Objective:To explore the immunogenecity of four Dengue virus-specific peptides in mice.Methods:Each peptide of four Dengue virus-specific peptides (C45-57KLVMAFIAFLRFL,E396-408SSIGKMFEATARG,NS323-35YRILQRGLLGRSQ,and NS3141-155NREGKIVGLYGNGVV) was used to immunize BALB/c mice and C57BL/6 mice,respectively.Three weeks later,mice were killed and splelocytes were prepared.Splelocytes were stimulated by the same peptide and intracellular cytokine staining (ICS) assay was used to detect the percentages of peptide-specific IFN-? or IL-4 producing CD4+ T cells in total CD4+ T cells.Results:Peptide C45-57 induced peptide-specific IFN-?+ CD4+ T cells(0.72%?0.04% vs 0.04%?0.02%,P

AIM To investigate the action of rosiglitazone on blood glucose,blood lipid,and the vascular remodeling in rats fed with high-fructose diet. METHODS After fed with high-fructose diet for one month,rats were randomly divided into two groups:High-fructose diet group that continued feeding with high-fructose diet for another month; High-fructose diet+rosiglitazone group fed with high-fructose diet and rosiglitazone (5 mg?kg -1?d -1, dissolved in water) for one month. At last,one half of each group were anesthetized,and the fasting blood, obtained from heart,was used to detect blood glucose,blood lipid,blood insulin. The aorta and mesenteric artery were gotten from the other half of each group, fixed in formalin,sliced and HE dyed. Pathology analysis system was used to measure the remodeling indexes of aorta and mesenteric artery:Lumen ,media,media/lumen,media cross-section area. RESULTS Comparing high-fructose diet+rosiglitazone group with high-fructose diet group, rosiglitazone decreased blood pressure(16.0 kPa vs 18.0 kPa, P

Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2~+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/10~4 PBMC vs (4±1 ) SFC/10~4 PBMC, P < 0.01 ; (10 ± 3 ) SFC/10~4 PBMC vs (2±1 ) SFC/10~4 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8~+ IFN-γ~+ T cells in total CD8~+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.

Objective To identify hepatitis C virus(HCV)-specific cytotoxicity T lymphocytes (CTL)epitopes by the combination of T epitopes prediction software and in vitro assays.Methods HCVspecific CTL epitopes were predicted by T epitope prediction software Rankpep and then candidate HCV-specific CTL epitopes were selected.Candidate HCV-specific CTL epitopes were used to stimulate PBMC of HCV-infected patients and healthy volunteers.and then enzyme-linked immunospot(ELISPOT)and intracellular cytokine staining(ICS)were used to measure the frequencies of IFN-γ-producing cells in total PBMC and the percentages of IFN-γ+CD8+T cells in total CD8+T cells,respectively.Results Five candidate CTL epitopes[NS3 450(TVPQDAVSR),NS3 594(GPTLLYRL),Ns4b 78(sMMAFSAAL),NS5a 416(SEENVSVVF)and NS5a 367(TVSSALAEL)]were used to stimulate PBMC of ten HCV-infected patients and two healthy volunteers.PBMC of seven HCV-infected patients secreted IFN-γ while PBMC of healthy volunteers did not produce IFN-γ.The frequencies of peptide-specific IFN-γ-producing cells ranged from 5 to 36 SFC/105 PBMC and the percentages of peptide-specific IFN-γ+CD8+T cells ranged from 0.02%-0.25%.Conclusion Resuhs of ELISPOT assay and ICS assay confirm that these five peptides NS3 450,NS3 594,NS4b 78,NS5a 416 and NS5a 367 are identified as Hovel HCV-specific CTL epitopes.

Objective To identify human leucocyte antigen (HLA)-A* 0201-restricted hepatitis C virus (HCV)-cytotoxic T lymphocyte (CTL) epitopes. Methods Based on the prediction results of RANKpep and SYFPEITHI prediction programs, six candidate CTL epitopes were selected and synthesized. The affinity of candidate CTL epitopes to HLA-A* 0201 molecules of T2 cells was explored. Subsequently, enzyme-linked immunosorbent spot (ELISPOT) assay and intracellular cytokine staining (ICS) were utilized to determine whether candidate CTL epitopes could induce the recall positive response in peripheral blood mononuclear cells (PBMC) of HLA-A* 0201 positive HCV-1b-infected patients. Results Among six candidate CTL epitopes, peptides C_181(LLSCLTTPV) and NS2_172 (VLQAGLIRV) had high affinity to HLA-A* 0201 molecules. Moreover, the affinity was proportional to the concentration of peptide. Furthermore, among ten HLA-A* 0201 positive HCV-1b-infected patients, the frequencies of C_181 and NS2_172-specific interferon (IFN)-γ-producing cells were 0-19 spots forming cells (SFC)/1 × 105 PBMC and 0-20 SFC/1 × 105 PBMC, respectively.The percentages of C_ 181 and NS2_172-specific IFN-γ+ CD8+ T lymphocytes in total CD8+ T lymphocytes were 0.006%-0.065% and 0.005%-0.080%, respectively. Conclusion Peptides C_181 (LLSCLTTPV) and NS2_172 (VLQAGLIRV) are identified as novel HLA-A* 0201-restricted HCV-CTL epitopes.

