RESUMO
Phosphatidyl-inositol mannosides (PIM) are glycolipids unique to mycobacteria and other related bacteria that stimulate host immune responses and are implicated in mycobacteria pathogenicity. Here, we found that the FcRγ-coupled C-type lectin receptor DCAR (dendritic cell immunoactivating receptor; gene symbol Clec4b1) is a direct receptor for PIM. Mycobacteria activated reporter cells expressing DCAR, and delipidation of mycobacteria abolished this activity. Acylated PIMs purified from mycobacteria were identified as ligands for DCAR. DCAR was predominantly expressed in small peritoneal macrophages and monocyte-derived inflammatory cells in lungs and spleen. These cells produced monocyte chemoattractant protein-1 (MCP-1) upon PIM treatment, and absence of DCAR or FcRγ abrogated MCP-1 production. Upon mycobacterial infection, Clec4b1-deficient mice showed reduced numbers of monocyte-derived inflammatory cells at the infection site, impaired IFNγ production by T cells, and an increased bacterial load. Thus, DCAR is a critical receptor for PIM that functions to promote T cell responses against mycobacteria.
Assuntos
Proteínas de Bactérias/imunologia , Lectinas Tipo C/imunologia , Fosfatidilinositóis/imunologia , Receptores Imunológicos/imunologia , Células Th1/imunologia , Animais , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium/imunologia , Infecções por Mycobacterium/imunologiaRESUMO
Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers.
Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Fatores Corda/administração & dosagem , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Fatores Imunológicos/administração & dosagem , Mycobacterium bovis , Adjuvantes Imunológicos , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/isolamento & purificação , Linfócitos T CD8-Positivos/metabolismo , Fracionamento Químico , Fatores Corda/química , Fatores Corda/isolamento & purificação , Citocinas/metabolismo , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Imunofenotipagem , Infusões Parenterais , Lipossomos , Ativação Linfocitária , Camundongos , Estrutura Molecular , Mycobacterium bovis/química , Solventes , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mycobacterium bovis Bacille Calmette-Guérin (BCG) has been shown to possess potent anti-tumor activity particularly in various animal models, while the cellular and molecular mechanisms underlying its activity are not well understood. We found that lipomannan (BCG-LM), a lipophilic component of the mycobacterial cell envelope, specifically inhibits tumor growth and induces the infiltration of eosinophils at local tumor invasion sites. In contrast, neither lipoarabinomannan (BCG-LAM) nor the cell wall of Mycobacterium bovis BCG (BCG-CW) exerted anti-tumor immunity. BCG-LM enhances cytotoxic activity of eosinophils via the increased production of superoxide. Global transcriptomic analyses of BCG-LM-pulsed dendritic cells identified C-C motif ligand (CCL) 5 as a crucial chemokine for the anti-tumor immunity induced by BCG-LM, indicating that CCL5 plays an important role for the accumulation of eosinophils in the tumor microenvironment. Furthermore, BCG-LM and memory Th2 cells exerted a synergetic effect on tumor progression by cooperatively enhancing the eosinophil function. Thus, this study revealed an un-identified BCG-LM-mediated anti-tumor mechanism via superoxide produced by infiltrated eosinophils in the tumor microenvironment. Since BCG-LM activates this unique pathway, it may have potent therapeutic potential as immune cell therapy for cancer patients.
Assuntos
Células Dendríticas/imunologia , Eosinófilos/imunologia , Imunoterapia Adotiva/métodos , Lipopolissacarídeos/imunologia , Mycobacterium bovis/imunologia , Neoplasias Experimentais/imunologia , Superóxidos/metabolismo , Células Th2/imunologia , Animais , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Parede Celular/imunologia , Quimiocina CCL5/metabolismo , Citotoxicidade Imunológica , Feminino , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/terapia , Transcriptoma , Carga Tumoral , Microambiente TumoralRESUMO
In the past 40 years, intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been carried out as the most effective treatment for preventing local recurrences and tumor progression of non-muscle-invasive bladder cancer. Bacillus Calmette-Guérin is a family of vaccines derived in 1921 by the in vitro attenuation of Mycobacterium bovis. Subsequently, bacillus Calmette-Guérin seed lots were spread around the world, and both phenotypic and genotypic differences among the strains have been compiled. In recent genomic comparisons, the evolution of the different bacillus Calmette-Guérin substrains has begun to emerge. However, some of these genetic alterations in bacillus Calmette-Guérin strains have yet to be shown to affect the therapeutic effects and/or adverse effects. There are thus ongoing research efforts to assess the effects of these genetic alterations on the properties of bacillus Calmette-Guérin strains, with the ultimate goal of identifying an ideal bacillus Calmette-Guérin strain for treatment of non-muscle-invasive bladder cancer and providing clues for the improvement of bacillus Calmette-Guérin strains. The present review provides a history of bacillus Calmette-Guérin immunotherapy, and discusses the genetic differences among bacillus Calmette-Guérin strains, the different clinical outcomes afforded by bacillus Calmette-Guérin strains and possible future developments.
