Temporal and spatial characterization of negative regulatory T cells in HIV-infected/AIDS patients raises new diagnostic markers and therapeutic strategies.

BACKGROUND: Negative regulatory T cells (Tregs) not only deplete effector T cells but also inhibit the clearance of HIV during infection, which may allow Tregs to be used as informative diagnostic markers. To facilitate both diagnosis and treatment, a thorough understanding of these regulators by characterizing them on temporal and spatial scales is strongly required. METHODS: Hundred HIV-infected/AIDS patients, including 87 males, with an average age of 35.8 years, as well as 20 healthy controls, were enrolled. Flow cytometry was used to analyze CD3+T cells, CD4+T cells, and CD8+T cells to evaluate the immune status of the participants. Then, a group of representative negative regulatory T cells, including CD4+PD-1+T cells, CD4+PD-1high T cells, CD8+PD-1+T cells, and CD4+CD25high Tregs was also analyzed to explore their effects on disease progression and intercorrelation. RESULTS: The percentages of CD4+ PD-1+ T cells and CD4+ CD25high Tregs increased in patients with the same ultrahigh significance. Temporally, the patients with both intermediate-stage and late-stage disease had higher percentages of CD4+ PD-1+ T cells; however, the percentage of CD4+ CD25high Tregs only increased in the patients with late-stage disease. In addition, CD4+ PD-1+ T cells but not CD4+ CD25high Tregs were negatively correlated with the absolute CD4+ T cell count. Spatially, no correlations between CD4+ PD-1+ T cells and CD4+ CD25high Tregs were observed, which suggests these Tregs function differently during immunosuppression. CONCLUSIONS: This study characterized negative regulatory T cells in HIV-infected/AIDS patients at both temporal and spatial scales and found that CD4+ CD25+ Tregs and CD4+ PD-1+ T cells could be used as potential diagnostic markers for identifying different disease stages and monitoring disease progression.

Risk factors of latent tuberculosis infection and immune function in health care workers in Suzhou, China.

BACKGROUND: The Chinese government has pay attention about tuberculosis infection among medical staff in infectious disease hospitals, but the effects have not yet been reported. This study will explore latent infection and immune function in the medical staff and systematically analyze the associated influencing factors. METHODS: Ninety-four medical staffs were enrolled and 20 medical staffs were defined as low risk group and others were high risk group. We used IFN-γ release assay and flow cytometry to analyze the latent TB infection status and immune function. Logistic regression analyses were performed to identify the independent risk factors of latent TB infection. RESULTS: This study explored and compared the infection status of medical workers and found that the rate of positive TB-IGRA results was higher among high risk group than in low risk group. Working environment, occupational history and work type were risk factors for TB infection in hospital. This study also found that high risk group had higher IFN-γ expression and a lower ratio of CD4+ to CD8+ T cells and further analysis found that this immune disorder is associated with wards and occupations. CONCLUSIONS: This study through rigorous sample collection and analysis found the risk factors of latent tuberculosis infection in health care workers. This finding may provide a theoretical basis to be used by the countries with a high TB burden to further improve their strategies for the prevention of TB infections in hospitals and may give an indication for improving the personal health of medical staff in infectious disease hospitals.

[Quality inspection of commercial Bupluri Radix samples in Guangzhou: an ELISA for determining saikosaponin A content using anti-saikosaponin A monoclonal antibody].

OBJECTIVE: To inspect the quality of commercially available Chaihu (Bupluri Radix, a common traditional Chinese herbal drug) in Guangzhou by determining the content of saikosaponin A (SSa) using enzyme-linked immunosorbent assay (ELISA). METHODS: A competitive ELISA system using mouse anti-saikosaponin A monoclonal antibody was established for determining SSa content. Commercial samples of Chaihu were obtained from 10 drug stores in Guangzhou, and SSa contents in the methanol extracts of these samples were determined using the ELISA system. RESULTS: The detection range of this competitive assay was 0.16-2.5 µg/ml for determining SSa contents. In the 10 commercial Chaihu samples, SSa contents in the methanol extract determined by this method ranged from 0.32 µg/mg to 6.87 µg/mg, and 3 samples showed a SSa content lower than the minimum requirement documented in the Chinese Pharmacopeia. CONCLUSION: This competitive ELISA is sensitive, rapid, economic and environment-friendly for SSa determination, especially suitable for batch determination. The results of SSa detection for the commercial Chaihu samples demonstrate an uneven quality of Chaihu in Guangzhou market, suggesting the necessity of more rigorous quality control measures for this drug.

