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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.
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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.
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The ablated aerosols of biological matrix sample were studied using 213 nm nanosecond laser ablation system.The stable signal intensity and high sensitivity were obtained when the laser energy was 25%, the spot size was 200 μm, the scan rate was 20 μm/s, the frequency was 20 Hz and the carrier gas was 700 mL He + 700 mL Ar.Relative fractionation index of 56 elements were investigated and 31P as the internal standard element was selected under the optimized laser ablation conditions.The results showed that particle size of the biological sample was 3 μm, which was larger compared with NIST 610 sample.Element fractionation in biological sample was smaller than in glass sample, and relative fractionation index of most elements attained 1.0 ± 0.1.Element fractionation mechanism of biological sample was discussed.The possible reason why the relative fractionation index in biological sample with large particle size did not significantly increase compared to the glass sample is that the 3-μm particles entered into ICP can be atomized.On the other hand, enrichment effect for large ablation particles was relatively small.Further study of the influence factors of fractionation effect indicated that, the fractionation effect had relations with laser ablation energy, laser frequency and scan rate, negatively relation with the oxide boiling point, and positively relation with oxide bond energy and ionization energy.
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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.
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TheLutetium-YttriumOrthosilicate(LYSO)isakindofscintillatingcrystalmaterialwiththebest comprehensive properties. In this work, the trace elements Ca, Fe, K, Li, Mg and Na in LYSO were determined by solution-cathode glow discharge-atomic emission spectrometry ( SCGS-AES ) . The optimal conditions included 0. 1 mol/L HNO3 sample solutions and operation at a voltage at 1080 V with a flow rate of 2. 0 mL/min. The LYSO matrix concentration tolerance of the SCGD source was determined to be 10 g/L. Sample solutions dissolving from several LYSO samples with HF, HNO3 and HClO4 were examined by SCGD-AES. For LYSO samples, the values obtained by SCGD-AES agree well with those obtained by axial inductively coupled plasma-atomic emission spectroscopy ( ICP-AES ) and inductively coupled plasma-mass spectroscopy (ICP-MS). The emissions of Lu and Y were not observed, so that the determination of trace elements in LYSO matrices could be conducted with little interference. The detection limits of Ca, Fe, K, Li, Mg and Na in LYSO were 1. 0, 3. 0, 0. 02, 0. 01, 0. 02 and 1. 0 mg/kg, respectively.