RESUMEN
Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.
Asunto(s)
Vacuna BCG/administración & dosificación , Inflamación/tratamiento farmacológico , Gripe Humana/tratamiento farmacológico , Neumonía/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Humanos , Inflamación/complicaciones , Inflamación/prevención & control , Inflamación/virología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/patogenicidad , Gripe Humana/complicaciones , Gripe Humana/prevención & control , Gripe Humana/virología , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/patología , Ratones , Fagocitos/efectos de los fármacos , Fagocitos/patología , Fagocitosis/efectos de los fármacos , Neumonía/complicaciones , Neumonía/prevención & control , Neumonía/virologíaRESUMEN
BACKGROUND: Since adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza. METHODS: Granulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production. RESULTS: Overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV. CONCLUSION: We also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Neumonía/prevención & control , Sustancias Protectoras/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pulmón/diagnóstico por imagen , Pulmón/metabolismo , Pulmón/virología , Macrófagos Alveolares/citología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Fagocitosis/efectos de los fármacos , Sustancias Protectoras/uso terapéutico , Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Tomografía Computarizada por Rayos XRESUMEN
Seasonal and especially pandemic influenza predispose patients to secondary bacterial pneumonias, which are a major cause of deaths and morbidity. Staphylococcus aureus is a particularly common and deadly form of post-influenza pneumonia, and increasing staphylococcal drug resistance makes the development of new therapies urgent. We explored an innate immune-mediated model of the lung to define novel mechanisms by which the host can be protected against secondary staphylococcal pneumonia after sub-lethal influenza infection. We found that stimulating the innate immunity in the lung by overexpression of GM-CSF will result in resistance to S. aureus pneumonia after sublethal influenza infection. Resistance was mediated by alveolar macrophages and neutrophils, and was associated with increased production of reactive oxygen species (ROS) by alveolar macrophages. Resistance was abrogated by treatment with agents that scavenged ROS. We conclude that stimulating innate immunity in the lung markedly reduces susceptibility to post-influenza staphylococcal pneumonia and that this may represent a novel immunomodulatory strategy for prevention and treatment of secondary bacterial pneumonia after influenza.
Asunto(s)
Infecciones por Orthomyxoviridae/complicaciones , Fagocitos/fisiología , Neumonía Estafilocócica/complicaciones , Especies Reactivas de Oxígeno/metabolismo , Anfirregulina , Animales , Regulación de la Expresión Génica/inmunología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Subtipo H1N1 del Virus de la Influenza A , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Macrófagos Alveolares , Neutrófilos , Infecciones por Orthomyxoviridae/microbiología , Infecciones por Orthomyxoviridae/virología , Neumonía Estafilocócica/microbiología , Staphylococcus aureusRESUMEN
Aging increases susceptibility to infection, in part because thymic involution culminates in reduced naïve T-lymphocyte output. Thymic epithelial cells (TECs) are critical to ensure normal maturation of thymocytes and production of peripheral T cells. The forkhead-class transcription factor, encoded by FoxN1, regulates development, differentiation, and function of TECs, both in the prenatal and postnatal thymus. We recently showed that expression of FoxN1, by keratin 14 (K14)-expressing epithelial cells is essential for maintenance of thymic medullary architecture, and deletion of FoxN1 in K14 promoter-driven TECs inhibited development of mature TECs and reduced the number of total thymocytes. These findings are reminiscent of changes observed during normal thymic aging. In the current report, we compared the effects of K14-driven FoxN1 deletion on peripheral T cell function in response to influenza virus infection with those associated with normal aging in a mouse model. FoxN1-deleted mice had reduced numbers of peripheral CD62L+CD44- naïve T-cells. In addition, during influenza infection, these animals had reduced antigen-specific CD8+ T-cell and IgG responses to influenza virus, combined with increased lung injury, weight loss and mortality. These findings paralleled those observed in aged wild type mice, providing the first evidence that K14-mediated FoxN1 deletion causes changes in T-cell function that mimic those in aging during an immune response to challenge with an infectious agent.
