Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis.

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5'-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5'nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5'-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.

[Propagation of hepatitis E virus in several cell lines including human embryo lung diploid cell KMB17].

OBJECTIVE: To investigate the hepatitis E virus (HEV) sensitive cells and its tissue culture conditions. METHODS: The HEV from dejecta supernatant of patients with acute hepatitis E was amplified and activated by passaged in Rhesus. Then, the positive dejecta samples of infected monkeys were dealt with super-centrifugation and virus for culture was obtained. Various human-derived (including KMB17, A549, BEL7402, and Hela) and non-human primates derived cells (Vero) were inoculated with HEV. Sensitivity of cells to HEV was measured by CPE (cytopathic effect), RT-PCR and immunofluorescence. RESULTS: CPE in KMB17, A549 and BEL7402 cells appeared during 7-9 days, meanwhile, cells shelled during 11-13 days on the first filial generation. The existence of HEV genome +RNA and replicated -RNA was still detectable by RT-PCR after the tenth filial generation. Neither CPE nor amplification of HEV genome RNA could be detected in Hela and Vero cells after the second to fourth filial generation. HEV could also be detected from inoculated KMB17 cells by immunofluorescence and RT-PCR. CONCLUSIONS: It indicates that KMB17, A549 and BEL7402 cells are sensitive to HEV under the experimental culture conditions, while Hela and Vero cells are insensitive. Tissue culture system of HEV in certain filial generation is established.

[Expression of hepatitis E virus ORF3 gene fragment in baculovirus system and its immunological character].

OBJECTIVE: To express hepatitis E virus (HEV) ORF3 protein by baculovirus system and provide basis for immunological character research. METHODS: Hepatitis E Virus ORF3 gene fragment was obtained by RT-PCR, ligated with vector pThioHisA for sequencing and then inserted into transfected vector pVL1393 to construct recombinant plasmid. Mediated by Lipofectin Reagent, the recombinant vector and baculovirus linearized DNA (BaculoGold) co-transfected insect cell Sf9 to make recombinant baculovirus. Expressed ORF3 was analyzed for its immunological character by Western blotting, and immunized Kunming Mice. RESULTS: Recombinant ORF3 protein could be recognized by the known positive serum and promoted organism to produce HEV-specific antibody. CONCLUSIONS: Recombinant baculovirus can express effectively HEV ORF3, which has HEV specific immunogenic character.

[Purification and immunological characterization of hepatitis E virus recombinant chimeric antigen encoded by ORF2 fragments and ORF3].

OBJECTIVE: To study immunological characteristics of recombinant chimeric HEV antigen. METHODS: Constructed recombinant plasmids pThioHisORF(2.1 + 2.2 + 3), which contains three HEV antigen gene fragments (ORF2.1:6287-6403nt, ORF2.2:6743-7126nt, ORF3), was transformed into E. coli and induced with IPTG. Expressed product P(2.1 + 2.2 + 3) existed in inclusion bodies, was purified by denature SP Sepharose FF cation exchange chromatography. Rabbits and rats were immunized with renatured P(2.1 + 2.2 + 3). The level of IgG in sera from experimental animals and clinical patients were examined with P(2.1 + 2.2 + 3) by ELISA. The characteristics of IgG of immunized animals interacted with recombinant antigen expressed by baculovirus system as well as recombinant chimeric antigen interacted with clinical patients sera were evaluated by Western-blotting. RESULTS: High titer of IgG antibodies, 1:25,600 in rabbits and 1:12,800 in rats, were detected after immunized with P(2.1 + 2.2 + 3). Furthermore, recombinant antigen expressed by baculovirus system was specifically recognized by IgG of experimental animal immunized with P(2.1 + 2.2 + 3), and the purified recombinant chimeric antigen P(2.1 + 2.2 + 3) was specifically reacted with the IgG of clinical patients. CONCLUSIONS: Recombinant chimeric antigen appears a promising strategy for detection of and prevention from HEV infection.