[Effect of adjusting the immunosuppressants on the prognosis of the infection in the patients with liver transplantation].

OBJECTIVE: To explore the method of adjusting the immunosuppressants in serious infection after liver transplantation. METHODS: With reference to sepsis-related organ failure assessment (SOFA), 2005.1-2007.12, when the patient's score > or =15, the immunosuppressants were withdrawn, and the patients were given powerful antibiotics and the other treatments in combination. They were further divided into two groups, SOFA 15-17 (group A, 10 cases) and > or =18 (group B, 16 cases). They were compared, and also with the patients without stoppage of immunosuppressants (group C, 13 cases, 2003.3-2004.12). After withdrawing the immunosuppressant, the rejection incidence and times, the changes in SOFA score and mortality and their relationships were analyzed. RESULTS: After adjusting the immunosuppressant and with control of serious infections, rejection occurred in 9 patients, with 5 cases in group A (50.0%), 4 in B (25.0%), none in C. The differences among groups showed statistically significant difference (chi(2)=8.0, P=0.02), but no difference was seen between group A and B (chi(2)=1.70, P=0.19). When the rejection developed, the SOFA score decreased obviously (9.78+/-3.14 vs. 17.22+/-1.86, t=6.10, P=0.00). The time of rejection was (17.56+/-2.60) days after stopping the immunosuppressant. All 25 deaths were due to serious infection with multiple organ dysfunction syndrome, but not rejection. Five deaths occurred in group A (50.0%), 7 in B (43.8%), 13 in C (100.0%). Not a single patient with rejection died from infection. Proper adjustment of the immunosuppressants could decrease the mortality (chi(2)=7.60, P=0.02). CONCLUSION: SOFA score could be used to guide the adjustment of the immunosuppressants, when SOFA> or =15, the immunosuppressants could be stopped, which would not increase the rejection incidence and decrease mortality. The lower the SOFA score is, the faster the patients recuperate better, but more rejection develops. In order to adjust the immunosuppressant in time, the period with high SOFA score should be shortened.

[Construction and expression of non-fusional expression vector of the multi-epitope gene of hepatitis B virus].

AIM: To study the cloning, expression and antigen of therapeutic multi-epitope gene of hepatitis B virus. METHODS: The multi-epitope gene of hepatitis B virus(BPT) was designed, synthesized and cloned into the prokaryotic expression vector pBAD/gIIIA. Then it was transformed into E.coli Top10 and multi-epitope protein of hepatitis B virus(B-BPT) was expressed under the induction of Arabinose. The immunogenicity of the protein was analyzed by Western blot detection. RESULTS: The recombinant plasmid pBAD/BPT was constructed successfully and the protein of multi-epitope gene of hepatitis B virus was expressed in E.coli. Western blot detection showed the protein had ideal antigenicity. CONCLUSION: The design of therapeutic multi-epitope gene of hepatitis B virus is proved to be correct. The expressed protein may be a good therapeutic vaccine.

[Impact of adaptive positive end expiratory pressure and mechanical ventilation on hemodynamics and oxygen kinetics in post-liver transplantation patients].

OBJECTIVE: To determine the impact of adaptive positive end expiratory pressure (PEEP) and mechanical ventilation on hemodynamics and oxygen kinetics in post-liver transplantation patients. METHODS: The study included 11 patients who accepted mechanical ventilation after piggyback liver transplantation. Swan-Ganz catheter and radial artery catheter were used to monitor the cardiac output (CO), mean pulmonary arterial pressure (MPAP), mean arterial blood pressure (MABP) and central venous pressure (CVP) and airway pressure. After transplantation, PEEP of 0, 5, 10 and 15 cm H(2)O (1 cm H(2)O=0.098 kPa) was instituted to support the ventilation alternately. After 30 minutes, pressure regulated volume controlled ventilation (PRVCV) and pressure controlled synchronized intermittent mandatory ventilation+pressure support ventilation (PC-SIMV+PSV) were used to support the ventilation alternately and the indexes of hemodynamics and oxygen kinetics were analyzed. RESULTS: The data showed that differences existed in peak airway pressure, mean airway pressure, CVP and MPAP when different levels of PEEP were used. These indexes were significantly higher in PEEP of 15 and 10 cm H(2)O than those in PEEP of 0 and 5 cm H(2)O.There were no differences in pH, partial pressure of carbon dioxide in arterial blood (PaCO(2)), pressure of oxygen in arterial blood (PaO(2)), arterial oxygen saturation (SaO(2)), oxygen delivery (DO(2)), oxygen consumption (VO(2)) and oxygen extraction rate (O(2)ER) at different levels of PEEP. The airway pressure was significantly lower under PRVCV pattern than those under PC-SIMV+PSV pattern [(8.78+/-1.53) cm H(2)O vs. (11.64+/-3.30) cm H(2)O, P<0.05]. There were no differences in other indexes between these two mechanical ventilation patterns. CONCLUSION: These date suggested that a low level of PEEP (5 cm H(2)O) during mechanical ventilation should be used in post-liver transplantation patients in order to decrease the influence of PEEP on systemic circulation and hepatic regurgitation. PRVCV could be a more suitable mechanical ventilation pattern for patient after liver transplantation.

[Effect of HBsAg pulsed dendritic cells on the functions of cytokine-induced killer cells].

AIM: To explore the effects of HBsAg-pulsed dendritic cells (DCs) on the proliferation and killing functions of cytokine-induced killer (CIK) cells. METHODS: The peripheral blood mononuclear cells (PBMCs) were isolated from the patients with chronic hepatitis B by the routine method, and induced into specific DCs with HBsAg pulsed. The (3)H-TdR incorporation method was used to determine the stimulation effect of HBsAg-pulsed DCs on the proliferation of CIK cells. LDH release assay was used to measure the specific killing activity of CIK cells on HepG2215 cells. RESULTS: The HBsAg-pulsed DCs could induce the memory proliferation of CIK cells and strengthen the killing activity of CIK cells (P<0.05). CONCLUSION: HBsAg-pulsed DCs can enhance the proliferation and killing functions of CIK cells.

[Expression of a human single-chain Fv antibody against HBsAg in Pichia pastoris].

To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.

[Influence of the reductase deficient Escherichia coli on the solubility of recombinant proteins produced in it].

The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli.

[The expression of humanized Fab fragment of the anti-HBsAg antibody in methylotropic yeast Pichia pastoris].

Using of two-step integrating technology, transducted the H and L chain gene of humanized Fab fragment of anti-HB-sAg antibody into the genome of methylotropic yeast P. pastoris. Constructed a engineering yeast to produce humanized Fab fragment of the anti-HBsAg antibody. The Fab fragment was efficiently secreted into the medium at a concentration of 50-80 mg/L. The Fab fragment was purified from culturing supernatant of the recombinant yeas by affinity chromatography. The ELISA analysis showed the high affinity of the expressed humanized Fab fragment to the HBsAg.