Objective To evaluate the preventive effect of different portal vein flush solutions in no heart beating donor harvest on biliary cast syndrome (BCS) after orthotopic liver transplantation (OLT), and the application of choledochoscopy in treating BCS. Methods The incidence of biliary complications in 137 consecutive recipients undergoing OLT in our liver transplant center from May. 2002 to Dec. 2003 was retrospectively analyzed. The incidence and risk factors of BCS were compared in 2 groups with different types of portal vein flush solution in no heart beating donor liver harvest: group 1 (n = 65). University of Wisconsin (UW) solution; and group 2 (n = 72), hypertonic citrate adenine ( HCA) solution combined with UW solution. Therapeutic experience and clinical value of choledochoscopy for BCS were summarized. Results Seventeen out of 137 cases (12.4 %) developed BCS in the first three months after operation. In the group 1, the incidence of BCS was 20. 0 % (13/ 65), while that in the group 2 5. 56 % (4/72) with the difference being significant (P

Objective To investigate the treatment of inverted nipple by continual extension with S tractors made of Kirschner wires on the base of preliminary clinical obervation.Methods Under local anesthesia,with the inverted nipples being dragged out by a presutured thread or a pair of towel clips,a Kirschner wire of 10 cm long and 0.5-1.0 mm in diameter was transversly penetrated through the base of inverted nipple.Then the Kirschner wire was bended at both points of 1.5 cm distantly from the nipple center in clockwise cemicircularly.Two cemicircular Kirschner wires were folded downward hingelikely and the center axis was supported by two cemicircles.This folded S tractor pushed and supported the inverted nipple forwardly and constantly.The hinge angle was adjusted during the treatment according to the traction requirment.It was nessessary for wearing these S tractors for at least 6 months before the Kirschner wires were being taken out.During referrals,the hinge angle was revised for mentaining the extension efforts.Results We treated 32 cases of inverted nipple,total 58 moderate and severe lesions,with this method effectively.No infection was observed during the treatment.The nipple form was naturally and stably outstanding after Kirschner wires taken out.No relapses were observed during a mean 6 monthe following up.Conclusions This procedure is simple and convenient without the need of complicated surgical procedures or perplexed equipments.The nipple shaps are observed natural and stable after Kirschner wire is removed.No recurrences are observed.There are no damages to breastfeeding function.The obvious drawback of this method is its reqairing of long continuously wearing and its inconvenience.

Objective To express and purify recombinant and biologically active Clostridium difficile toxin B (rTcdB). Methods The genes of TcdB were amplified by polymerase chain reaction (PCR) using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522.The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5-10 mg of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megaterium.

Objective To evaluate the cosmetic outcomes of nasal aesthetic sub-unit principle. Methods From May 2007 to March 2015,19 patients with traumatic soft tissue defects of nose had a Single-stage reconstruction with suitable reconstructive method. Full-thick-ness skin transplantation was performed in 1 case,forehead nasal flap in 1 case,bilobed flaps in 2 cases,nasolabial flaps in 9 cases,a folded nasolabial flap in 2 cases,free auricular composite flap in 1 case,the temporal flap pedicled with the orbicular oculi muscle in 3 cases. All pa-tients were followed up for 5~27 months,evaluation items were recorded and analyzed. Results Skin transplantation and local flap were all survived. But 1 case with nasolabial flap infected was healing by second intention,the rest were primary healing,free auricular composite flap was gloomy,and it became normal 5 months after operation. The radian and the curve of the folded nasolabial flap to repair the nasal alar area were slightly poor. Condusion Good aesthetic effets come from nasal aesthetic sub-unit principle in Single-stage soft tissue reconstruction of a traumatic nasal defects.

Objective To observe the clinical effect of Xiaoke-Yuzu decoction on diabetic peripheral neuropathy (DPN). Methods A total of 100 DPN inpatients were recruited and randomly divided into the treatment and control groups. The two groups were both received basic therapy, while the treatment group additionally received Xiaoke-Yuzu decoction. Toronto clinical scores and Chinese medicine symptom scores of both groups were collected to evaluate the clinical effect before and after the therapy. Results The Toronto scores of treatment group were significantly lower than control group after treatment (symptoms score 1.50 ± 0.94 vs. 2.23 ± 1.01, reflection score 3.60 ± 1.77 vs. 4.27 ± 1.72, feeling test score 1.53 ± 0.63 vs. 2.10 ± 0.84,all P<0.05). Meanwhile, the Chinese medicine symptom scores of treatment group were also significantly lower than the control group (main symptom score 1.77 ± 1.17 vs. 3.17 ± 1.82, posterior symptom score 2.23 ± 1.59 vs. 4.27 ± 1.57, the tongue and pulse score 1.83 ± 0.65 vs. 2.47 ± 0.51, all P<0.05). Conclusion Xiaoke-Yuzu decoction plus basic therpy could improve the clinical symptoms of DPN patients.

