New molecular diagnostic and immunological tools for tuberculosis research

摘要: Tuberculosis (TB) remains one of the world’s leading causes mortality due to a single infectious agent, with approximately 1.5 million deaths and 9.2 new cases per year as estimated in 2006. It is assumed that about 5-10% individuals infected M. tuberculosis develop TB remaining 90-95% contain through their immune systems, but have latent infection (LTBI). To effectively control TB, it essential detect LTBI reliably diagnose active TB. Conventional diagnosis continues rely on smear microscopy culture several known limitations terms both speed sensitivity delay and, consequently, hold-up treatment increase spread community. M. widespread, disease generally limited primary stage. Patients an defect or impaired immunity are more prone disease. In LTBI, host response capable controlling by release chemokines cytokines produced T helper (Th) cells, critical for outcome infection. Several cells system involved from macrophages dendritic called antigen presenting (APC) CD4, CD8, gamma delta cells. Activation these excessive pro inflammatory responses can lead tissue damage, need mechanisms counteract this, such Th2 regulatory (Treg)-mediated responses. The optimal scenario would therefore seem balanced Th1, Treg response, suited challenge. balance between types reflected resultant resistance against Therefore aims thesis were find approaches (First Part) (Second Part). this work we wanted explore pathogenesis particular focus impact suppressing tuberculosis-specific (Third Part). For describe alternative PCR methodology based amplification small DNA fragments, originated dying throughout body (transrenal DNA; Tr-DNA) detected urine. was found fragments specifically cell-free fraction urine specimens pulmonary patients. To compared performances two short-incubation interferon (IFN)-g assays (IGRAs), commercial QuantiFERON TB-Gold in-house whole blood stimulation region difference (RD)-1 proteins, those 7-day tuberculin skin test (TST). effort markers diagnosis, also evaluated production pro-inflammatory [interleukin (IL)-1, IL-2, IL-6 Tumor Necrosis Factor (TNF)-alfa], anti-inflammatory (IL-4, IL-10, IL-13) [inducible protein (IP)-10, Macrophage Inflammatory Protein (MIP)-alfa, MIP-1beta, IL-8] after specific stimulation. results raise hypothesis IGRAs mainly recent ongoing tuberculosis, while prolonged-incubation be sensitive past Moreover IL-2 IP-10 may additional RD1 stimulation. Finally evaluate response. Using classical recognition, discordant expansion during Recently CD39 has been shown accurate marker detection. Objectives part were: 1) identify expressing patients compare obtained standard phenotypic markers; 2) if expanded vitro exogenous antigen-specific stimulation; 3) characterize function modulation study demonstrated useful because within CD4+CD25high identifies cell subset characterized high transforming growth factor (TGF)-beta1 absence IFN-gamma expression. Moreover, showed CD39+