Asunto(s)
Células Epiteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Linfocitos T/microbiología , Timo/citología , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T/metabolismoRESUMEN
RATIONALE: Alveolar macrophages contribute to host defenses against influenza in animal models. Enhancing alveolar macrophage function may contribute to protection against influenza. OBJECTIVES: To determine if increased expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the lung increases resistance to influenza. METHODS: Wild-type mice and transgenic mice that expressed GM-CSF in the lung were infected with influenza virus, and lung pathology, weight loss, and mortality were measured. We also administered GM-CSF to the lungs of wild-type mice that were infected with influenza virus. MEASUREMENTS AND MAIN RESULTS: Wild-type mice all died after infection with different strains of influenza virus, but all transgenic mice expressing GM-CSF in the lungs survived. The latter also had greatly reduced weight loss and lung injury, and showed histologic evidence of a rapid host inflammatory response that controlled infection. The resistance of transgenic mice to influenza was abrogated by elimination of alveolar phagocytes, but not by depletion of T cells, B cells, or neutrophils. Transgenic mice had far more alveolar macrophages than did wild-type mice, and they were more resistant to influenza-induced apoptosis. Delivery of intranasal GM-CSF to wild-type mice also conferred resistance to influenza. CONCLUSIONS: GM-CSF confers resistance to influenza by enhancing innate immune mechanisms that depend on alveolar macrophages. Pulmonary delivery of this cytokine has the potential to reduce the morbidity and mortality due to influenza virus.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Administración Intranasal , Animales , Apoptosis/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pérdida de Peso/inmunologíaRESUMEN
Smoking is associated with increased susceptibility to tuberculosis and influenza. However, little information is available on the mechanisms underlying this increased susceptibility. Mice were left unexposed or were exposed to cigarette smoke and then infected with Mycobacterium tuberculosis by aerosol or influenza A by intranasal infection. Some mice were given a DNA vaccine encoding an immunogenic M. tuberculosis protein. Gamma interferon (IFN-γ) production by T cells from the lungs and spleens was measured. Cigarette smoke exposure inhibited the lung T-cell production of IFN-γ during stimulation in vitro with anti-CD3, after vaccination with a construct expressing an immunogenic mycobacterial protein, and during infection with M. tuberculosis and influenza A virus in vivo. Reduced IFN-γ production was mediated through the decreased phosphorylation of transcription factors that positively regulate IFN-γ expression. Cigarette smoke exposure increased the bacterial burden in mice infected with M. tuberculosis and increased weight loss and mortality in mice infected with influenza virus. This study provides the first demonstration that cigarette smoke exposure directly inhibits the pulmonary T-cell response to M. tuberculosis and influenza virus in a physiologically relevant animal model, increasing susceptibility to both pathogens.
Asunto(s)
Virus de la Influenza A/fisiología , Pulmón/citología , Mycobacterium tuberculosis/fisiología , Nicotiana , Humo/efectos adversos , Linfocitos T/efectos de los fármacos , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica/fisiología , Interferón gamma/genética , Interferón gamma/metabolismo , Pulmón/efectos de los fármacos , Pulmón/microbiología , Ratones , Ratones Transgénicos , Infecciones por Orthomyxoviridae/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Tuberculosis Pulmonar/inmunologíaRESUMEN
Development of an effective vaccine against tuberculosis hinges on an improved understanding of the human immune response to Mycobacterium tuberculosis. Work in this area at the University of Texas Health Science Center at Tyler has led to advances in four areas: (1) natural killer cells contribute to innate immunity by lysing M. tuberculosis-infected mononuclear phagocytes, and to adaptive immunity by enhancing the CD8+ T-cell effector function and inhibiting expansion of T regulatory cells; (2) Interferon-gamma plays a central role in resistance to many intracellular pathogens, including M. tuberculosis, and we have identified three transcription factors that bind to the Interferon-gamma proximal promoter and increase Interferon-gamma transcription in live T-cells that are activated by M. tuberculosis antigens; (3) A DNA vaccine that encodes the M. tuberculosis 10fts;kDa culture filtrate protein and the lysosomal integral membrane protein-2 was produced to direct vaccine antigens to the MHC class II processing and presentation pathway. When this vaccine was coated with polyethylenimine and administered to mice, it yielded a remarkably potent pulmonary immune response that reduced the bacillary burden by 90% after M. tuberculosis challenge; (4) The early secreted antigenic target of 6fts;kDa (ESAT-6) is a putative vaccine antigen. We found that high concentrations of this antigen markedly inhibit Interferon-gamma production by T-cells and are working to understand the molecular mechanisms underlying this effect. Developing methods to enhance NK cell functions that favor protective immunity, increase interferon-gamma transcription, elicit protective pulmonary immune responses and prevent ESAT-6 from inhibiting T-cell function will contribute significantly to development of antituberculosis vaccines.