Objective To investigate the phenotype, frequency of Th17 cells and the association between Th17 cells and viral clearance in patients with H1N1 influenza A. Methods Three groups including 70 confirmed patients with H1N1 influenza A, 30 patients with seasonal influenza as well as 68 healthy subjects as controls were enrolled in this study. The percentages of Th1, Th2, Treg and Th17 lymphocytes in the peripheral blood were determined by intracellular staining and flow cytometry. The levels of interferon-γ (IFN-γ), transforming growth factor-beta (TGF-β),interleukin-6 (IL-6) in plasma and supernatant of the peripheral blood mononuclear cell (PBMC)culture were quantified by enzyme-linked immunosorbent assay (ELISA). Viral load in nasopharyngeal swabs was detected by real time quantitative reverse transcription-polymerase chain reaction (RTPCR). Data were analyzed by one way ANOVA and liner correlation analysis. Results The percentage of Th17 cells in H1N1 influenza A patients was (2. 740±0. 210)%, which the percentage of was significantly decreased compared to healthy subjects (3. 443 ±0. 154)% and seasonal influenza patients (3. 443±0. 277) % (F=4. 242, P＜0. 05); while the percentage of Thl, Th2 and Treg cells were not significantly different among these groups. Moreover, the TGF-β level in plasma of H1N1 influenza A patients was (10±8) ng/mL, which was significantly lower than healthy subjects (43 ±32 ) ng/mL and seasonal influenza patient ( 18 ± 10) ng/mL ( F= 17.72, P＜0.01 ). The TGF-β level in the supernatant of PBMC culture of H1N1 influenza A patients was (782 ± 736) pg/mL, which was significantly lower than healthy subjects (1462±315) pg/mL and seasonal influenza patients (1481 ±348) pg/mL (F=5. 730, P＜0.01). Additionally, the viral clearance period was inversely correlated with the percentage of Th17 cells (r=-0.38, P=0.02). Conclusions The proportion of Th17 cells in patients with H1N1 influenza A is significantly decreased, which is closely correlated with the level of TGF-β. This decrease may results in the delayed viral clearance.

Objective To develop an ELISA(Enzyme-Linked Immunosorbent Assay)diagnostic kit for early rapid detection of sarum anti-EV71 antibody and evaluate its clinical application value.Methods Recombinant protein VP1 of EV71 were prepared and purified as an immobilized antigen for establishment of an indirect ELISA for detection of serum anti-EV71 IgM and anti-EV71 IgG.Compared with RT-PCR.isolation of EV71 and micro-neutralizing assay.the clinical application value of anti-EV71 IgM and anti-EV71 ISG in the diagnosis of EV71 disease was evaluated.Results In comparison with RT-PCR.the sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgM antibody were 83%,85%,81%and 87%,respectively.The sensitivity,specificity,positive predictive value and negative predictive value of anti-EV71 IgG antibody were 72%,74%,68%and 77%.respectively.Compared with viral isolation assay.the sensitivity and specificity of anti-EV71 IgM antibody were 85%and 97%,respectively.The sensitivity and specificity of anti-EV71 IsG antibody were 75%and 77%,respectively.In addition.the titers of anti-EV71 IgG antibody were significantly correlated with the titers of neutralizing antibody to EV71 by linear regression analysis(r=0.72,P＜0.05).Finally,the serum titers of anti-IgG from patients with EV71 associated hand food and mouth disease at convalescent stage exhibited significantly higher than that of the same patients at acute stage(P＜0.01),but the titers of anti-IgM had no significant difference(P＞0.05).Conclusions With VP1 recombinant protein used as an immobilized antigen,an indirect ELISA diagnostic kit was successfully develooed for detection of serum anti-human EV71 IgM and anti-human EV71 IgG antibodies.