Assuntos
Vacina BCG/uso terapêutico , Carcinoma de Células de Transição/terapia , Mycobacterium bovis/genética , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias da Bexiga Urinária/terapia , Administração Intravesical , Vacina BCG/história , Citocinas/imunologia , História do Século XX , Humanos , Imunoterapia/métodos , Mycobacterium bovis/imunologia , Resultado do Tratamento , Neoplasias da Bexiga Urinária/patologiaRESUMO
Mycobacterium avium complex (MAC) pulmonary disease is prevalent in middle-aged to elderly women with a thin body habitus. By comparing the rate of serologically diagnosed asymptomatic MAC infection and body mass index among 1033 healthy subjects, we find that middle-aged to elderly women became infected with MAC, regardless of their body habitus.
Assuntos
Infecções Assintomáticas/epidemiologia , Índice de Massa Corporal , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/epidemiologia , Adulto , Idoso , Feminino , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores Sexuais , Adulto JovemRESUMO
We report a case of recurrent disseminated Mycobacterium avium complex (DMAC) disease with anti-gamma interferon autoantibodies. To our knowledge, this is the first reported case caused by reinfection with a separate isolate of M. avium. DMAC disease activity was monitored using serum IgG antibody titers against lipid antigens extracted from a MAC strain.
Assuntos
Autoanticorpos/imunologia , Interferon gama/imunologia , Complexo Mycobacterium avium , Infecção por Mycobacterium avium-intracellulare/imunologia , Infecção por Mycobacterium avium-intracellulare/microbiologia , Idoso , Feminino , HumanosRESUMO
Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.
Assuntos
Carga Bacteriana/imunologia , Ácidos Graxos/metabolismo , Macrófagos/microbiologia , Sphingobacterium/fisiologia , Esfingolipídeos/metabolismo , Receptor 4 Toll-Like/fisiologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Proliferação de Células , Feminino , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Baço/microbiologia , Receptor 2 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A micro-organism resembling members of the genus Dermatophilus, strain W254(T), which was isolated from the submandibular lymph node of a pig, and an additional 16 strains isolated from swine tonsils, were studied to establish their taxonomic status. Although all 17 strains were isolated anaerobically under an atmosphere of 100 % CO2, all of them were aerotolerant anaerobes. The micro-organisms showed at least five cellular morphologies: (i) a radially protrusive thallus, which proliferated into tuber-like cells; (ii) segmentation in both tubers and thallus followed by multilocule formation, (iii) development of coccoid forms in the locules; (iv) a change from the coccoid forms to zoospores; (v) resting cells, which were able to develop into protrusive thalli again. The micro-organisms were positive for nitrate reduction, but negative for catalase, indole production, hydrolysis of urea and gelatin liquefaction. Milk was not decomposed and none of the strains was haemolytic. A total of 16 compounds, including glucose, were utilized as sole carbon sources and seven compounds, including l-arabinose, were not utilized. Three out of the 17 strains were subjected to further studies. The micro-organisms had meso-diaminopimelic acid in their peptidoglycan and galactose, glucose, madurose and a trace of mannose in their whole-cell sugar patterns. The major phospholipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol.Cellular fatty acids were C15 : 0 (35.7-23.1 %), C16 : 0 (5.9-2.4 %) C17 : 0 (62.9-39.5 %), C17 : 1 (24.4-0 %) and C18 : 0 (3-1.6 %). The predominant menaquinone was MK-8 (H4). The G+C content of the DNA was 69.6-71.8 mol%. Analysis of the 16S rRNA gene sequences showed that the strains clustered with the type strains of members of the family Dermatophilaceae. Based on the polyphasic taxonomic characterization carried out, all 17 strains are considered to belong to a novel species in a new genus, for which the name Tonsilliphilus suis gen. nov., sp. nov. is proposed. The type strain of the type species is W254(T) ( = ATCC 35846(T) = CCM 3774(T) = DSM 21880(T) = JCM 15727(T)).
