly lower in the newly diagnosed SAA group (29.29 ± 9.23, 46.88 ± 14.30, and 15.15 ± 9.26%, respectively) than in the remission (47.23 ± 31.56, 67.22 ± 34.68, and 43.56 ± 26.85%, respectively; P < 0.05) and normal control (51.25 ± 38.36, 72.06 ± 39.88, 50.38 ± 39.88%, respectively; P < 0.05) groups. FasL expression of CD8(+) cells was significantly higher in the newly diagnosed SAA group (89.53 ± 45.68%) than the remission (56.39 ± 27.94%; P < 0.01) and control (48.63 ± 27.38%; P <0.01) groups. No significant differences were observed between the remission and control groups. FasL expression in CD8(+) T cells was significantly higher in newly diagnosed patients, and CD34(+), CD33(+), CD14(+), and GlycoA(+) cells all showed Fas antigen expression. The Fas/FasL pathway might play an important role in excessive hematopoietic cell apoptosis in SAA bone marrow. Furthermore, CD34(+) cells are likely the main targets of SAA immune injury.
Assuntos
Anemia Aplástica/genética , Proteínas Reguladoras de Apoptose/genética , Células da Medula Óssea/metabolismo , Proteína Ligante Fas/genética , Células-Tronco Hematopoéticas/metabolismo , Adolescente , Adulto , Anemia Aplástica/imunologia , Anemia Aplástica/patologia , Antígenos CD34/biossíntese , Antígenos CD34/genética , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/biossíntese , Células da Medula Óssea/imunologia , Antígenos CD8/biossíntese , Antígenos CD8/genética , Proteína Ligante Fas/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Humanos , Antígenos Comuns de Leucócito/biossíntese , Antígenos Comuns de Leucócito/genética , MasculinoRESUMO
Abstract. Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by defects in superoxide (O2-) production, which result from mutations in one of the four NADPH oxidase components, predisposing to bacterial and fungal infections. Besides the O2-defect, it has been described that neutrophils from CGD patients are resistant to cell death, a phenomenon that has been connected to chronic inflammation and predisposition to autoimmune diseases. A diminished expression of Fas and its counterpart FasL, molecules known to play a major role in cell death, has been described in lymphocytes depleted of O2-reactive oxygen species (ROS), suggesting an involvement of ROS in Fas/FasL expression. In this work, Fas and FasL expressions were analyzed in T cells and neutrophils from two CGD families, previously known to harbor two different molecular defects: absence of either p47-phox or p67-phox. We found that T lymphocytes from CGD patients express low levels of Fas and FasL, while a diminished FasL expression was observed on neutrophils from a CGD A470 patient. These defects may contribute to understand altered cell death in CGD patients.
Assuntos
Proteína Ligante Fas/biossíntese , Doença Granulomatosa Crônica/metabolismo , Leucócitos/metabolismo , Receptor fas/biossíntese , Adolescente , Adulto , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by defects in superoxide (O2-) production, which result from mutations in one of the four NADPH oxidase components, predisposing to bacterial and fungal infections. Besides the O2-defect, it has been described that neutrophils from CGD patients are resistant to cell death, a phenomenon that has been connected to chronic inflammation and predisposition to autoimmune diseases. A diminished expression of Fas and its counterpart FasL, molecules known to play a major role in cell death, has been described in lymphocytes depleted of O2-reactive oxygen species (ROS), suggesting an involvement of ROS in Fas/FasL expression. In this work, Fas and FasL expressions were analyzed in T cells and neutrophils from two CGD families, previously known to harbor two different molecular defects: absence of either p47-phox or p67-phox. We found that T lymphocytes from CGD patients express low levels of Fas and FasL, while a diminished FasL expression was observed on neutrophils from a CGD A470 patient. These defects may contribute to understand altered cell death in CGD patients.
