Extraction of the anti-sepsis component from Terminaliachebula Retz and evaluation of its biological activities.

Many clinical experiments and studies have demonstrated that traditional Chinese medicines possess the capacity for being used in anti-sepsis. In this paper, we screened 78 herbs based on biosensor technology by targeting of lipid A. Terminaliachebula Retz was found to possess the highest capability of binding lipid A. With CER (cation-exchange resin) and HPLC, we obtained three active components extracted from Terminaliachebula Retz, and named them TCR1, TCR2 and TCR3 respectively. These three components were evaluated with the biosensor, and it was found that the TCR3 was the most capable candidate to bind lipid A. We also studied the biological activities of TCR3 against sepsis in vitro and in vivo. in vitro, TCR3 could significantly inhibit LPS (lipopolysaccharide)-induced LAL (Limulus amoebocyte lysate)) from agglutination and decrease TNFalpha (tumour necrosis factor alpha) release from RAW264.7 cells induced by LPS in a dose-dependent manner. in vivo, TCR3 could significantly protect mice against a lethal challenge with LPS and heat-killed Escherichia coli 35218 in a dose-dependent manner. These results demonstrate that Terminaliachebula Retz is an important herb to neutralize LPS and it has the potential to serve as a treatment for sepsis.

Rapid analysis of alpha-fetoprotein by chemiluminescence microfluidic immunoassay system based on super-paramagnetic microbeads.

A rapid and sensitive chemiluminescence microfluidic immunoassay system based on super-paramagnetic microbeads for determination of alpha-fetoprotein (AFP) is described. In this system we use CO(2) laser to fabricate microfluidic chip, and use super-paramagnetic microbeads as solid carrier of antibody, chemiluminescence as detection signal. With this system we can perform AFP analysis within 20 min, and the linear range of AFP concentration is 1 approximately 800 ng/mL, the detection limit is 0.23 ng/mL. By this method no separation or preconcentration steps are needed. Most of all, the chip can be reused.

[Influence of signal transduction modulators on the secretory function of T lymphocytes in severely scalded mice and its mechanism].

OBJECTIVE: To investigate the influence of various signal transduction modulators on the splenic T lymphocytes secretion of IL-2 and IL-10 in severely scalded mice, and to explore its mechanism. METHODS: The mice were inflicted with 18% TBSA full-thickness scald by high-pressure heat vapour, and T lymphocytes were isolated from murine splenocytes through nylon wool column at 12 and 96 post-scald hours (PSH). Then the cells were divided into following groups: i. e. control, scald, scald and modulator [1 ml of 50 micromol/L PKC inhibitor ( H-7) , 30 micromol/L tetradecanoylphorbol-13-acetate (TPA) , 10micromol/L nonreceptor tyrosine protein kinase inhibitor (herbimycin) , 25 microg/ml of mitogen activated protein kinase kinase inhibitor (PD098059) , 100 nmol/L Calcium ionophore ( A23187) were added to the cells, respectively] groups. The scald group was subdivided into S1 (with scald at 12 PSH) and S2 (with scald at 96 PSH) groups. The modulator group was subdivided into modulator, S1 and modulator( the modulators were added into cells at 12 PSH) , and S2 and modulator( the modulators were added to cells at 96 PSH) groups. The influence of modulators to T lymphocyte secretion of IL-2 and IL-10 were observed. RESULTS: After the addition of H-7, the IL-2 and IL-10 levels in each group were obviously lower than that in controls( P <0. 05 or 0.01) , and that in S1 and H7 group, S2 and H7 group were obviously lower than that in scald group at corresponding time-points( P <0.01). The levels of IL-10, and especially IL-2 were elevated by TPA, but they were markedly lower than that in control group after PD098059 pretreatment. The secretion of IL-2 and IL-10 was significantly suppressed by herbimycin in S1 and herbimycin, and S2 and herbimycin groups, but those in Sl and A21387[ (2 417+/-39) pg/ml, (2 793+/-25)pg/ml] , S2 and A21387 [ (921+/-50) pg/ml, (2 633+/-35)pg/ml] groups were evidently higher than those in S1[ (1 542+/-40)pg/ml, (2 390+/-15)pg/ml] , S2 [(328+/-19)pg/ml, (1 618+/-21)pg/ml,( P <0.05 or <0.01)]groups. CONCLUSION: PKC, calcium, MAPKK and TPK play critical roles in the dysfunction of splenic T lymphocyte secretion of IL-2 and IL-10 in severely scalded mice, among which TPK and PKC are mainly targeted to IL-2 secretion, and MAPKK is targeted to IL-10 secretion. TPA and A23187 can markedly rectify the disturbance of IL-2/IL-10 secretion ratio by increasing the IL-2 secretion after scald.