Assuntos
Actinomycetales/classificação , Tonsila Palatina/microbiologia , Filogenia , Suínos/microbiologia , Actinomycetales/genética , Actinomycetales/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/análise , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosfolipídeos/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Doenças dos Suínos/microbiologia , Vitamina K 2/análogos & derivados , Vitamina K 2/análiseRESUMO
Bacillus Calmette-Guérin (BCG) Tokyo 172 is a predominant World Health Organization Reference Reagent for the BCG vaccine. Recently, the BCG Tokyo 172 substrain was reported to consist of two subpopulations with different colony morphologies, smooth and rough. Smooth colonies had a characteristic 22-bp deletion in Rv3405c of the region of difference (RD) 16 (type I), and rough colonies were complete in this region (type II). We hypothesized that the morphological difference is related to lipid phenotype and affects their antigenicity. We determined the lipid compositions and biosynthesis of types I and II. Scanning electron microscopy showed that type I was 1.5 times longer than type II. Phenolic glycolipid (PGL) and phthiocerol dimycocerosate (PDIM) were found only in type I. Although it has been reported that the RD16 is involved in the expression of PGL, type II did not possess PGL/PDIM. We examined the ppsA-E gene responsible for PGL/PDIM biosynthesis and found that the existence of PGL/PDIM in types I and II is caused by a ppsA gene mutation not regulated by the RD16. PGL suppressed the host recognition of total lipids via Toll-like receptor 2, and this suggests that PGL is antigenic and involved in host responses, acting as a cell wall component. This is the first report to show the difference between lipid phenotypes of types I and II. It is important to clarify the heterogeneity of BCG vaccine substrains to discuss and evaluate the quality, safety, and efficacy of the BCG vaccine.
Assuntos
Vacina BCG/metabolismo , Mycobacterium bovis/genética , Animais , Carboidratos/química , Cromatografia em Camada Fina/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ácidos Graxos/metabolismo , Genótipo , Glicolipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Mycobacterium bovis/imunologia , Ácidos Micólicos/metabolismo , Fenótipo , Piruvato Sintase/genéticaRESUMO
We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6'-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.
Assuntos
Fatores Corda/toxicidade , Granuloma do Sistema Respiratório/imunologia , Pulmão/imunologia , Pneumonia/imunologia , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T/imunologia , Animais , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Citocinas/imunologia , Feminino , Granuloma do Sistema Respiratório/induzido quimicamente , Granuloma do Sistema Respiratório/patologia , Pulmão/patologia , Depleção Linfocítica , Camundongos , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Pneumonia/induzido quimicamente , Pneumonia/patologia , Linfócitos T/patologia , Fatores de TempoRESUMO
OBJECTIVE: ⢠To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guéin (BCG)-cell wall skeleton (CWS) treatment. MATERIALS AND METHODS: ⢠The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. ⢠Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. ⢠The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. RESULTS: ⢠Major histocompatibility complex class I-related chain B (MICB) expression was increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. ⢠UL-16-binding protein (ULBP) 1 expression was also increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. ⢠ULBP1 expression was increased ≈2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. ⢠T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an ≈1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an ≈1.4-fold increase at an E : T ratio of 2. CONCLUSIONS: ⢠In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. ⢠T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. ⢠The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.
Assuntos
Vacina BCG/farmacologia , Esqueleto da Parede Celular/farmacologia , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias da Bexiga Urinária/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ligantes , Mycobacterium bovis/química , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais Cultivadas/imunologia , Regulação para CimaRESUMO
Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.
