[Inhibition of expression of CCR5 and CXCR4 on cells by adenovirus-mediated antisense RNA].

OBJECTIVE: To suppress the expression of CCR5 and CXCR4, the co-receptors for human immunodeficiency virus type 1 ( HIV-1), and thus inhibit HIV-1 from entering cells. METHODS: DNA fragments encoding either CCR5 or CXCR4 were amplified from healthy human peripheral blood mononuclear cells (PBMCs) by reverse transcript polymerase chain reaction (RT-PCR) and sequencing was performed. Correct fragments were inserted into Shuttle plasmid inversely, which was recombined with backbone plasmid containing homologous adenoviral genome in E. coli BJ5183. The recombinant plasmids were transfected into 293 cells in which they were packaged and amplified. Recombinant adenoviruses containing antisense RNA of CCR5 or CXCR4 were obtained and identified by RT-PCR, and the titres of them were determined by cytopathic effect (CPE) method. The U937 and MT4 cells were infected by recombinant adenoviruses containing antisense RNA of CCR5 (multiplicity of infection, MOI = 100) and CXCR4 (MOI = 200), respectively. The expression of co-receptors on infected cell was measured by fluorescence activated cell sorter at 24, 48, 72 hours and 10 days after infection. In addition, the chemotactic activity and proliferation of infected cells were detected with Boyden chamber and 3H incorporation respectively. RESULTS: We constructed the recombinant plasmids and obtained the recombinant adenoviruses which contained antisense RNA of CCR5 or CXCR4 and were designated as pAd-antiR5 and pAd-antiX4 respectively. The titers of recombinant adenoviruses pAd-antiR5 and pAd-antiX4 were 5 x 10" PFU/ml and 7 x 10(10) PFU/ml, respectively. The expression rate of CCR5 on U937 cells decreased from 82. 10% (blank control) to 1.12% (Ad-antiR5 infected) , and that of CXCR4 on MT4 cells decreased from 42% (blank control) to 1.03% (Ad-antiX4 infected) 24 hours later. The expression rates of CCR5 on Ad-antiR5 infected U937 cells were 1.02% , 1.26% , 1.23% at 48 hours, 72 hours, and 10 days later, respectively. The expression rates of CXCR4 on Ad-antiX4 infected MT4 cells were 1.13%, 1.17%, 1.22% at 48 hours, 72 hours, and 10 days later, respectively. Moreover, the recombinant adenovirus had no effects on chemotactic activity and proliferation of the cells. CONCLUSION: The recombinant adenovirus containing antisense CCR5 or CXCR4 can remarkably decrease the expression of co-receptors for HIV-1 on U937 or MT4 cells without affecting their chemotactic activities and proliferative abilities.

[Study on extraction technology for extract and flavonoids in Chrysanthemum morifolium by orthogonal design].

OBJECTIVE: To ascertain extraction technology condition for extract and flavonoids from Chrysanthum morifoliwn. METHOD: The optimizing ultrasonic extraction condition on the basis of extractive yield and flavonoids were determined by orthogonal design. RESULT: The order of factors which affected the flavonoid extraction was extraction times > ethanol concentration > ultrasonic time > solvent quantity. CONCLUSION: The optimum ultrasonic extractions are A2B3C3D3. Compared with traditional extraction, ultraction method is timesaving, simple to operate, stable and need not be heated.

Association of interleukin-12 p40 gene 3'-untranslated region polymorphism and outcome of HCV infection.

AIM: To investigate the effect of interleukin-12 p40 gene (IL12B) 3'-untranslated region polymorphism on the outcome of HCV infection. METHODS: A total of 133 patients who had been infected with HCV for 12-25 (18.2+/-3.8) years, were enrolled in this study. Liver biochemical tests were performed with an automated analyzer and HCV RNA was detected by fluorogenic quantitative polymerase chain reaction. B-mode ultrasound was used for liver examination. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the detection of IL12B (1188A/C) polymorphism. RESULTS: Self-limited infection was associated with AC genotype (OR = 3.48; P = 0.001) and persistent infection was associated with AA genotype (OR = 0.34; P = 0.014) at site 1188 of IL12B. In patients with persistent HCV infection, no significant differences were found regarding the age, gender, duration of infection and biochemical characteristics (P>0.05). According to B-mode ultrasound imaging and clinical diagnosis, patients with persistent infection were divided into groups based on the severity of infection. No significant differences were found in the frequency of IL-12 genotype (1188A/C) between different groups (P>0.05). CONCLUSION: The polymorphism of IL12B (1188A/C) appears to have some influence on the outcome of HCV infection.

[Nine-year follow-up of hepatitis C virus infection in a rural area of Hebei province, China].

OBJECTIVE: To investigate natural history of hepatitis C virus infection and related factors among plasma donors in China. METHODS: 172 plasma donors in a rural area of Hebei province had been diagnosed as HCV infection in 1993. No antiviral treatment was applied to them during the period of infection. In the present study, we investigated the outcome of HCV infection nine years later and related factors affecting the outcome. In fact, only 142 cases were followed up in the investigation. The mean age of 142 cases of blood donors was 46 +/- 9 and the mean age of infection was 37 +/- 9 years old. RESULTS: After nine-year follow-up, 1.2% died of end-stage liver disease. 130 (91.6%) of 142 cases under investigation were still positive for HCV RNA or anti-HCV in their blood and 12 cases (8.4%) were negative for both HCV RNA and anti-HCV. 3.1% developed liver cirrhosis among the patients with persistent infection. The mean level of ALT, AST, GGT among HCV RNA positive cases were significantly higher than that of HCV RNA negative cases (P < 0.001). The abnormal rates of ALT and/or AST in male patients were significantly higher than those of female patients (P = 0.005). The rate of progression to liver cirrhosis from chronic hepatitis C virus was significantly higher in patients co-infected with HCV/HBV than that of the cases of single HCV infection. CONCLUSION: Higher chronic rate was observed in this research. Superinfection of HBV/HCV may have worse clinical outcomes.

Effect of cytokines on liver necrosis.

AIM:To investigate the effect of cytokines on the liver necrosis.METHODS: rIL (interleukin)-1, rIL-6, rIFN (interferon), rTNF (tumor necrosis factor) -alpha with or without D-galactosamine (D-GAL) were injected into the abdominal cavity of mice separately.ALT, TBIL (total bilirubin) and histological changes were observed.RESULTS: There was no effect on hepatocyte of normal mice after injection of rIL-1, rIL-6, rIFN alone or together. The serum total bilirubin (TBIL) and liver necrosis of mice increased after rTNF-alpha, rIL-6 or rIFN were used separately with D-GAL. The TBIL level (&mgr;mol/L)was 46.19 plus minus 10.62, 44.55 plus minus 12.9 and 41.94 plus minus 14.9, higher than that caused by D-GAL alone (TBIL, 26.67&mgr;mol/L plus minus 11.14&mgr;mol/L). The serum TBIL of mice and the degree of liver necrosis increased after injection of IL-1, IL-6 with D-GAL and rTNF-alpha.CONCLUSION: Cytokines, like IL-1, IL-6, IFN&agr and TNF-&agr;joined in the process of hepatocyte necrosis.They can enhance the degree of liver necrosis induced by D-GAL.