Objective To develop a multiple PCR-based reverse line blot hydrization assay (mPCR-RLB)to simutaneously detect several STD pathogens:Neisserria gonorrhoeae,Chlamydia trachomatis,Ureaplasma urealyticum,U.parvum,Mycoplasma genitalium,M,hominis and Trichomonas vaginalis.Methods Seven pairs of biotin-labelled primer were designed and synthesized to target the 16S rRNA-23S rRNA intergenic spacer regions of Neisserria gonorrhoeae,Chlamydia trachomatis,Mycoplasma,and repetitive DNA sequence of Trichomonas to identify and subtype thesc pathogens.DNA was extracted from the referrence strains of seven pathogens and used as templates.mPCR was performed to simutaneously amplify the target regions of these pathogens.Then,the biotin-labelled amplicons were hybridized with membrane-bound specific oligonucleotide probes followed by the detection of bound amplicons with chemiluminescence assay.Serially diluted plasmids containing the target genes of pathogens were amplified with this method to detect its sensitivity.Two-hundred and eleven specimens,including 104 male urethral swabs and 107 female cervical swabs,were collected from the STD clinic of Wuhan First Hospital;mPCR-RLB and single-primer PCR were performed.For specimens with inconsistent results,nested PCR was performed to confirm the results.Results The assay sensitively and specifically identified referrence strains of the tested pathogens.The detection limit of mPCR-RLB was 100 copies for all the pathogens.Of the 211 clinical specimens,2.8%(6/21)were negative for single-primer PCR,but positive for mPCR-RLB,and nested-PCR results were consistent with those of mPCR-RLB.Conclusion mPCR-RLB is a sensitive,specific and rapid method for the detection of STD pathogens from clinical specimens.

ObjectiveTo investigate the relationship of lymphotoxin β receptor (LTβR) and classical nuclear factor-κB (NF-κB) activation pathway in the pathogenesis and progress of cystitis and bladder cancer.MethodsThe LTβR and P65 mRNA expression were detected by Real-time quantitative PCR in 108 cases of fresh bladder tissue specimens (75 cases of bladder cancer,10 cases of inflammation and 23 normal bladder mucosa cases grouped by the tissue classification ),and protein expression were analyzed by immunohistochemistry assay in 118 cases of paraffin-embedded biopsy specimens (73 cases of bladder cancer,30 cases of cysitis and 15 normal bladder mucosa cases).The correlation analysis between the expressions of LTβR and P65 with clinical pathological data was then performed.Differences between LTβR and P65 mRNA and protein expression level were compared in different groups of bladder tissues using Kruskal-Wallis H test and the Chi-square test.Results( 1 )The mRNA expressions of LTβR and NF-κB/P65were higher in bladder cancer than those in normal group ( LTβR:29.4 ( 14.2 - 46.7 ) × 10 - 3/1.2 ( 0.3 -7.0) ×10-3,Z=-5.508; P65:9.7 (2.7 -21.1) ×10-3/1.0(0.8 ～1.8) ×10-3,Z=-5.030,P＜0.05 ).There were significantly differences between bladder cancer with different histological grades ( LTβR:18.2(2.1-31.3) × 10-3/ 28.4(16.6-36.2) × 10-3/47.9(34.3 -70.5) ×10-3,x2K-W=20.378;P65:4.9(1.3 - 12.0) × 10-3/7.4(3.0-21.9) × 10-3/17.0(10.0 ～28.3)× 10-3 ,x2K-W2 =15.219,P all ＜0.05) and lymph node metastasis (LTβR:27.2(9.7-40.1) ×10-3/39.4(26.7 -52.6) ×10-3,Z=-2.552; P65:7.4(2.3-15.6) ×10-3/13.4(6.7-23.3) ×10-3,Z=-2.026,P＜0.05).(2)The positive rates of LTβR and phosphorylated P65 ( p-P65 ) protein in cancer were higber than those of normal group (LTβR:69.8％/13.3％,x2 =16.600 ; p-P65:56.2％/6.7％,x2 =12.220,P ＜ 0.05 ).Upregulation of LTβR and p-P65 were associated with the histological grade (LTβR:56.3％/70.0％/90.4％,x2 =7.055; p-P65:40.6％ /60.0％/76.2％,x2 =6.679,P ＜0.05) and with lymph node metastasis (LTβR:58.3％/92.0％,x2 =8.849; p-P65:52.1％/64.0％,x2 =5.088,P ＜0.05).(3)There was a positive correlation between LTβR and P65 expression ( mRNA:r =0.654,P ＜ 0.05,protein:r =0.399,P ＜ 0.05 )in the bladder cancer and cystitis (r =0.521,P＜0.05).ConclusionsThe activation of LTβR and P65 was associated with progression and metastasis of bladder cancer.The activation of classical NF-κB pathway by LTβR may be achieved by P65.