Asunto(s)
Células Asesinas Naturales/inmunología , Mycobacterium tuberculosis/inmunología , Fagocitos/inmunología , Linfocitos T Reguladores/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Vacuna BCG/inmunología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Fagocitos/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Texas , Tuberculosis/tratamiento farmacológico , Tuberculosis/inmunologíaRESUMEN
To determine the immunogenicity and protective efficacy of the Mycobacterium tuberculosis 10 kD culture filtrate protein (CFP10), and to evaluate strategies that enhance local immunity, we used C57Bl/6 DR4 mice that were transgenic for human HLA DRB1 0401, because CFP10 contains epitopes for DRB1 0401 but not for C57Bl/6 mice. Intramuscular immunization with a DNA vaccine encoding CFP10 elicited production of IFN-gamma by systemic CD4+ T cells, and one intravenous dose of the CFP10-based DNA vaccine coated with polyethylenimine (PEI) stimulated IFN-gamma production by lung CD4+ cells and reduced the pulmonary bacillary burden. We conclude that CFP10 is a potential vaccine candidate and that coating vaccines with PEI enhances local protective immunity to tuberculosis
Asunto(s)
Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/prevención & control , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígeno HLA-DR1/inmunología , Inmunización , Interferón gamma/inmunología , Pulmón/citología , Pulmón/inmunología , Proteína 2 de la Membrana Asociada a los Lisosomas/inmunología , Proteínas de Membrana de los Lisosomas/inmunología , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunología , Plásmidos/inmunología , Polietileneimina/química , Bazo/citología , Bazo/inmunología , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-gamma and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-gamma production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-alpha but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-gamma. ESAT-6 decreased IFN-gamma transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-gamma proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-gamma secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70.
Asunto(s)
Antígenos Bacterianos/fisiología , Proteínas Bacterianas/fisiología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Mycobacterium tuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiología , Antígenos Bacterianos/genética , Antígenos CD/biosíntesis , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/metabolismo , Proteínas Bacterianas/genética , Proliferación Celular , Células Cultivadas , Humanos , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Lectinas Tipo C , Fosforilación , Receptores de Antígenos de Linfocitos T/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos CD1/inmunología , Células Dendríticas/inmunología , Lípidos/inmunología , Mycobacterium tuberculosis/inmunología , Péptidos/inmunología , Tuberculosis/inmunología , Transporte Biológico Activo/inmunología , Membrana Celular/inmunología , Células Cultivadas , Células Dendríticas/microbiología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Cinética , Fagocitosis/inmunología , Fagosomas/inmunología , Linfocitos T/inmunologíaRESUMEN
CD4+ T cells are believed to play a dominant role in human defenses against Mycobacterium tuberculosis through production of interferon (IFN)-gamma, cytolytic T-cell (CTL) activity, and inhibition of intracellular mycobacterial growth. Most functional studies of CD4+ cells have used bulk T-cells that recognize crude mycobacterial antigens, and the functional capacity of individual human T cells is not well defined. We studied the functional capacity of human CD4+ T-cell clones that recognize a specific mycobacterial peptide. Clone B9 produced high concentrations of IFN-gamma and exhibited potent CTL activity, whereas clone D3 produced IFN-gamma but showed poor CTL activity. The CTL activity of clone B9 was inhibited by SrCl(2) and concanamycin A but not by anti-Fas antibodies. Clone B9 also reduced the mycobacterial burden in dendritic cells by more than 90%, and this antimycobacterial activity was inhibited by SrCl(2) and concanamycin A. We conclude that: (1) individual human peptide-specific CD4+ T-cell clones have differential capacity to produce Th1 cytokines and to lyse M tuberculosis-infected target cells; and (2) both granulysin and perforin contribute to the capacity of human CD4+ T-cells to lyse infected targets and to inhibit intracellular mycobacterial growth.
