Study On Immune Tolerance Mechanism Of Brucellosis And The Method Of Intracellular Pathogens Detection By Fluorescence

Background:Brucellosis is caused by Brucella which is a kind of zoonotic infectious diseases,which can cause multiple organ system damage and thus seriously reduce the quality of life of patients,so the exact diagnosis and treatment is particularly important.Brucellosis was detected mainly through serology in our country and Bacteria culture as the gold standard.But these traditional diagnostic methods in Brucellosis easily lead to delays,or even misdiagnosed.At the same time,the evaluation of the efficacy of treatment can only be through the serum level of antibody and the clinical symptoms,but the intracellular bacteria can not be informed.Therefore,the prevention and treatment of Brucellosis,as well as the evaluation of therapeutic effects,are urgently needed a more accurate,safe and easy to operate pathogen diagnostic method.Foxp3+regulatory T cells(Treg)is an essential T cell subtype for human immune homeostasis,which is also an important cause of immunologic tolerance/inhibition.It was reported that the regulation of Tregs lead to the inability of the pathogen to be cleared in time then form a chronic infection.Brucellosis easily switch to chronic infection and relapse,difficult to cure.So far,there is no clinical investigation on the immunity of Brucellosis in the process of infection or treatment,and it is still unknown the role of Treg-mediated immunologic tolerance in this process.Aims:1.To investigate the changes of Treg content in peripheral blood of brucellosis patients,and study the correlation between Treg-mediated immunologic tolerance and chronic brucellosis infection providing clinical data for individual immunotherapy.2.To establish an accurate,safe and easy-to-use method for the detection of Brucella antigen in order to provide a new reference standard for the diagnosis and treatment of brucellosis,and to guide the clinical treatment.Methods1.Object of study:Brucellosis patients from General Hospital of Heilongjiang Land Reclamation2.Experimental material:Anti human CD3,CD4&Foxp3 flow cytometry antibody;Brucella outer membrane protein monoclonal antibodies;293A cell line,THP-1 cell line;fluorescein isothiocyanate(FITC).3.Methods:flow cytometry,ELISA,immunofluorescence,FITC-labeled antibody,Simulation of Brucella infection in THP-1 cell.Resu t1.The level of Tregs in PBMCs of Brucellosis patients was closely related to the duration of infection and recovered normal situation.But different treatment regimens may not have a significant improvement in the therapeutic effect for acute Brucellosis.2.Tregs in PBMCs of chronic brucelosis patients will always maintain at high levels.3.A monoclonal antibody 5H3 was successfully screened and labeled with FITC,which can be used for flow cytometry and immunofluorescence.The results of antibody activity assay showed that 5H3-FITC had high sensitivity to detect Brucella antigen eukaryotic expressed in 293FT cells by both of above methods.4.5H3-FITC was used to detect simulate Brucella infectious THP-1 cells by immunofluorescence and flow cytometry.The results showed that both of these methods could detect the bacteria in the cells.Conclusion1.Foxp3+Treg rised in Brucellosis patients,so Treg-mediated immunologic tolerance might exist and relate with chronic brucellosis,difficult cute and relapse.2.A new method to detect brucella pathogens was established using FITC-labeled monoclonal antibody.