AIM: To observe the effects of high-dose hepatitis B surface antigen (HBsAg) vaccine on cellular immune response in BALB/C mice. METHODS: The mice were immunilized separately with low-dose and high-dose HBsAg vaccine by intramuscular injection two times. The specific proliferative activities of T lymphocytes were measured by [ 3H]-TdR incorporation assay. IL-2 as well as IFN-? levels in the culture supernatant of T cells and anti-HBs IgG2a lever in sera were detected by enzyme-linked immunoabsorbent assay. RESULTS: After first vaccination with high-dose HBsAg, the proliferative activities of T cells in the experimental group were significantly stronger, both levels of IL-2 and IFN-? were markedly higher than that in the control group and the percentage of mice to produce serum anti-HBs IgG2a was significantly higher compared to that of mice immunilized by low-dose HBsAg. All data in experimental groups were further increased after second dose of vaccine. CONCLUSION: Vaccination of mice with high-dose HBsAg can induce cellular immune responses tended to Th1(T helper 1 subset) response.

Objective: To screen candidate antigen for therapeutic HBV vaccine with a novel adjuvant DC-Chol. Methods: BALB/c mice were injected with DC-Chol liposome and HBsAg prepared from CHO and Yeast respectively. One week later, IL-4, IL-2, IFN-?were measured by ELISA or ELISPOT. Results: The levels of IL-2, IFN-?of HBsAg from Yeast with DC-Chol liposome were 20 and 119 times higher respectively than those of HBsAg from CHO with DC-Chol liposome. ELISPOT assay showed that the counts of spot-forming cells of IL-4 and IFN-?of HBsAg from Yeast with DC-Chol liposome were 2.8 and 46.3 times higher respectively than those of HBsAg from Yeast with Al(OH)3. Conclusion: HBsAg prepared from Yeast together with DC-Chol liposome may be an appropriate candidate for therapeutic HBV vaccine .

Dipeptides ; administration & dosage ; therapeutic use ; End Stage Liver Disease ; drug therapy ; virology ; Female ; Hepatitis B virus ; Humans ; Male

Adenoma ; pathology ; Aged ; Carcinoma, Papillary ; metabolism ; pathology ; Cytokines ; metabolism ; Diagnosis, Differential ; Ear Neoplasms ; metabolism ; pathology ; Endolymphatic Sac ; pathology ; Humans ; Male ; Vimentin ; metabolism

NS2 and NS3 are two post-transcriptional gene silencing suppressors that are encoded by Rice stripe virus. Gene silencing suppressors are always related to the pathogenicity of viruses. In this study, the cDNA of NS2 and NS3 were recombined by overlapping PCR assays, ligated to the RNAi vector, and inserted into the PXQ expression vector using Pst I; the expressed vector was transferred into calluses induced from seeds of the japonica rice cultivar, 'Nipponbare', using an Agrobacterium-mediated method. Thirty-one T0 transgenic plants were selected by G418 screening. PCR and southern blot analyses confirmed that the target gene was transformed into transgenic rice successfully, and different transgenic plants contained various copies of the gene. The disease resistance assay revealed that T0 transgenic rice had a delayed onset of RSV for approximately 10-20 d, and the accumulation of virus in the transgenic plants was reduced by 30%-50%. This was related to the delayed onset of disease.
Disease Resistance ; Oryza ; genetics ; immunology ; virology ; Plant Diseases ; genetics ; immunology ; virology ; Plants, Genetically Modified ; genetics ; immunology ; virology ; RNA Interference ; Tenuivirus ; genetics ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

Adolescent ; Anticonvulsants ; administration & dosage ; adverse effects ; therapeutic use ; Child ; Child, Preschool ; Epilepsy ; drug therapy ; Female ; Fructose ; administration & dosage ; adverse effects ; analogs & derivatives ; therapeutic use ; Humans ; Infant ; Male ; Time Factors ; Treatment Outcome

Objective To study the expression of vascular endothelial growth factor(VEGF)-C, VEGF-D and their receptor-3(VEGFR-3)in patients with colorectal cancer and their clinicopathological value.Methods Eighty specimens of the colorectal cancer and thirty normal adjacent bowels were stud- ied.The expression of VEGF-C,VEGF-D and VEGFR-3 proteins and mRNAs in specimens of colorectal cancers and normal colorectal tissues was studied by Strept-avidin-biotin complex method and RT-PCR. Clinicopathological data and survival of each patient were recorded and analyzed.Results①The staining of brown or filemot in cytoplast were observed as the positive expression of VEGF-C,VEGF-D and VEGFR-3 proteins.The positive rate(48.8%,56.3%,38.8%)and mean value(1.09?1.20,1.13?1.09,0.90?1.19)of VEGF-C,VEGF-D,VEGFR-3 expressions in specimens of colorectal cancer were significantly higher than those of the normal bowel tissues(P＜0.05).The expression of VEGF- C,VEGF-D and VEGFR-3 mRNAs by RT-PCR was correlated with that of VEGF-C,VEGF-D and VEGFR-3 proteins in colorectal carcinomas and normal bowel tissues.②Significant correlation between VEGF-C(P=0.0069),VEGF-D(P=0.0024)and VEGFR-3 expression was observed in colorectal cancers;moreover,no correlation between VEGF-C and VEGF-D.③The expression of VEGF-C, VEGF-D and VEGFR-3 in colorectal cancers was not correlated with age,gender,site and dimension of lesion,types of gross and histological,degree of differentiation and liver and pulmonary metastasis,but correlated significantly with Dukes' stage(P=0.0234,P=0.0003,P=0.0429)and lymph node me tastasis(P=0.0059,P＜0.01,P=0.0068).The increased death rate(P=0.0374,P=0.0127) and poor survival(P＜0.01,P＜0.01)were observed in the colorectal cancer patients with positive ex- pression of VEGF-C and VEGFR-3 when comparing with the patients of the negative expressions,but the expression of VEGF-D in colorectal cancers was not correlated with prognosis of the patients.Con- clusions Colorectal cancer cells may secrete lymphangiogenetic growth factors VEGF-C,VEGF-D and their receptor VEGFR-3,which induce the growth of lymphatic vessel endothelium and lymphangiogene- sis by VEGF-C,VEGF-D/VEGFR-3 signaling pathway,further accelerate lymphatic metastasis of colo- rectal cancers.VEGF-C,VEGF-D and VEGFR-3 might be acted as molecular phenotypes of lym- phangiogenesis in coiorectal cancers and important markers for evaluating lymphatic metastasis and prog- nosis in patients with coloreetaI carcinoma.

The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.

Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.

Objective To evaluate the expression of Toll-like receptor(TLR)on the monocyte- derived dendritic cells(DC)from chronic hepatitis B(CHB)patients and to analyze the expression pro- file and significance of the TLR such as TLR3,TLR4,TLR?,TLR8 and TLRg,which are associat- ed with immune response to viral infection.Methods Peripheral blood mononuclear cell(PBMC) centrifugated by the hydroxyethyl starch(HES)centrifugation were cultured and induced into DC by granulocyte-maerophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4),and their mor- phology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus(HBV)-infected patients and 15 healthy volunteers.TLR3,TLR4,TLR7,TLRS,TLR9 expression on immature and mature DC were analyzed by FACS Calibur.DC was pulsed with HBcAg on day 3 and 5,then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated.Results Monocyte- derived DC developed different TLR expression patterns as they went through different maturation stages.TLR7,TLR8 expressions on immature DC and TLR3,TLR7 expressions on mature DC were lower in CHB than in control(for TLR7,TLR8 expression on immature DC:75.9%,1.0%vs 98.4%,15.4%,P