Caracterização de biomoléculas e padrão de expressão de citocinas na tuberculose

Abstract

Tuberculosis (TB) is a heterogeneous infection caused by Mycobacterium tuberculosis (Mtb), with high mortality rates. The main diagnostic method consists of bacteriological findings, lacking more effective alternatives. Accuracy, agility and accessibility in diagnosis are crucial for the containment of infectious flow. In this work, we aimed to characterize the expression pattern of 27 cytokines in the saliva and serum of patients with Tb in association with the time of treatment, to evaluate the application of synthetic peptides, TBC10 and TB2C10, in immunoenzymatic and biosensor assays for the diagnosis of Tb by saliva and select specific protein binding nucleic acids. 27 cytokines, in serum and saliva of 86 individuals, were simultaneously dosed by multiplex kit. The individuals were grouped into TB (untreated patients), TBT (patients undergoing treatment), TBTDO (patients at the end of treatment), PPD + (healthy individuals reactive tuberculin test PPD) and C- (healthy non-reactive to PPD). Nine cytokines presented greater expression in TB than in C-. The expression IL-2, IL-15 and IL-4 in saliva was significantly different between patients and PPD +. All Tb-positive groups had higher expression of IL-2, IL-15, IL-8, IL-9 in saliva than healthy individuals. The values obtained for IP-10 in the serum allowed to distinguish the positive groups from each other and negative, presenting potential for monitoring the treatment of Tb. Six cytokines, IL-13, IL-10, IL-6, IL-2, IL-15 and IL-8 were significantly more expressed in serum than the positive groups. The score associated with the four cytokines with better representativeness in the saliva and the six in the serum allowed to predict Mtb infection with assertiveness of 77% in saliva and 96% in serum. Quantification of cytokines may aid in the diagnosis of Tb. For the validation of the synthetic peptides, TBC10 and TB2C10 were tested in immunoassays for the detection of specific IgA of tuberculosis present in saliva. Both were able to discriminate healthy patients with sensitivity of 94.12% and specificities 76.92% and 84.62%, with values of p <0.0001. An antibody biopanning was performed for molecular characterization of Tb-specific IgA ligands from a C10 phage. A scFv (single chain variable fragment) was obtained capable of binding to TBC10, TB2C10 and Mtb proteins, which allowed to delineate the likely epitopes of Mtb proteins recognized by IgA. SELEX was used to select TBC10 and TB2C10 binding DNA molecules. Four aptamers were able to recognize the biomarkers and also Mtb proteins. Both the scFv and the DNA aptamers selected from the biomarkers were able to recognize Mtb proteins, confirming the fidelity of protein epitopes and biomarkers. The biomarkers TBC10 and TB2C10 presented high potential of application for diagnosis of Tb through saliva, the aptamers, useful for confirming the identity of IgA binding epitopes, presented potential for diagnostic and therapeutic purposes, as well as the scores used from the expression of cytokines in serum and saliva allow prediction of active tuberculosis infection.