[Immunodetection of ginsenoside Rb1 in rat serum].

OBJECTIVE: To establish a novel immunoassay for qualitative detection of ginsenoside Rb1 in rat serum. METHODS: Anti-G-Rb1 monoclonal antibody (mAb) was through a hybridoma approach. Rat serum containing G-Rb1 was deproteinized with methanol to prepare the sample for testing, which was loaded onto polyethersulfone (PES) membrane and developed in the mixture of acetonitrile, water and acetic acid (25:75:1). After treatment with NaIO(4), the membrane was transferred to 1% BSA solution for immobilization of G-Rb1. The membrane was subsequently treated with anti-G-Rb1 mAb solution, followed by addition of peroxidase-labeled goat anti-mouse IgG and color development using 4-chloro-1-naphthol-0.03% H(2)O(2). RESULTS: On the PES membrane, a clear blue spot representing G-Rb1 occurred where the rat serum for testing and the standard G-Rb1 samples were blotted. The limit of this immunodetection was 0.25 microg. CONCLUSION: This immunoassay has greater specificity and reliability than thin-layer chromatography with a sensitivity similar to that of high-performance liquid chromatography, and does not require sophisticated equipment for convenient G-Rb1 detection in rat serum.

[Determination of stachydrine and leonurine in Herba Leonuri by ion- pair reversed-phase high-performance liquid chromatography].

OBJECTIVE: To establish a high-performance liquid chromatography (HPLC) for determining stachydrine and leonurine contents in the crude drug of Yimu Cao, a Chinese motherwort herb(Herba Leonuri, the aerial part of Leonurus japonicus). METHODS: The sample was obtained from the crude drug by ultrasonic extraction with ethanol after decolorization with ethyl acetate. YMC-Park CN column (250.0 mm x 4.6 mm) was used with the mobile phase for stachydrine of 0.00125 mol/L SDS:C2H5CN (90:10) at the flow rate of 1.5 ml/min with the wavelength for detection of 201.7 nm, and that for leonurine of 0.00125 mol/L SDS (containing 0.05% HClO4):CH4OH(90:10) at the flow rate of 1 ml/min with the wavelength for detection of 282 nm. The sensitivity was 0.1 AUFs and the column temperature was 20 degrees Celsius;. RESULTS: With the injection amount (mg) as the abscissa and the peak area as the ordinate, the regression equation of the calibration curve for stachydrine was Y=1,187.542 3X-168.9822, and that for leonurine was Y=5,202.654X-221.141 (r=0.9998) with the linearity scope of 2.5-12.5 mg, detective limit of 0.15 mg, and recovery of (99.03+/-2.744) %. The analysis of extracted drug samples from 18 regions indicated that the alkaloids contents in Yimu Cao varied significantly, i.e. stachydrine within a range of 0.1%-0.2% while leonurine content, which was much lower, within 0.01%-0.05%. In general, the alkaloid contents were higher in the drug produced in northern China than in those produced in southern China. L. japonicus and the variant L. japonicus albiflorus had total alkaloid contents around 0.3%, which was higher than the contents of other species. CONCLUSION: As a convenient and feasible means for determining the alkaloid contents in Yimu Cao, HPLC produces accurate and reliable results and can be more effective than the formerly used methods.

Detection of autoantibodies in the serum of primary hepatocarcinoma patients.

OBJECTIVE: To study the significance of detecting autoantibodies in primary hepatocarcinoma (PHC) patients. METHODS: Autoantibodies were detected by indirect immunofluorescence assay. Antigens and antibodies of HBV were determined by enzyme immune assay. Antibody to HCV IgG was detected by enzyme-linked immunoabsorbent assay. RESULTS: The positive rate of autoantibody was 27.3% (38/139) in 139 PHC patients. The main type of autoantibodies in PHC was anti-nuclear antibody (36/38, 94.7%), others included anti-smooth muscle antibody(2/38, 5.3%), anti-mitochondria antibody (1/38, 2.6%), anti-midbody antibody (1/38, 2.6%, and anti-liver cell membrane antibody (2/38, 5.3%). CONCLUSIONS: Detecting autoantibodies in PHC patients is of significance in studying the mechanism of autoimmune reaction and etiology in PHC. The diversity of autoantibodies might result from a wide variety of etiological factors involved in PHC development, and from a wide variety of overexpressed or mutated proteins involved in repeated cycles of necrosis and regeneration in hepatocarcinoma development.