Assuntos
Evasão da Resposta Imune , Tuberculose Latente/imunologia , Glicoproteínas de Membrana/metabolismo , Mycobacterium tuberculosis/imunologia , Ácidos Micólicos/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Parede Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glicolipídeos/metabolismo , Humanos , Tuberculose Latente/microbiologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Cultura Primária de Células , Receptores de IgG/metabolismo , Receptores Imunológicos/genética , Fatores de Virulência/metabolismoRESUMO
Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1â4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Glicolipídeos/biossíntese , Glicopeptídeos/biossíntese , Complexo Mycobacterium avium/enzimologia , Proteínas de Bactérias/genética , Configuração de Carboidratos , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Dados de Sequência Molecular , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/genética , Complexo Mycobacterium avium/metabolismo , SorotipagemRESUMO
BACKGROUND: The tuberculosis (TB) still increases in the number of new cases, which is estimated to approach 10 million in 2010. The number of aged people has been growing all over the world. Ageing is one of risk factors in tuberculosis because of decreased immune responses in aged people. Mycobacterium bovis Bacillus Calmette Guérin (BCG) is a sole vaccine currently used for TB, however, the efficacy of BCG in adults is still a matter of debate. Emerging the multidrug resistant Mycobacterium tuberculosis (MDR-TB) make us to see the importance of vaccination against TB in new light. In this study, we evaluated the efficacy of BCG vaccination in aged mice. RESULTS: The Th1 responses, interferon-γ production and interleukin 2, in BCG inoculated aged mice (24-month-old) were comparable to those of young mice (4- to 6-week-old). The protection activity of BCG in aged mice against Mycobacterium tuberculosis H37Rv was also the same as young mice. CONCLUSION: These findings suggest that vaccination in aged generation is still effective for protection against tuberculosis.
RESUMO
Introduction. The incidence of Mycobacterium avium complex (MAC) pulmonary disease (MAC PD), a refractory chronic respiratory tract infection, is increasing worldwide. MAC has three predominant colony morphotypes: smooth opaque (SmO), smooth transparent (SmT) and rough (Rg).Aim. To determine whether colony morphotypes can predict the prognosis of MAC PD, we evaluated the virulence of SmO, SmT and Rg in mice and in human macrophages.Methodology. We compared the characteristics of mice and human macrophages infected with the SmO, SmT, or Rg morphotypes of M. avium subsp. hominissuis 104. C57BL/6 mice and human macrophages derived from peripheral mononuclear cells were used in these experiments.Results. In comparison to SmO- or SmT-infected mice, Rg-infected mice revealed severe pathologically confirmed pneumonia, increased lung weight and increased lung bacterial burden. Rg-infected macrophages revealed significant cytotoxicity, increased bacterial burden, secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CCL5 and CCL3), and formation of cell clusters. Rg formed larger bacterial aggregates than SmO and SmT. Cytotoxicity, bacterial burden and secretion of IL-6, CCL5 and CCL3 were induced strongly by Rg infection, and were decreased by disaggregation of the bacteria.Conclusion. M. avium Rg, which is associated with bacterial aggregation, has the highest virulence among the predominant colony morphotypes.
Assuntos
Macrófagos/metabolismo , Mycobacterium avium/genética , Mycobacterium avium/metabolismo , Animais , Citocinas , Feminino , Humanos , Incidência , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium avium/patogenicidade , Complexo Mycobacterium avium/metabolismo , Complexo Mycobacterium avium/patogenicidade , Infecção por Mycobacterium avium-intracellulare/epidemiologia , Infecção por Mycobacterium avium-intracellulare/metabolismo , Fenótipo , Virulência/fisiologiaRESUMO
BACKGROUND: Although the incidence of pulmonary tuberculosis is decreasing, the number of immunocompetent patients with Mycobacterium avium complex (MAC) pulmonary disease is steadily increasing. Therefore, albeit not contagious, MAC pulmonary disease needs to be diagnosed rapidly and accurately. We examined the serodiagnostic contributions of serum immunoglobulin G antibody titers against the species-specific and -common mycobacterial lipid antigens in the diagnosis of MAC pulmonary disease. METHODS: Serum samples were obtained from 65 patients with MAC pulmonary disease, 15 patients with suspected MAC disease, 25 patients with pulmonary tuberculosis, 10 patients with Mycobacterium kansasii disease, and 100 healthy volunteers (control subjects). We measured the serum immunoglobulin G antibody