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Bacillus Calmette-Guérin (BCG) remains the only licensed vaccine against human tuberculosis (TB). BCG is a live-attenuated strain of Mycobacterium bovis, with limitations in efficacy against respiratory TB, the most common form of the disease responsible for transmission. However, continues to be used in the immunization programmes of different countries in the absence of another alternative. In order to improve BCG efficacy against pulmonary TB, in the current clinical TB vaccine pipeline, there are live-attenuated TB vaccines to replace BCG. This review discusses the current status of the development of live vaccine candidates designed to replace BCG from the rational strategies and immunological challenges to its clinical trial and identify key areas in the next years considered essential to confer improved safety and efficacy over BCG.
Assuntos
Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/fisiologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/imunologia , Vacinas Atenuadas/imunologia , Animais , Humanos , VacinaçãoRESUMO
Tuberculosis (TB) remains one of a major health problem worldwide. Tuberculosis vaccine research has made an extraordinary progress over the past few years. However, there is still no replacement for the Bacillus Calmette-Guérin vaccine, the only TB vaccine licensed for human use. Therefore, the discovery and development of new TB vaccines remains a priority. This article discusses current strategies used to diversify TB vaccines and includes discussion of the status of efforts to improve protection against Mycobacterium tuberculosis (M tb) infection or TB disease by developing new and safe TB vaccines. This article also highlights the current research efforts in immune-enhancing approaches to improve vaccination efficacy. The development of more effective TB vaccines might have significant impact on global TB control.
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Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Humanos , Tuberculose Pulmonar/imunologia , Vacinação , Vacinas Atenuadas/imunologiaRESUMO
Antibiotic resistance is an increasing public health concern around the world. Rapid increase in the emergence of multidrug-resistant bacteria has been the target of extensive research efforts to develop a novel class of antibiotics. Antimicrobial peptides (AMPs) are small cationic amphiphilic peptides, which play an important role in the defense against bacterial infections through disruption of their membranes. They have been regarded as a potential source of future antibiotics, owing to a remarkable set of advantageous properties such as broad-spectrum activity, and they do not readily induce drug-resistance. However, AMPs have some intrinsic drawbacks, such as susceptibility to enzymatic degradation, toxicity, and high production cost. Currently, a new class of AMPs termed "peptidomimetics" have been developed, which can mimic the bactericidal mechanism of AMPs, while being stable to enzymatic degradation and displaying potent activity against multidrug-resistant bacteria. This review will focus on current findings of antimicrobial peptidomimetics. The potential future directions in the development of more potent analogs of peptidomimetics as a new generation of antimicrobial agents are also presented.
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PURPOSE: Since few reports had been published on the prevalence of toxocariasis in ankylosing spondylitis (AS) patients with acute non-granulomatous anterior uveitis (ANGAU), the aim of this work was to determine the presence of antibodies against Toxocara canis in AS patients with ANGAU. METHODS: Thirty-six patients (14 female and 22 male) with AS were enrolled in the study. The history of ANGAU was accepted only if diagnosed by an ophthalmologist. The detection of IgG antibodies to T. canis was determined by enzyme-linked immunosorbent assay. In addition, antibodies to Ascaris lumbricoides were also tested to verify non-specific reactions. RESULTS: The prevalence of ANGAU in the AS patients was 58% (21 / 36), and 38% (8 / 21) of the patients with ANGAU were positive for antibodies to Toxocara, while 7% (1 / 15) of AS patients without ANGAU were positive for T. canis (p = 0.038, two tails; mid-p exact). No antibodies were detected to A. lumbricoides antigens in the serum samples of patients with AS. CONCLUSIONS: These data suggest that the seroprevalence of antibodies to T. canis is high in Mexican patients with AS-associated uveitis, suggesting a chronic asymptomatic toxocariosis, which could be associated with the pathogenesis of ANGAU; however, further larger-scale studies are needed to confirm this observation.
