ABSTRACT
Objective:Using the monoclonal antibody to Brucella Omp31, flow cytometry (FCM) method for detecting Brucella antigens is established, and to analyze its potential value in clinical diagnosis. Methods:The supernatants of sonicated proteins (SSPs) from Brucella abortus (2308, 104M and S19), Brucella melitensis (M5-90), and Brucella suis (S2) were identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 5H3 to Brucella Omp31, which were prepared by breaking Brucella species with ultra-sonication. The recombinant eukaryotic plasmid (pcDNA3.1-Omp31) was constructed and transfected in 293FT cells, and the expression of Omp31 was detected by Western blotting. THP-1 cells were infected by Brucella melitensis M5-90 strain to simulate mononuclear phagocytes carrying with Brucella spp. To identify the ability of mAb 5H3, FCM for detecting intracellular Brucella was established, mAb 5H3 was labeled with fluorescein isothiocyanate (FITC-5H3) or P-phycoerythrin (PE-5H3), and then the transfected 293FT cells and THP-1 cells invaded by M5-90 strain were individually identified by FCM with FITC-5H3, and sensitivity of FITC-5H3 in FCM was tested. The PBMCs collected from brucellosis patients or normal blood donors were tested by FCM with double mAbs including PE-5H3 and FITC-CD14 to evaluate this method's feasibility in clinical practice. Results:MAb 5H3 was able to identify Brucella melitensis (M5-90) and Brucella suis (S2), as well as Brucella abortus (2308, 104M and S19) with Omp31 gene deletion. The mAb 5H3 labeled with FITC or PE was used for identifying Brucella antigen in various cells by FCM. The results revealed that the proportion of 293FT positive cells expressing Omp31 was about 59.3%, and the proportion of THP-1 positive cells infected by vaccine strain M5-90 was about 6.2%. In addition, the sensitivity of FCM with FITC-5H3 for the 293FT cells transfected with pcDNA3.1-Omp31 was about 4%. The FCM based on double mAbs staining of PE-5H3 and FITC-CD14 was preliminarily established. For brucellosis patients, the proportion of cells (1.93%) stained with the double mAbs in PBMCs was higher than that of normal blood donors (< 0.30%, negative) in FCM. Conclusions:A FCM assay is preliminary established basing on mAb 5H3 against Omp31 for detecting intracellular Brucella. Moreover, we have found that mAb 5H3 could recognize Brucella abortus originally lacking Omp31, which reduces the defect of Omp31 applied in all Brucella species detection. The development of this FCM assay provides a new strategy and usable reagents for brucellosis pathogens diagnosis.
ABSTRACT
Objective To investigate the percentage of regulatory T cells (Treg) in peripheral blood lymphocytes of patients with chronic brucellosis and the percentage change before and after treatment of different regimens,and to analyze the influence of Treg cell-induced immunosuppression on the therapeutic effect of chronic stage brucellosis.Methods Using case-control study,35 patients with chronic brucellosis who were hospitalized in Heilongjiang General Hospital of Agriculture Bureau [28 males,7 females,aged (45.37 ± 20.16) years old] were selected as case group.According to the treatment regimen,they were divided into standard treatment group (15 cases) and immune enhancer group (20 cases),the treatment was 20 d;30 cases of in-hospital health examinations were selected [16 males and 14 females,aged (35.53 ± 11.38) years old] as control group.Peripheral blood sample of the subject was collected before and after the treatment,the Treg cells as a percentage in peripheral blood lymphocytes were detected by flow cytometry.And the percentage change of Treg cells of brucellosis patients who underwent different treatment regimens was analyzed.Results Before treatment,the percentage of Treg cells in peripheral blood lymphocytes of the control group,the standard therapy and the immune enhancer groups [(1.69 ± 0.38)％,(3.12 ± 0.86)％,(3.05 ± 1.07)％] was significantly different (F =25.89,P ＜ 0.05);compared with the control group,the percentage of Treg cells in the peripheral blood lymphocytes of the standard treatment group and the immune enhancer group increased (P ＜ 0.05);there was no significant difference between the standard treatment group and the immune enhancer group (P ＞ 0.05).After treatment,the percentage of Treg cells in peripheral blood lymphocytes of the control group,the standard therapy and the immune enhancer groups [(1.69 ± 0.38)％,(3.06 ± 0.76)％,(2.85 ± 0.89)％] was significantly different (F =30.84,P ＜ 0.05);compared with the control group,the percentage of Treg cells in the peripheral blood lymphocytes of the standard treatment group and the immune enhancer group increased (P ＜ 0.05);there was no significant difference between the standard treatment group and the immune enhancer group (P ＞ 0.05),and compared with the same group before the treatment,respectively,the differences were not statistically significant (P ＞ 0.05).Conclusions The percentages of Treg cells in peripheral blood lymphocytes of the chronic brucellosis patient are not significantly changed before and after different treatment regimens.It suggests that the immunesuppression induced by Treg cells may be one of the reasons why the host organism cannot effectively remove residual Brucella in the body,which leads to chronic infection.
ABSTRACT
Brain renin-angiotensin system (RAS) is closely associated with many pathophysiological processes of cardiocerebrovascular diseases,including stroke.The activation of the different components in RAS will produce specific biological effects.This article reviews the roles of brain RAS in the pathophysiological processes of ischemic stroke,especially the neuroprotective effect of ACE2/Ang-(1-7)/Mas axis.
ABSTRACT
Objective To investigate the humoral and cellular immune responses in patients with acute brucellosis, and evaluate dynamic changes of regulatory T-lymphocytes (Foxp3+ Treg) in the peripheral blood of patients during treatment, in order to clarify the relationship between immunosuppression and the therapeutic effect in human brucellosis.Methods Sixty-five patients with brucellosis hospitalized at the Third Department of Infectious Diseases, Heilongjiang Agriculture and Reclamation Bureau General Hospital between July 2015 and November 2015 were included.Twenty-eight patients were treated with conventional therapy (group A: patients received 3 courses of treatment.Each lasted for 20 days with one-week interval), and 37 patients were treated with conventional therapy in combination with immunopotentiator (group B).Thirty healthy volunteers were enrolled as the controlled group.The ratio of CD3+CD4+ Foxp3+ Treg cells in the peripheral blood of brucellosis patients were measured by flow cytometry (FCM) at the end of each course of treatment.Data in accordance with normal distribution were described as mean±standard deviation.Comparison between two groups was done by two sample t test.Comparison among multiple groups was performed by analysis of variance and SNK test.Data that did not fit the normal distribution were analyzed by multiple-sample nonparametric test.Results After the first (20 d), second (50 d) and third course of treatment (80 d), the ratios of Foxp3+Treg in the peripheral blood of 65 acute brucellosis patients were 2.83%, 3.77% and 4.03%, respectively, which were all significantly higher than control group (1.69%;t=5.97, 9.05 and 5.66, respectively, all P0.05), while those were both higher than control group (t=7.09 and 4.94, respectively;both P<0.01).At the end of the second course, the ratio of Foxp3+ Treg in group B was higher than group A (t=2.22, P<0.01), and both of them were higher than control group (t=10.79 and 7.25, respectively;both P<0.01).At the end of treatment, Foxp3+ Treg in group A was also significantly higher than the other two groups (t=6.02 and 6.45, respectively;both P<0.01).Conclusions In patients with acute brucellosis treated with the standard antibiosis treatment in combination with immunopotentiator, the ratio of Foxp3+Tregs significantly increases and maintains at a high level, which suggests that extra immunopotentiator may be not helpful for the treatment of brucellosis at the very early stage.