Asunto(s)
Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Citotoxicidad Inmunológica , Interferón gamma/biosíntesis , Mycobacterium tuberculosis/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Clonales , Citocinas/biosíntesis , Células Dendríticas/microbiología , Humanos , Interferón gamma/inmunología , Macrólidos/farmacología , Mycobacterium tuberculosis/crecimiento & desarrollo , Fragmentos de Péptidos/inmunología , Perforina/fisiologíaRESUMEN
IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Antígenos Bacterianos/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Interferón gamma/metabolismo , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/farmacología , Complejo CD3/inmunología , Supervivencia Celular , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Humanos , Interferón gamma/genética , Mycobacterium tuberculosis/inmunología , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Interferente Pequeño/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Advancement in technology and science and our detailed knowledge of immunology, molecular biology, microbiology, and biochemistry among other basic science disciplines have defined new directions for vaccine development strategies. The applicability of genetic engineering and proteomics along with other new technologies have played pivotal roles in introducing novel ideas in vaccinology, and resulted in developing new vaccines and improving the quality of existing ones. Subunit vaccines, recombinant vaccines, DNA vaccines and vectored vaccines are rapidly gaining scientific and public acceptance as the new generation of vaccines and are seriously considered as alternatives to current conventional vaccines. The present review focuses on recent advances in veterinary vaccinology and addresses the effects and impact of modern microbiology, immunology, and molecular biology.
Asunto(s)
Enfermedades de los Animales/prevención & control , Vacunación/veterinaria , Vacunas , Medicina Veterinaria/métodos , Bienestar del Animal , Animales , Predicción , Medicina Veterinaria/tendenciasRESUMEN
RATIONALE: Diagnosis of latent tuberculosis infection (LTBI) is currently based on the tuberculin skin test. The enzyme-linked immunospot assay (ELISPOT) is a new blood test to diagnose LTBI. OBJECTIVE: To compare the ELISPOT and the tuberculin skin test for detecting LTBI in contacts of patients with tuberculosis. METHODS: Prospective study of 413 contacts of patients with tuberculosis. MEASUREMENTS AND MAIN RESULTS: Because there is no gold standard for LTBI, the sensitivity and specificity of the ELISPOT and tuberculin skin test cannot be directly measured. For each contact, we therefore estimated the likelihood of having LTBI by calculating a contact score that quantified exposure to and infectiousness of the index case. We analyzed the relationship of contact score to ELISPOT and tuberculin skin test results. The likelihood of a positive ELISPOT (p = 0.0005) and a tuberculin skin test (p = 0.01) increased significantly with rising contact scores. The contact score was more strongly related to the ELISPOT than to the tuberculin skin test results, although this difference was not statistically significant. Among U.S.-born persons and those who were not vaccinated with bacille Calmette-Guérin, approximately 30% had positive ELISPOT or tuberculin skin test results. Foreign-born, bacille Calmette-Guérin-vaccinated persons were significantly more likely to have a positive tuberculin skin test than a positive ELISPOT result (p < 0.0001). CONCLUSIONS: Compared with the tuberculin skin test, the ELISPOT appears to be at least as sensitive for diagnosis of LTBI in contacts of patients with tuberculosis.
Asunto(s)
Portador Sano/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Prueba de Tuberculina , Tuberculosis/diagnóstico , Adolescente , Adulto , Vacuna BCG , Niño , Trazado de Contacto , Emigración e Inmigración , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Tuberculosis/transmisiónRESUMEN
IFN-gamma is essential for resistance to many intracellular pathogens, including Mycobacterium tuberculosis. Transcription of the IFN-gamma gene in activated T cells is controlled by the proximal promoter element (-73 to -48 bp). CREB binds to the IFN-gamma proximal promoter, and binding is enhanced by phosphorylation of CREB. Studies in human T cell lines and in transgenic mice have yielded conflicting results about whether CREB is a positive or a negative regulator of IFN-gamma transcription. To determine the role of CREB in mediating IFN-gamma production in response to a microbial pathogen, we evaluated the peripheral blood T cell response to M. tuberculosis in healthy tuberculin reactors. EMSAs, chromatin immunoprecipitation, and Western blotting demonstrated that stimulation of PBMC with M. tuberculosis induced phosphorylation and enhanced binding of CREB to the IFN-gamma proximal promoter. Neutralization of CREB with intracellular Abs or down-regulation of CREB levels with small interfering RNA decreased M. tuberculosis-induced production of IFN-gamma and IFN-gamma mRNA expression. In addition, M. tuberculosis-stimulated T cells from tuberculosis patients, who have ineffective immunity, showed diminished IFN-gamma production, reduced amounts of CREB binding to the IFN-gamma proximal promoter, and absence of phosphorylated CREB. These findings demonstrate that CREB positively regulates IFN-gamma production by human T cells that respond to M. tuberculosis.