Assuntos
Anticorpos Anti-Idiotípicos/isolamento & purificação , Infecções Oculares Parasitárias/imunologia , Imunoglobulina G/imunologia , Espondilite Anquilosante/complicações , Toxocara canis/imunologia , Toxocaríase/imunologia , Uveíte Anterior/imunologia , Doença Aguda , Adulto , Idoso , Animais , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Parasitárias/complicações , Infecções Oculares Parasitárias/parasitologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/parasitologia , Toxocara canis/isolamento & purificação , Toxocaríase/complicações , Toxocaríase/parasitologia , Uveíte Anterior/complicações , Uveíte Anterior/parasitologia , Adulto JovemRESUMO
Antimicrobial peptides are cationic molecules, which participate in multiple aspects of the immune response including the control of inflammatory diseases, characteristic that make these molecules attractive as therapeutic tools. These peptides are produced in bacteria, insects, plants and vertebrates, and are classified together due to their capacity to directly inhibit the growth of microorganisms, and to regulate the immune response by inducing the secretion of chemokines and cytokines. Various families of antimicrobial peptides have been identified including the cathelicidins and defensins, the most investigated human antimicrobial peptides. This review will cover the main biological functions of antimicrobial and cell-penetrating peptides in inflammation, and describe the importance and utility of antimicrobial peptides as therapeutics for inflammatory diseases.
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Antimicrobial peptides are predominantly small cationic polypeptides that are classified together on the basis of these molecules to directly kill or inhibit the growth of microorganisms including mycobacteria, and to activate mechanisms of cellular and adaptive immunity. Various families of antimicrobial peptides have been identified, including the cathelicidins. The cathelicidin family is characterised by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain that can be released from the precursor protein after cleavage by proteinases. LL-37 is the C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP18), which is mainly expressed by neutrophils and epithelial cells. The cathelicidin hCAP18/LL-37 is a multifunctional molecule that may mediate various host responses, including bactericidal action, chemotaxis, epithelial cell activation, angiogenesis, epithelial wound repair and activation of chemokine secretion. The antimicrobial peptide LL-37 is produced from human cells during infection of mycobacteria and exerts a microbicidal effect. The discussion will (1) describe recent work on the antimicrobial and immunomodulatory functions of the cathelicidin hCAP18/LL-37, (2) highlight the effectiveness of the cathelicidin hCAP18/LL-37 as a potent component in antimycobacterial immune responses and (3) summarise current progress in the understanding of the therapeutic application of hCAP18/LL-37 and its derivates antimicrobial peptides in mycobacterial infection.
Assuntos
Catelicidinas/fisiologia , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Fragmentos de Peptídeos/fisiologia , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/metabolismo , Catelicidinas/farmacologia , Catelicidinas/uso terapêutico , Humanos , Imunidade Inata , Fatores Imunológicos/farmacologia , Fatores Imunológicos/fisiologia , Fatores Imunológicos/uso terapêutico , Mycobacterium/efeitos dos fármacos , Mycobacterium/imunologia , Infecções por Mycobacterium/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêuticoRESUMO
The human cathelicidin LL-37 is one of the major antimicrobial peptides of the non-specific innate immune system in Mycobacterium tuberculosis infection. Its expression has been reported in epithelial cells infected with mycobacteria. However, the underlying molecular mechanisms by which Mycobacterium bovis bacillus Calmette-Guérin (BCG) triggers gene transcription of cathelicidin have not been elucidated. The objective of this study was to investigate the role of reactive oxygen species (ROS) in the M. bovis BCG-mediated up-regulation of the antimicrobial peptide cathelicidin LL-37 in human epithelial cells. Infection of A549 cells with M. bovis BCG led to a rapid ROS production. Importantly, blockade of ROS by preincubation of cells with the general ROS scavenger N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor DPI significantly reduced M. bovis BCG-induced up-regulation of cathelicidin LL-37 mRNA expression as determined by semi-quantitative RT-PCR or real-time PCR. In contrast, the xanthine oxidase inhibitor allopurinol did not affect M. bovis BCG-mediated up-regulation of cathelicidin LL-37 mRNA. Moreover, M. bovis BCG-mediated cathelicidin LL-37 mRNA expression was significantly blocked by the effect of the mitochondrial electron transfer chain subunit I inhibitor rotenone and H(2)O(2) scavenging enzyme catalase. In addition, M. bovis BCG-induced cathelicidin LL-37 protein secretion was inhibited by the addition of NAC, DPI, and the selective inhibitor of NADPH oxidase apocynin. Our results collectively indicate that M. bovis BCG-mediated up-regulation of cathelicidin is influenced by NADPH/ROS signaling pathways. In conclusion, these findings demonstrate a novel regulatory mechanism for the expression of cathelicidin LL-37 in human epithelial cells stimulated with M. bovis BCG.