titers against trehalose monomycolate (TMM-M) and apolar-glycopeptidolipid (GPL), lipid antigens extracted from MAC. RESULTS: In patients with MAC pulmonary disease, the antibody titers against TMM-M and apolar-GPL were significantly higher than those in the other patient groups or in the control subjects. By receiver operator characteristic curve analysis, an optical density of 0.27, corresponding to the optimal cutoff antibody titer against TMM-M, was associated with a sensitivity of 89.2% and a specificity of 97.0%, and an optical density of 0.33, corresponding to the optimal cutoff antibody titer against apolar-GPL, was associated with a sensitivity of 89.2% and a specificity of 94.0%. CONCLUSIONS: Measurements of antibody titers against TMM-M and apolar-GPL would be useful in the diagnosis of MAC pulmonary disease and in the differential diagnosis of mycobacterial pulmonary disease.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Lipídeos/imunologia , Complexo Mycobacterium avium/imunologia , Tuberculose Pulmonar/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Fatores Corda/imunologia , Feminino , Glicolipídeos/imunologia , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Mycobacterium tuberculosis (tubercle bacilli) and the related acid-fast bacteria including Mycobacterium bovis Bacille Calmett-Guerin (BCG) have a characteristic cell wall (CW) containing various lipoglycans and glycolipids. Such lipoglycans have been reported to activate type-I inflammatory responses via dendritic cells (DCs) through Toll-like receptor 2. In this study, lipoglycans, lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinositol mannoside (PIM), were purified from the CW fractions of M. bovis BCG Tokyo-172, and the effect on the differentiation of human peripheral blood naive CD4 T cells into T(h)1 and T(h)2 was examined. LAM/LM molecules enhanced T(h)1 differentiation under both T(h)1 and T(h)2 conditions, whereas some other glycolipids and phospholipid enhanced T(h)2 differentiation under T(h)2 conditions. Other components had little effect under the given conditions. Even in highly purified CD4 T cell cultures, LAM/LM enhanced T(h)1 generation only under T(h)1 culture conditions. These results indicate that LAM/LM possesses a potent augmenting activity in T(h)1 differentiation in human CD4 T cells. LAM/LM appeared to act directly on naive CD4 T cells to enhance T(h)1 differentiation under T(h)1 culture conditions, while acting indirectly to up-regulate the generation of T(h)1 cells via IL-12/DCs under T(h)1 and T(h)2 conditions. Therefore, these results provide the first evidence indicating that LAM/LM from M. bovis BCG may possess a potent modulating activity in the human system, and thus supporting the strategy for the use of BCG components in the vaccine development for such T(h)2 diseases as allergic asthma and rhinitis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Mycobacterium bovis , Células Th1/citologia , Apresentação de Antígeno/efeitos dos fármacos , Antígenos de Bactérias/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunidade Celular/efeitos dos fármacos , Lipopolissacarídeos/imunologia , Fosfatidilinositóis/imunologia , Fosfatidilinositóis/farmacologia , Células Th1/imunologiaRESUMO
OBJECTIVE: To develop a prototype of a non-live bacterial agent that consists of a cell wall (CW) preparation from heat-killed bacillus Calmette-Guérin (BCG-CW) incorporated into octaarginine-modified cationized liposomes as a vector (R8-liposome-BCG-CW), and to evaluate its immunoprotective potentiation in mice, as although BCG is an established effective immunotherapy for nonmuscle-invasive bladder cancer, more active and less toxic treatments are needed. MATERIALS AND METHODS: The cellular interaction of R8-liposome-BCG-CW co-cultured with mouse bladder cancer cell line (MBT-2) was examined by confocal laser scanning microscopy. MBT-2 cells (7 x 10(5)) were subcutaneously inoculated with 1 mg BCG, 0.1 mg or 1 mg BCG-CW, 0.1 mg or 1 mg R8-liposome-BCG-CW in female C3H/HeN mice. The MBT-2 cells pretreated with BCG or R8-liposome-BCG-CW were re-challenged at 6 weeks. The sizes of the primary and re-challenged tumours were evaluated at 4 and 10 weeks, respectively. RESULTS: Confocal laser scanning microscopy showed the enhanced incorporation of R8-liposome-BCG-CW into MBT-2 cells after 1 h of co-incubation. 0.1 mg R8-liposome-BCG-CW completely inhibited the growth of MBT-2 tumours while 0.1 mg BCG-CW alone did not (P = 0.002). Mice vaccinated with a mixture of MBT-2 cells and R8-liposome-BCG-CW inhibited the growth of re-challenged tumour of MBT-2 cells pretreated with BCG or R8-liposome-BCG-CW but did not inhibit that of MBT-2 cells with no pretreatment at 10 weeks, with mean (sd) tumours sizes of 54 (60) mm(2) (P < 0.001) or 69 (43) mm(2) (P = 0.003) compared with 309 (125) mm(2), respectively. CONCLUSION: The immunotherapeutic potential of BCG-CW was enhanced by improving cellular association using the R8-liposomes delivery system. Development of this non-live bacterial agent may contribute to providing a more active and less toxic tool as a substitute for live BCG as immunotherapy against nonmuscle-invasive bladder cancer in the future.