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Interferón gamma/biosíntesis , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Linfocitos T/microbiología , Regulación hacia Arriba/inmunología , Proteína de Unión a CREB , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación hacia Abajo/inmunología , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/genética , Interferón gamma/metabolismo , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Líquido Intracelular/microbiología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Mycobacterium tuberculosis/patogenicidad , Proteínas Nucleares , Fosforilación , Regiones Promotoras Genéticas/inmunología , Unión Proteica/inmunología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Serina/metabolismo , Linfocitos T/metabolismo , TransactivadoresRESUMEN
The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors. T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos HLA/inmunología , Mycobacterium tuberculosis/inmunología , Fragmentos de Péptidos/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Alelos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citotoxicidad Inmunológica , Epítopos/química , Epítopos/inmunología , Antígenos HLA-DQ/inmunología , Cadenas beta de HLA-DQ , Antígenos HLA-DR/inmunología , Cadenas HLA-DRB1 , Humanos , Interferón gamma/biosíntesis , Fragmentos de Péptidos/química , Eliminación de Secuencia , Vacunas contra la TuberculosisRESUMEN
The mobile insertion sequence, IS6110, is an important marker in tracking of Mycobacterium tuberculosis strains. Here, we demonstrate that IS6110 can upregulate downstream genes through an outward-directed promoter in its 3' end, thus adding to the significance of this element. Promoter activity was orientation dependent and was localized within a 110 bp fragment adjacent to the right terminal inverted repeat. Transcripts from this promoter, named OP6110, begin approximately 85 bp upstream of the 3' end of IS6110. Use of green fluorescent protein (GFP) expression constructs showed that OP6110 was upregulated in M. tuberculosis during growth in human monocytes and in late growth phases in broth. Analysis of natural insertion sites in M. tuberculosis showed that IS6110 upregulated expression of several downstream genes during growth in human monocytes, including Rv2280 in H37Rv and the PE-PGRS gene, Rv1468c, in the clinical strain 210, which is a member of the Beijing family. Transcription between IS6110 and downstream genes was confirmed by reverse transcription polymerase chain reaction. The ability to activate genes during infection suggests that IS6110 has the potential to influence growth characteristics of different strains, and indicates another mechanism by which IS6110 can impact M. tuberculosis evolution.
Asunto(s)
Elementos Transponibles de ADN , Monocitos/microbiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Regiones Promotoras Genéticas , Fusión Artificial Génica , Línea Celular , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , ARN Bacteriano/análisis , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Secuencias Repetidas Terminales , Sitio de Iniciación de la Transcripción , Regulación hacia ArribaAsunto(s)
Enfermedades de los Bovinos/metabolismo , Parálisis de la Parturienta/metabolismo , Animales , Calcio/metabolismo , Bovinos , Femenino , Homeostasis/fisiología , Concentración de Iones de Hidrógeno , Parálisis de la Parturienta/etiología , Parálisis de la Parturienta/prevención & control , Fósforo/metabolismo , EmbarazoRESUMEN
The ability of Mycobacterium tuberculosis to grow in macrophages is central to its pathogenicity. We found previously that the widespread 210 strain of M. tuberculosis grew more rapidly than other strains in human macrophages. Because principal sigma factors influence virulence in some bacteria, we analysed mRNA expression of the principal sigma factor, sigA, in M. tuberculosis isolates during growth in human macrophages. Isolates of the 210 strain had higher sigA mRNA levels and higher intracellular growth rates, compared with other clinical strains and the laboratory strain H37Rv. SigA was also upregulated in the 210 isolate TB294 during growth in macrophages, compared with growth in broth. In contrast, H37Rv sigA mRNA levels did not change under these conditions. Overexpression of sigA enhanced growth of recombinant M. tuberculosis in macrophages and in lungs of mice after aerosol infection, whereas recombinant strains expressing antisense transcripts to sigA showed decreased growth in both models. In the presence of superoxide, sense sigA transformants showed greater resistance than vector controls, and the antisense sigA transformant did not grow. We conclude that M. tuberculosis sigA modulates the expression of genes that contribute to virulence, enhancing growth in human macrophages and during the early phases of pulmonary infection in vivo. This effect may be mediated in part by increased resistance to reactive oxygen intermediates.