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Peptídeos Catiônicos Antimicrobianos/imunologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Mycobacterium bovis/imunologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima , Peptídeos Catiônicos Antimicrobianos/biossíntese , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Humanos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , CatelicidinasRESUMO
Chemokines are the key molecules that recruit immune cells by chemotaxis and act in leukocyte activation during mycobacterial diseases. Currently, tuberculosis is a leading infectious disease affecting millions of people worldwide. The purpose of this review is to describe a series of recent scientific evidence concerning to the protective role of some members of the CC- and the CXC chemokine subfamilies for the control of mycobacterial infection. The discussion will (1) highlight the effectiveness of some chemokines as potent immunoprophylactic tool for controlling the mycobacterial establishment within the host, (2) describe recent work on the relevance of cellular signaling pathways by which mycobacterial antigens mediate chemokine induction, and (3) summarize current progress in the understanding of the potential use of chemokines as potent adjuvants in antimycobacterial immune responses.
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Quimiocinas CC/fisiologia , Quimiocinas CXC/fisiologia , Infecções por Mycobacterium/imunologia , Adjuvantes Imunológicos , Quimiotaxia de Leucócito , Humanos , Transdução de Sinais , Tuberculose/imunologiaRESUMO
Human beta-defensin (HBD)-2 is an inducible antimicrobial peptide that plays an important role in innate immunity. Induction of this peptide by mycobacteria in epithelial cells has been reported. However, the mechanism(s) by which Mycobacterium bovis bacillus Calmette-Guérin (BCG) triggers gene transcription of HBD-2 remains poorly understood. In the present work we found that treatment of human epithelial cells with Ro32-0432 or Gö6976, two selective inhibitors of protein kinase C (PKC), significantly reduced the effect of M. bovis BCG on induced HBD-2 mRNA expression (65 and 80% inhibition by 10microM Ro32-0432, and 1microM Gö6976 as assessed by real-time PCR, respectively). Moreover, there was increased activation of c-Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K)/Akt in A549 cells infected with M. bovis BCG, and this JNK and PI3K activation was mediated through PKC. Finally, we found that M. bovis BCG-induced HBD-2 mRNA gene expression in A549 cells was dependent on JNK, and PI3K determined by real-time PCR analysis, which was attenuated by inhibitors of JNK (SP600125 and AG126), and PI3K (wortmannin and Ly294002). These studies are the first to show that M. bovis BCG-induced HBD-2 mRNA expression in A549 cells is regulated at least in part through activation of signaling proteins of PKC, JNK and PI3K.
