RESUMO
OBJECTIVES: To characterize at high resolution DNA methylation changes of cytokines which occur in the genome of macrophages in association with Mycobacterium tuberculosis (MTB) infection METHODS: We studied the methylation profiles of THP-1 derived macrophage cells infected with clinical MTB strains [Beijing/W & non-Beijing/W lineage, sensitive (INH(S), RIF(S)) & resistant (INH(R), RIF(R)) strains] and of host macrophages from MTB infected cohorts (active & latent patients) with the human methylation CpG islands microarrays. RESULTS: Methylated modification on the promoter sequences of cytokines and their receptors were found to be associated with MTB infection in a strain- and host-dependent manner. Our epigenetic analyses revealed that infection with Beijing/W MTB strains enhanced IL6R, IL4R and IL17R hyper-methylations in infected macrophages. Validation of IL6R methylated sequence confirmed that MTB infection induced DNA methylation of CpG67 region in the IL6R promoter. In addition, studies on the human macrophage methylation profiles from the patient cohorts indicated that the methylation rate of IL17 family members and related genes were significantly altered in patients with active MTB infections. CONCLUSIONS: Our study offered novel insights into the epigenetic changes in the interaction of host macrophages in MTB infections and warrant further explorations into these changes in modulating the immune response in active and latent MTB infections.
Assuntos
Citocinas/genética , Metilação de DNA , Epigênese Genética , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Receptores de Interleucina/genética , Tuberculose/genética , Tuberculose/microbiologia , Estudos de Casos e Controles , Linhagem Celular , Ilhas de CpG , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Subunidade alfa de Receptor de Interleucina-4/genética , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Tuberculose Latente/genética , Tuberculose Latente/metabolismo , Tuberculose Latente/microbiologia , Macrófagos/metabolismo , Regiões Promotoras Genéticas , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Tuberculose/metabolismoRESUMO
Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.
Assuntos
DNA Bacteriano/isolamento & purificação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Manejo de Espécimes/métodos , Escarro/microbiologia , Tuberculose/diagnóstico , Adulto , Idoso , Reações Cruzadas , Elementos de DNA Transponíveis , DNA Bacteriano/genética , DNA Intergênico , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Fatores de Tempo , Tuberculose/microbiologiaRESUMO
Prostate carcinoma and metastasis are common among male subjects worldwide. CKBM is a drug product targeting prostate cancer in multiple ways. Prostate cancer cell lines PC3 and DU145 were treated with CKBM. The effect of CKBM on the cell's viability, cell cycle, adhesive and invasive properties and its growth in an animal model were assessed. Results indicated that CKBM inhibited PC3 and DU145 cell growth in vitro at IC(50 )values 3.923 and 4.697% respectively, and it brought about cell cycle arrest at G2/M phase. CKBM also attenuated DU145 cells to invade and adhere to extracellular matrices including Matrigel, laminin, fibronectin and collagen IV. Moreover, PC3 tumor xenograft growth was inhibited by over 60% after 28-day of 0.2, 0.4 or 0.8 ml/day CKBM treatment. The present study indicates that CKBM is effective against prostate cancer cell growth in vitro and in vivo. Further studies are required to elucidate its mechanism of action.