Objective To study the anatomical characteristics of the medial sural vessels and the clinical effects of treatment for leg skin and soft tissue defect with free flaps.Methods 6 adult lower limbs were treated by latex perfusion and then observed the origin of the blood supply to gastrocnemius muscle,measure the number of the arteries and veins,the length and diameter of the medial sural vessels.From July 2009 to May 2013,15 clinical cases of serious skin and soft tissue defect were repaired by using free flap with medial sural vessels.The areas of wound surface were ranging from 13 cm × 7 cm-24 cm × 12 cm.10 of them were treated by anterolateral thigh flap,the other of them were treated by latissimus dorsi flap.The donor site were directly sutured or repaired by free skin graft.Results The blood supply of gastrocnemius mucle was multifocal.The medial sural arteries and lateral sural arteries were both origin from popliteal artery.The medial sural vessels include 1 artery and 2 veins.With the length 4-7 cm (average,5.4 cm),the arterial diameter of the origin 2.6-2.9 mm (average,2.7 mm) and the entry point 1.9-2.3 mm (average,2.1 mm),the vein diameter of the origin 1.8-2.2 mm (average,2.0 mm)and the entry point 2.7-3.4 mm(average,2.9 mm).Totally 15 cases flaps were survived with primary healing.The mean follow-up period was 16.5 months(range,11-21 months),The flaps had satisfactory appearance,soft texture,good elasticity and achieved protective sensation at the last followed-up.Conclusions The medial sural vessels are with constant anatomical position,deeply position,hardly damage,long pedicle.Thus,the medial sural vessels combine with free flap is an good choice for the reconstruction of leg skin and soft tissue defect.

Objective To study the anatomical characteristics of the medial sural vessels and the clinical effects of treatment for leg skin and soft tissue defect with free flaps.Methods 6 adult lower limbs were treated by latex perfusion and then observed the origin of the blood supply to gastrocnemius muscle,measure the number of the arteries and veins,the length and diameter of the medial sural vessels.From July 2009 to May 2013,15 clinical cases of serious skin and soft tissue defect were repaired by using free flap with medial sural vessels.The areas of wound surface were ranging from 13 cm × 7 cm-24 cm × 12 cm.10 of them were treated by anterolateral thigh flap,the other of them were treated by latissimus dorsi flap.The donor site were directly sutured or repaired by free skin graft.Results The blood supply of gastrocnemius mucle was multifocal.The medial sural arteries and lateral sural arteries were both origin from popliteal artery.The medial sural vessels include 1 artery and 2 veins.With the length 4-7 cm (average,5.4 cm),the arterial diameter of the origin 2.6-2.9 mm (average,2.7 mm) and the entry point 1.9-2.3 mm (average,2.1 mm),the vein diameter of the origin 1.8-2.2 mm (average,2.0 mm)and the entry point 2.7-3.4 mm(average,2.9 mm).Totally 15 cases flaps were survived with primary healing.The mean follow-up period was 16.5 months(range,11-21 months),The flaps had satisfactory appearance,soft texture,good elasticity and achieved protective sensation at the last followed-up.Conclusions The medial sural vessels are with constant anatomical position,deeply position,hardly damage,long pedicle.Thus,the medial sural vessels combine with free flap is an good choice for the reconstruction of leg skin and soft tissue defect.

Objective To evaluate the clinical efficacy of modified Bascom cleft lift procedure in the treatment of chronic sinus.Methods Modified Bascom cleft lift procedure was performed in 53 patients admitted from Oct 2012 to Jul 2016.49 cases were male and 4 were female.The average age was (25.4± 2.3) years.Results All patients were satisfied with the operation.There were 49 cases of primary healing and 4 cases of incision complications.The average follow-up was (12.1 ±4.3) months,no recurrence was observed.Conclusion The modified Bascom cleft lift technique is effective and reliable,with less complications and a lower recurrence rate.