Assuntos
Anti-Infecciosos/metabolismo , Células Epiteliais/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mycobacterium bovis/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , beta-Defensinas/metabolismo , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Transdução de Sinais/fisiologia , beta-Defensinas/genéticaRESUMO
Worldwide, tuberculosis remains the most important infectious disease causing morbidity and death. Currently, at least one-third of the world's population is infected with Mycobacterium tuberculosis. In addition, the World Health Organization estimates that about 8-10 million new tuberculosis cases occur annually worldwide and this incidence is currently increasing. Moreover, multidrug-resistant tuberculosis has been increasing in incidence in many areas during the past decade. These situations underscore the importance of the development of new therapeutic agents against mycobacterial infectious diseases. In this article, it is review current progress in the understanding of antimicrobial peptides as potential candidates to develop an alternative/adjunct therapeutic strategy against tuberculosis. This immunoadjunctive therapy might be evaluated in the context of possible drug resistance. This review also summarizes the knowledge about the functions of antimicrobial peptides in the pulmonary innate host defense system and their role in mycobacterial infection, and at the same time outlines recent advances in our understanding of the combined effect of antimicrobial peptides and anti-tuberculosis drugs against intracellular mycobacteria. A concerted effort should now focus on the clinical application of antimicrobial peptides for their practical use.
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Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Infecções por Mycobacterium/tratamento farmacológico , Tuberculose/tratamento farmacológico , Animais , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Defensinas/metabolismo , Defensinas/uso terapêutico , HumanosRESUMO
Infection of human cells with mycobacteria has been shown to result in the production of anti-inflammatory cytokines. However, the signaling pathways that regulate the Mycobacterium bovis BCG-induced interleukin (IL)-10 production are currently unknown. In the present study, we investigated the involvement of phosphatidylinoditol 3-kinase (PI3K)/Akt and the p38 MAPK signaling pathways in the secretion of IL-10 in human lung epithelial cells (A549) after infection with M. bovis BCG. Treatment of A549 cells with LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and wortmannin, two PI3K inhibitors, inhibited M. bovis BCG-induced IL-10 production. Stimulation of cells with M. bovis BCG caused an increase in Akt phosphorylation in a time-dependent manner, which was inhibited by wortmannin. In addition, treatment of A549 cells with an Akt inhibitor significantly blocked M. bovis BCG-induced IL-10 production. Moreover, the p38 inhibitor SB203580 significantly decreased IL-10 production in a dose-dependent manner, whereas M. bovis BCG-induced IL-10 secretion was completely unaffected by the MEK inhibitor PD98059. Finally, the inhibition of PI3K did not significantly affect p38 MAPK activation in M. bovis BCG-infected cells, indicating that PI3K activity is not required for the M. bovis BCG-induced phosphorylation of p38 MAPK. Collectively, these data suggest that the PI3K/Akt and p38 MAPK signaling pathways play an important role in the regulation of M. bovis BCG-induced IL-10 secretion in A549 cells.
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Células Epiteliais/imunologia , Interleucina-10/biossíntese , Mycobacterium bovis/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Interleucina-10/metabolismo , Pulmão/citologia , Transdução de Sinais/imunologiaRESUMO
Upon contact with airway epithelial cells, mycobacteria activate several signal transduction events that are required for induction of NF-kappaB-dependent chemokine gene expression. However, downstream signaling pathways, especially that of Ca(2+)-dependent protein kinase C alpha (PKCalpha), and in particular, the identity of the IKKalphabeta signal pathway for CXCL8 secretion in Mycobacterium bovis BCG-induced epithelial cells are still unknown. In this study, we demonstrated that the phosphoinositide-phospholipase C (PI-PLC) downstream signaling pathway is involved in M. bovis BCG-induced CXCL8 release, since A549 cells pretreated with U73122, a PI-PLC inhibitor, inhibited CXCL8 release, whereas U73343 the inactive analog had no effect. In addition, our results demonstrated that M. bovis BCG-induced CXCL8 production by A549 cells was significantly blocked by using neomycin (another well-described inhibitor of PI-PLC with a different mechanism of action), Ro-32-0432 and Ro-31-8220 (two PKCalpha inhibitors), PP1 and PP2 (two potent and selective inhibitors of the Src-family tyrosine kinases), and Bay 11-7082 (an IkappaB phosphorylation inhibitor). We also demonstrated that M. bovis BCG can rapidly induce translocation of PKCalpha from the cytosol to the membrane, and that treatment of cells with M. bovis BCG caused time-dependent increases in phosphorylation of c-Src at tyrosine 416. Finally, our studies revealed that M. bovis BCG induced the association of c-Src and IKKalphabeta during the interaction of PKCalpha and IKKalphabeta. Altogether, these results represent the first evidence to date suggesting that M. bovis BCG activates the PI-PLC/PKCalpha/c-Src/IKKalphabeta signaling pathway to induce CXCL8 release in human epithelial cells.