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacina BCG/uso terapêutico , Parede Celular/imunologia , Oligopeptídeos/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adjuvantes Imunológicos/efeitos adversos , Animais , Vacina BCG/efeitos adversos , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Lipossomos , Camundongos , Camundongos Endogâmicos C3H , Microscopia Confocal , Mycobacterium bovis , Neoplasias da Bexiga Urinária/imunologiaRESUMO
Intravesical therapy using Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most established cancer immunotherapy for bladder cancer. However, its underlying mechanisms are unknown. Mycolic acid (MA), the most abundant lipid of the BCG cell wall, is suspected to be one of the essential active components of this immunogenicity. Here, we developed cationic liposomes incorporating three subclasses (α, keto, and methoxy) of MA purified separately from BCG, using the dendron-bearing lipid D22. The cationic liposomes using D22 were efficiently taken up by the murine bladder cancer cell line MB49 in vitro, but the non-cationic liposomes were not. Lip-kMA, a cationic liposome containing keto-MA, presented strong antitumor activity in two murine syngeneic graft models using the murine bladder cancer cell lines MB49 and MBT-2 in comparison to both Lip-aMA and Lip-mMA, which contained α-MA and methoxy-MA, respectively. Interestingly, Lip-kMA(D12), which was made of D12 instead of D22, did not exhibit antitumor activity in the murine syngeneic graft model using MB49 cells, although it was successfully taken up by MB49 cells in vitro. Histologically, compared to the number of infiltrating CD4 lymphocytes, the number of CD8 lymphocytes was higher in the tumors treated with Lip-kMA. Antitumor effects of Lip-kMA were not observed in nude mice, whereas weak but significant effects were observed in beige mice with natural killer activity deficiency. Thus, a cationized liposome containing keto-MA derived from BCG induced in vivo antitumor immunity. These findings will provide new insights into lipid immunogenicity and the underlying mechanisms of BCG immunotherapy.
Assuntos
Vacina BCG/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Ácidos Micólicos/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Animais , Vacina BCG/administração & dosagem , Vacina BCG/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/química , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia , Cetoácidos/administração & dosagem , Cetoácidos/isolamento & purificação , Cetoácidos/uso terapêutico , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/ultraestrutura , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Nus , Estrutura Molecular , Ácidos Micólicos/administração & dosagem , Ácidos Micólicos/isolamento & purificação , Tamanho da Partícula , Neoplasias da Bexiga Urinária/patologiaRESUMO
The Mycobacterium avium complex is distributed ubiquitously in the environment. It is an important cause of pulmonary and extrapulmonary diseases in humans and animals. The species in this complex produce polar glycopeptidolipids (GPLs); of particular interest is their serotype-specific antigenicity. Several reports have described that GPL structure may play an important role in bacterial physiology and pathogenesis and in the host immune response. Recently, we determined the complete structure of the GPL derived from Mycobacterium intracellulare serotype 7 and characterized the serotype 7 GPL-specific gene cluster. The structure of serotype 7 GPL closely resembles that of serotype 12 GPL, except for O methylation. In the present study, we isolated and characterized the serotype 12-specific gene cluster involved in glycosylation of the GPL. Ten open reading frames (ORFs) and one pseudogene were observed in the cluster. The genetic organization of the serotype 12-specific gene cluster resembles that of the serotype 7-specific gene cluster, but two novel ORFs (orfA and orfB) encoding putative methyltransferases are present in the cluster. Functional analyses revealed that orfA and orfB encode methyltransferases that synthesize O-methyl groups at the C-4 position in the rhamnose residue next to the terminal hexose and at the C-3 position in the terminal hexose, respectively. Our results show that these two methyltransferase genes determine the structural difference of serotype 12-specific GPL from serotype 7-specific GPL.