| Mycobacterium is a kind of acid-fast bacilli, which contains Mycobacterium tuberculosis (M. tuberculosis, Mtb), Mycobacterium leprae (M.leprae), nontuberculous Mycobacterium(NTM). The occurrence of mycobacterial infection is the consequence of cross-reaction between bacilli and host immunity. However, the mechanism of host defense against mycobacterial infection is still not very clear. Which has been understood is that granuloma is a special histological structure formed in the local sites after the entry of mycobacteria. So far the immune function of granuloma remains disputed, and its formation shows a close relation with immune reaction of the host. The methods used in the previous studies to investigate the special structure, including animal models, in vitro methods applying single kind of human immune cells, were found to be different from the actual process of granuloma formation in human beings in vivo.Chaper one in this study is to build an in vitro simulacrum simulating the actual pathogenesis in mycobacterial infection, and to investigate the ability of certain common mycobacteria to induce immune response. In Section one, based on the known structure of granuloma, an in vitro simulacrum was built by co-culturing macrophages, PBMCs with bacilli. MOI value and temperatures were adjusted according to the characteristics of various mycobacteria. During co-culturing, the formation of granulomatous structure was observed, the vital antigens on the cell surface were tested to confirm the activation and polarization of macrophages, and the significant cytokines related with Th1and Th2cellular immunity were investigated by mRNA expression and secretion levels. All the aspects were observed to validate the success of simulacrum building, after which the simulacrum could be used to screen and evaluate certain vaccine candidates. The simulacrums contained four common mycobacteria, and showed cellular aggregates similar with granuloma in7-9days, the antigens CD14, CD68, CD86were positively expressed on the immune cells in the simulacrum, which also expressed and produced different amount of cytokines, such as TNF-a, IFN-y, IL-1β,IL-10. After the successful construction of the simulacrum, it was applied to investigate and compare the ability of various mycobacteria to induce immune response. Compared to the BCG and live-attenuated Mtb, M.marinum induced more expression of cytokines, TNF-a, IL-1B, IL-10, IL-12p40and chemokines, CXCL8, CXCL10, XCL1and also more secretion of cytokines, TNF-a, IL-1β and IL-10. These results indicated that M.marinum could induce stronger Thl and Th2type immune response. Meanwhile, the viability of M.marinum was inhibited in the simulacrum, revealing the safety of infection with this bacilli. All these results indicated that M.marinum could be a promising vaccine candidate.About12potential vaccines have undergone clinical trials until2011. These vaccine candidates were categorized into two:mycobacterial priming vaccines of an improved whole organism and subunit-boosting vaccines. However, all vaccine candidates have been proven to control the infection but fail to eradicate it. Therefore, therapeutic vaccines are designed as an adjunctive therapy in addition to antibiotics, and the indication of vaccine therapy mostly contains the population with refractory mycobacterial infection, including cases with resistance or side-effect (hypersensitivity) to various drugs. To investigate the epidemiologic characteristics of the aforementioned population can provide the foundation for translational researches on therapeutic vaccines. These special populations with tuberculosis has been studied and monitored throughout the world, but the populations with leprosy in China remains unknown.Therefore, in the second chapter, we investigated the two populations (drug-resistence, dapsone hypersensitivity syndrome) causing refractory leprosy which could be the indication of therapeutic vaccines all over the country. We developed two cross-sectional studies to observe the incidence of two factors in Chinese leprosy patients. In Section one, the information and skin specimens of the relapsed and therapy resistant leprosy patients were obtained by the local institutes after being diagnosed. The three genetic fragments (folP1/rpoB/gyrA), which encoded the proteins associated with the therapeutic effect of three drugs (dapsone/rifampicin/ofloxacin), of M.leprae in the specimens were amplified and directly sequenced, the BLAST program was then used to identify the mutant sites associated with drug resistance. Results showed that3specimens from1relapse and2therapy resistant patients were found to have the same point mutation in the folP1region. Another relapsed patient had a missense mutation of rpoB, while gyrA turned to be negative for mutation. In Section two, the incidence and clinical characteristics of dapsone hypersensitivity syndrome (DHS) among MDT-treated leprosy patients from2006to2009in China were studied in a retrospective survey using a specially designed questionnaire. According to the diagnostic criteria, there were63patients diagnosed to have DHS with an incidence of1.0%. Among these patients,13were complete types of DHS. All the patients presented with various symptoms in14days-2months after MDT. Seven patients died with a death rate of11.1%.Overall, we established an in vitro simulacrum of mycobacterial granuloma, and investigated the ability of various mycobacteria to induce host immune response, then we found that Mycobacterium marinum was a special bacilli which could induce stronger immune response and might be a promising vaccine candidate. Theoretically therapeutic vaccines acted as an adjunctive therapy in addition to antibiotics, especially for the population with refractory mycobacterial infection, the epidemiological information of whom with leprosy was also investigated in this study, to value the in-depth study and translational researches on therapeutic vaccines. |
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