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Vacina BCG/imunologia , Células Epiteliais/imunologia , Quinase I-kappa B/metabolismo , Interleucina-8/metabolismo , Mycobacterium bovis/imunologia , Proteína Quinase C-alfa/metabolismo , Vacina BCG/farmacologia , Proteína Tirosina Quinase CSK , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Humanos , NF-kappa B/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Quinases da Família srcRESUMO
CCL5 is a key in limiting mycobacterial infection. Although NF-kappaB has been implicated, signaling cascades involved in CCL5 production by epithelial cells following infection with Mycobacterium bovis BCG are still not defined. Here we show that using pharmacological inhibition of sphingosine kinase (SPK), striking inhibition of M. bovis BCG-induced CCL5 protein was observed. Phosphatidylinositol 3-kinase (PI3K) and Akt were also important for CCL5 production by epithelial cells infected with M. bovis BCG. Moreover, there was increased activation of PI3K, IKK/alphabeta and NF-kappaB in A549 cells infected with M. bovis BCG. Importantly, the PI3K activation was dependent on SPK. Finally, M. bovis BCG increases the recruitment of p300 with NF-kappaB in A549 cells. Together, these studies are the first to show that M. bovis BCG-induced CCL5 secretion is dependent on the SPK/PI3K/Akt/NF-kappaB and p300 signaling pathway. The regulatory pathways of M. bovis BCG-induced CCL5 production can potentially be exploited therapeutically.
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Vacina BCG/imunologia , Quimiocina CCL5/biossíntese , Células Epiteliais/microbiologia , Transdução de Sinais/fisiologia , Tuberculose/prevenção & controle , Western Blotting , Linhagem Celular , Proteína p300 Associada a E1A/metabolismo , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Humanos , Imunoprecipitação , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tuberculose/imunologiaRESUMO
Induction of human beta defensin-2 (HBD-2) by mycobacteria has been reported. However, the molecular mechanism(s) by which mycobacteria up-regulates HBD-2 gene expression in epithelial cells remains poorly understood. In this work, we provide evidence that the induction of HBD-2 mRNA in response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) was inhibited in a dose-dependent manner by pretreatment with a cell-permeable BAPTA-AM, which chelates intracellular calcium. Our data also demonstrate that HBD-2 mRNA induction by M. bovis in A549 lung epithelial cells requires activation of calmodulin. Interestingly, HBD-2 mRNA expression in response to M. bovis BCG was attenuated by pretreatment with SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]), but not by an inhibitor of extracellular signal-regulated kinase (ERK): PD98059. Furthermore, we found that a second p38 MAPK inhibitor (SB202190) significantly blocked M. bovis BCG-mediated HBD-2 induction in A549 lung epithelial cells. Together, these data suggest that M. bovis BCG induces HBD-2 mRNA expression in A549 lung epithelial cells at least in part mediated through intracellular calcium flux as well as activation of signaling protein of p38MAPK, but not ERK.