La Enfermedad Granulomatosa Crónica (EGC) es una inmunodeficiencia primaria caracterizada por un defecto en la producción de superóxido (O2-), que se genera como consecuencia de mutaciones en uno de los cuatro componentes del complejo NADPH oxidasa y predispone a infecciones por bacterias y hongos. Además de los defectos en la producción de O2-, se ha descrito que los neutrófilos de los pacientes con EGC exhiben una resistencia a la muerte celular, evento que se asocia con la inflamación crónica y predisposición a enfermedades autoinmunes. Se ha descrito que linfocitos en medios desprovistos de O2-especies reactivas del oxigeno (ROS), muestran reducida expresión de Fas y FasL, moléculas que juegan un papel relevante en el control de la muerte celular, sugiriendo la participación de los ROS su regulación. En este trabajo analizamos la expresión de Fas y FasL en linfocitos T y neutrófilos en dos familias portadores de dos defectos genéticos diferentes asociados con EGC: ausencia de p47-phox o de p67-phox. Evidenciamos una baja expresión de Fas y FasL en los linfocitos T de los pacientes con EGC, pero solo los neutrófilos de los pacientes con defecto de p47-phox, fueron incapaces de expresar FasL. Estos defectos pudieran contribuir a entender la alteración de la muerte celular observada en los pacientes con EGC.
Assuntos
Adolescente , Adulto , Feminino , Humanos , Adulto Jovem , /biossíntese , Proteína Ligante Fas/biossíntese , Doença Granulomatosa Crônica/metabolismo , Leucócitos/metabolismoRESUMO
Glucocorticoids (GCs) and cAMP-dependent signaling pathways exert diverse and relevant immune regulatory functions, including a tight control of T cell death and homeostasis. Both of these signaling molecules inhibit TCR-induced cell death and FasL expression, but the underlying mechanisms are still poorly understood. Therefore, to address this question, we performed a comprehensive screening of signaling pathways downstream of the TCR, in order to define which of them are targets of cAMP- and GC-mediated inhibition. We found that cAMP inhibited NF-κB and ERK pathways through a PKA-dependent mechanism, while Dexamethasone blocked TCR-induced NF-κB signaling. Although GCs and cAMP inhibited the induction of endogenous FasL mRNA expression triggered by TCR activation, they potentiated TCR-mediated induction of FasL promoter activity in transient transfection assays. However, when the same FasL promoter was stably transfected, the facilitatory effect of GCs and cAMP became inhibitory, thus resembling the effects on endogenous FasL mRNA expression. Hence, the endogenous chromatinization status known to occur in integrated or genomic vs. episomic DNA might be critical for proper regulation of FasL expression by cAMP and GCs. Our results suggest that the chromatinization status of the FasL promoter may function as a molecular switch, controlling cAMP and GC responsiveness and explaining why these agents inhibit FasL expression in T cells but induce FasL in other cell types.
Assuntos
Apoptose/imunologia , AMP Cíclico/imunologia , Proteína Ligante Fas/biossíntese , Glucocorticoides/imunologia , Transdução de Sinais/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Morte Celular , Separação Celular , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Citometria de Fluxo , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Humanos , Hibridomas , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Regiões Promotoras Genéticas/genética , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , TransfecçãoRESUMO
PURPOSE: To assess FasL mRNA levels in ejaculated sperm from adolescent patients with and without varicocele. METHODS: Semen was obtained by masturbation following 2-4 days of ejaculatory abstinence, from 14 adolescents with varicocele grades II and III (study group), and 20 adolescents without varicocele (control group). Seminal analysis was done according to World Health Organization guidelines and morphology using Kruger's strict criteria. The Fas-ligand (FasL) gene expression was performed using reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR) analysis, according to the expression level of the housekeeping cyclophilin A gene. A Student's t-test was applied to compare the groups, and Spearman's rank test in order to verify possible correlations (p < 0.05). RESULTS: Quantitative RQ-PCR demonstrated that the expression of FasL mRNA in sperm from the varicocele group was higher than in the control group. Also, sperm concentration was higher in the controls, when compared to the varicocele group. When submitted to correlation analysis, adolescents with varicocele presented a correlation between sperm concentration and FasL gene expression levels (r = -0.470), not observed in controls. CONCLUSION: Our results allow us to conclude that, in adolescents with varicocele presenting lower sperm concentration, FasL mRNA levels are higher than in adolescents without varicocele.
Assuntos
Proteína Ligante Fas/genética , RNA Mensageiro/análise , Espermatozoides/química , Varicocele/metabolismo , Adolescente , Apoptose , Sistemas Computacionais , Ejaculação , Proteína Ligante Fas/biossíntese , Humanos , Masculino , Estudos Prospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise do Sêmen , Varicocele/genética , Adulto JovemRESUMO
Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vß inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vß region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.