Objective To investigate the effects of IL-12 coexpression level on antigen expression and immune responses induced by HBsAg DNA vaccination. Methods DNA vaccine plasmid pHBV carrying codon-optimized preS2-S gene of reference sequence CHN-HBV07-C in China was constructed. Three DNA vaccine plasmids pHBV-12i, pHBV-12l and pHBV-12h were also constructed by subcloning three different IL-12 expression cassettes with various expression strengths to plasmid pHBV, respectively. Expression levels of IL-12 and HBsAg in vaccine plasmid-tranfected 293T cells were measured by quantitative ELISA. DNA vaccines were administered intramuscularly to BALB/c mice and HBsAg-specific cellular immune responses were determined by IFN-γ ELISPOT. HBsAg-specific antibodies were tested by Chemiluminescence Quantitative Immunoassay. Results The HBsAg expression level in 293T cells was 70 ng/ml when transfected by plasmid pHBV without IL-12 expression cassette, and the HBsAg level was 18 ng/ml when transfected by plasmid pHBV-12l carrying low-level IL-12 expression cassette, whereas the HBsAg level was only 6 ng/ml when transfected by plasmid pHBV-12h carrying high-level IL-12 expression cassette.Results of DNA vaccination revealed that HBsAg-specific humoral and cellular immune responses were significantly decreased in mice administering vaccine pHBV-12h carrying high-level IL-12 expression cassette. Although HBsAg-specific antibody responses in mice inoculated with pHBV-12l were also decreased when compared with those in pHBV-vaccinated mice without IL-12 expression, the HBsAg-specific cellular immune responses were significantly increased. Conclusion High-level coexpression of IL-12 may suppress the expression of HBsAg, Whereas modest coexpression of IL-12 significantly enhanced the HBsAg-specific T cell responses induced by DNA vaccination. Therefore, it is so important to balance the expression between adjuvant and antigen to enhance the immune response.

Objective To construct recombinant retroviral vector containing human hepatocellular carcinoma-related gene ANGPTL4 ( angiopoietin-like 4) cDNA and to evaluate antitumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfer. Methods ANGPTL4 cDNA was cloned in vitro from human liver cell lines HL-7702 and subcloned into plasmid vector pMSCV and sequenced. High-tiler recombinant retrovirus pMSCV-ANGPTLA and blank retrovirus pMSCV packaged under mediation of lipofectamine infected HepG2 cells in vitro, respectively. Flow cytometry and fluorescence microscopy detected expression of GFP (green fluorescence protein) in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2 cells was determined. Results Recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. Titer of recombinant retrovirus pMSCV-ANGPTL4 packaged is 1. 4 ? 106 infective viral grains /ml. Titer of blank retrovirus pMSCV packaged was 1. 5 ? 106 infective viral grains /ml. Positive cell rate of HepG2-ANGPTL4 cells group expressing GFP was 68.45% , and average intensity of fluorescence of HepG2-ANGPTL4 cells group was 31.67 -fold as that of HepG2 cells group. Positive cell rate of HepG2-pMSCV cells group expressing GFP was 77.72%, and average intensity of fluorescence of HepG2-pMSCV cells group was 64. 87 -fold as that of HepG2 cells group. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells group was higher than that in HepG2-pMSCV cells group (154%) and HepG2 cells group( 161%). The proliferation rate of HepG2-ANGPTL4 cells group in vitro was lower than HepG2-pMSCV cells group and HepG2 cells group (P

Objective To analyze clinical and laboratory features, viral load and viral shedding period of patients with mild or severe H1N1 influenza A infection. Methods Seventy mild cases and 16 severe cases with concurrent pneumonia were included from Shcnzhen area for analysis.Nasopharyngeal-swab specimens of patients were collected and viral load was detected by real-time quantitative polymerase chain reaction (PCR) assay during their hospitalization. The viral load and viral shedding period were compared between patients over 14 years old and less than 14 years old, and between 70 mild cases without pneumonia and 16 severe cases with pneumonia. The statistic analysis was performed using t test and chi square test. Results The most common symptoms and signs of the patients were fever, cough and enlargement of tonsils. However, the severe cases suffered more frequently from cough, dyspnea and high fever compared with the mild cases (x2 = 10. 9 and 14.3, respectively, t=3.65; both P＜0.01 ). The levels of white blood cell (WBC) count and alanine arninotransferase (ALT) of severe patients were both significantly higher than those of mild patients(t= 3.2, 2.4,respectively; both P＜0.05). The chest radiology of the severe cases showed interstitial pneumonia,mostly with ground glass image. The viral load of patients under 14 years was significantly higher than those over 14 years [(4.86± 1.23) lg vs (4. 17±0.89) lg; t=2.3, P＜0.05], and the viral shedding period of patients under 14 years was significantly longer than those over 14 years [(5.33±0. 49) d vs(3. 63±0.28) d; t=3.4, P＜0.01]. The severe patients also displayed significantly higher viral load and prolonged viral shedding period than the mild patients [(6. 36±1. 44) lg vs (4. 35±0.99) lg, t=6.1,P＜0.01; (5.75±1.77) d vs (4. 24±1. 96) d, t=3.2, P＜0.01]. Conclusion Age anddisease severity of patients with H1N1 influenza A infection are significantly associated with viral load and viral shedding period.