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Anti-Infecciosos/metabolismo , Cálcio/farmacologia , Células Epiteliais/microbiologia , Mycobacterium bovis/patogenicidade , beta-Defensinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/farmacologia , Linhagem Celular , Células Epiteliais/imunologia , Regulação da Expressão Gênica , Humanos , Transdução de Sinais , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Antimicrobial peptides produced by epithelial cells represent essential elements of innate immunity. Human beta-defensin-2 (hbetaD-2) is a major inducible antimicrobial peptide which plays an important role in host defense. The goal of this study was to determine the effect of Mycobacterium bovis bacillus Calmette-Guérin (BCG) on hbetaD-2 gene expression in epithelial cells, and to characterize further the signaling pathways involved in hbetaD-2 induction in response to M. bovis BCG. Using reverse transcription-polymerase chain reaction (RT-PCR), hbetaD-2 mRNA expression was detected in pulmonary epithelial cells infected with M. bovis BCG. Furthermore, we found that M. bovis BCG-induced hbetaD-2 mRNA expression was sustained by endogenous TNF-alpha in A549 cells. Of note, hbetaD-2 induction by M. bovis BCG was not blocked by pretreatment with anti-IL6 antibody. Moreover, hbetaD-2 mRNA expression was regulated at a transcriptional level, since hbetaD-2 induction by M. bovis BCG was blocked by two inhibitors of NF-kappaB. Taken together, these results suggest that M. bovis BCG infection of human epithelial cells induces hbetaD-2 mRNA expression which is up-regulated by TNF-alpha produced from M. bovis BCG-infected cells, and is modulated by NF-kappaB. Studies of hbetaD-2 mRNA regulation in epithelial cells infected with M. bovis BCG provide insight into how its expression may be enhanced to control Mycobacterium tuberculosis infection.
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Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Mycobacterium bovis/imunologia , Transcrição Gênica/genética , beta-Defensinas/genética , Linhagem Celular , Humanos , Interleucina-6/farmacologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Current knowledge of the cellular signaling by Mycobacterium bovis bacillus Calmette-Guerin (BCG) in epithelial cells is still limited. In this study, we provide evidence that the signaling events induced by M. bovis BCG in these cells included phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Our data also demonstrate that M. bovis BCG-induced CXC chemokine ligand (CXCL)8 release in epithelial cells was reduced by a mitogen-activated protein/ERK kinase (MEK) inhibitor (PD98059), but not by a p38 MAPK (SB203580) inhibitor. In addition, we found that a second and more potent MEK inhibitor (U0126) significantly blocked CXCL8 release in epithelial cells by M. bovis BCG. Evaluation of CXCL8 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of PD98059 and U0126 was associated with a reduction in this parameter. Moreover, the induction of CXCL8 secretion in epithelial cells by M. bovis BCG occurs at the transcription level. Collectively, the findings reported in the present study suggest that MEK signaling is essential for the induction of CXCL8 in epithelial cells in response to M. bovis BCG.
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Vacina BCG/imunologia , Células Epiteliais/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas/genética , Transcrição Gênica/genéticaRESUMO
BACKGROUND: A comprehensive understanding of the immune response induced by Mycobacterium bovis Bacille Calmette-Guérin in activation of protective T cells against tuberculosis is important to develop effective therapies to combat this disease. In this study, our experiments were designed to determine effects of transforming growth factor (TGF)-beta on M. bovis-induced T-cell activation and survival. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used for detection of apo-ptotic cells by three different methods: 1). scattered light change during early phase of apoptosis; 2). detection of hypodiploid DNA, or 3). terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) technique. Quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS: TGF-beta added at initiation of culture did not alter percentage of viable cells. By contrast, TGF-beta added 72 h post-activation decreased percentage of viable cells. This effect was statistically significant (p <0.05). Furthermore, addition of anti-TGF-beta MoAb together with TGF-beta abolished the ability of this cytokine to decrease survival in post-activated human T cells. Role of TGF-beta on post-activated human T cells was further confirmed by staining apoptotic nuclei with propidium iodide, which detects late events of apoptosis, and by DNA fragmentation determined using TUNEL assay. Interestingly, TGF-beta did not promote Fas-mediated killing. Finally, TGF-beta increased apoptosis of CD4(+) T cells after mycobacterial stimulation. CONCLUSIONS: This study indicated an important role for TGF-beta in suppression of protective immune response against M. bovis by promoting elimination of post-activated T cells. Furthermore, results showed that TGF-beta had no direct effect on M. bovis-induced up-regulation of Fas (CD95).