Transplantation tolerance mediated by regulatory T cells in mice.

BACKGROUND: With potent suppressive effect on responder T cells, CD(4)(+)CD(25)(+) regulatory T (Treg) cells have become the focus of attention only recently and they may play an important role in transplantation tolerance. However, the mechanism of action is not clear. This study was designed to assess the possibility of using CD(4)(+)CD(25)(+) Treg cells to induce transplantation tolerance and to investigate their mechanism of action. METHODS: CD(4)(+)CD(25)(+) Treg cells were isolated using magnetic cell separation techniques. Mixed lymphocyte reactions were used to assess the ability of Treg cells to suppress effector T cells. Before skin transplantation, various numbers of CD(4)(+)CD(25)(+) Treg cells, which have been induced using complex skin antigens from the donor, were injected into the host mice either intraperitoneally [0.5 x 10(5), 1 x 10(5), 2 x 10(5), 3 x 10(5), 4 x 10(5), or 5 x 10(5)] or by injection through the tail vein [5 x 10(3), 1 x 10(4), 2 x 10(4), 5 x 10(4), 1 x 10(5), 2 x 10(5)]. Skin grafts from two different donor types were used to assess whether the induced Treg cells were antigen-specific. The survival time of the allografts were observed. Single photon emission computed tomography was also used to determine the distribution of Treg cells before and after transplantation. RESULTS: Treg cells have suppressive effect on mixed lymphocyte reactions. Grafts survived longer in mice receiving CD(4)(+)CD(25)(+) Treg cell injections than in control mice. There was a significant difference between groups receiving intraperitoneal injection of either 2 x 10(5) or 3 x 10(5) CD(4)(+)CD(25)(+) Treg cells and the control group (P < 0.05, respectively). Better results were achieved when Treg cells were injected via the tail vein than when injected intraperitoneally. The transplantation tolerance induced by CD(4)(+)CD(25)(+) Treg cells was donor-specific. Analysis of the localization of Treg cells revealed that Treg cells mainly migrated from the liver to the allografts and the spleen. CONCLUSIONS: CD(4)(+)CD(25)(+)Treg cells can induce donor-specific transplantation tolerance. Cell-to-cell contact may be the primary mechanism by which Treg cells act on effector T cells.

[The influence of the application of cytotoxic lymphocyte antigen 4-Ig adenovirus on the burn wounds with alloskin grafting on the murine immune function].

OBJECTIVE: To investigate the influence of local application of cytotoxic lymphocyte antigen 4-Ig (CTLA4-Ig) adenovirus on the burn wound with alloskin grafting upon the murine immune function. METHODS: Sixty BALB/c mice were randomly divided into A (operation control), B (CTLA4-Ig transfection) and C (normal control) groups, with 20 mice in each group. Skin wounds (full-thickness loss) sized 1.5 cm x 1.5 cm were created on the backs of mice in A and B groups. Then the skin grafts of the same size obtained from C57BL mice were grafted into the skin wounds. 0.1 g of cross-linking polyacrylic resin (carbomer cream) without adenovirus was daubed onto the wounds in A group, and the same amount of carbomer cream with adenovirus in titers of 5 x 10(9)/L was daubed onto the wounds in B group, while no treatment was given in C group. 1 ml of 10% SRBC (sheep red blood cell) was injected intraperitoneally to all the mice of the three groups on the 1st post injury day (PID). Splenocytes from BALB/c, C57BL and Kunming mice were harvested for mixed lymphocyte culture on 7, 14, 21 and 28 PIDs. Agglutination assay was used in the same time to detect the SRBC antibody titers. RESULTS: The reaction of murine splenocytes in B group to the donor (C57BL) splenocytes was suppressed in a specific way (P < 0.05) within 14 PIDs. There was no difference in the titers of anti-SRBC antibody among the 3 groups (P > 0.05). CONCLUSION: Local application of CTLA4-Ig recombinant adenovirus exhibited no influence on the murine humoral immunity, but might induce systemic and specific T cell tolerance in immunity system.