Preliminary study of hemodynamic distribution in patient-specific stenotic carotid bifurcation by image-based computational fluid dynamics.

BACKGROUND: Regions prone to atherosclerosis, such as bends and bifurcations, tend to exhibit a certain degree of non-planarity or curvature, and these geometric features are known to strongly influence local flow patterns. Recently, computational fluid dynamics (CFD) has been used as a means of enhancing understanding of the mechanisms involved in atherosclerotic plaque formation and development. PURPOSE: To analyze flow patterns and hemodynamic distribution in stenotic carotid bifurcation in vivo by combining CFD with magnetic resonance angiography (MRA). MATERIAL AND METHODS: Twenty-one patients with carotid atherosclerosis proved by digital subtraction angiography (DSA) and/or Doppler ultrasound underwent contrast-enhanced MR angiography of the carotid bifurcation by a 3.0T MR scanner. Hemodynamic variables and flow patterns of the carotid bifurcation were calculated and visualized by combining vascular imaging postprocessing with CFD. RESULTS: In mild stenotic cases, there was much more streamlined flow in the bulbs, with reduced or disappeared areas of weakly turbulent flow. Also, the corresponding areas of low wall shear stress (WSS) were reduced or even disappeared. As the extent of stenosis increased, stronger blood jets formed at the portion of narrowing, and more prominent eddy flows and slow back flows were noted in the lee of the stenosis. Regions of elevated WSS were predicted at the portion of stenosis and in the path of the downstream jet. Areas of low WSS were predicted on the leeward side of the stenosis, corresponding with the location of slowly turbulent flows. CONCLUSION: CFD combined with MRA can simulate flow patterns and calculate hemodynamic variables in stenotic carotid bifurcations as well as normal ones. It provides a new method to investigate the relationship of vascular geometry and flow condition with atherosclerotic pathological changes.

[Expression in Escherichia coli and immunogenicity of rotavirus VP7].

Rotaviruses are the single most important cause of severe diarrhea in young children all over the world. VP7 is the major outer capsid and is a primary candidate for inclusion in a subunit or recombinant vaccine. Part of the VP7 gene containing all the three antigenic regions was expressed as a chimeric protein with glutathione S-transferase (GST) in E. coli. The chimeric protein, representing about 30% of the total protein of the recombinant-plasmid-carrying bacteria, reacted with polyclonal antibodies raised against whole virus. Immunization of sero-negative rabbits and mice with purified fusion-protein generated both virus-binding and neutralizing antibodies.

[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.

[Construction and expression of huGM-CSF (9-127)-IL-6(29-184) fusion protein gene].

OBJECTIVE: To construct and express huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities. METHODS: The novel gene coding for the fusion protein of huGM-CSF(9-127)-IL-6(29-184) was constructed by strategy of step by step cloning in pBV220 expression vector. The amino acids 1-8 of huGM-CSF and the amino acids 1-28 of huIL-6 were deleted by PCR technique. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence coding 15 amino acid residues (G-G-S-G-S)3. Fusion protein was expressed in E.coli host strain DH5 alpha. To obtain the fusion protein, Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration were performed. The biologic activities were detected by MTT method. RESULTS: Fusion protein was expressed in E.coli host strain DH5 alpha in the form of inclusion body. The expression level was more than 25% of the total cell lysate. Through Q Sepharose H.P. ion exchange chromatography and Sephacryl S-200 gel filtration, huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity was obtained. The protein showed both huGM-CSF and huIL-6 biologic activities. The specific activity of huGM-CSF was 1.08 x 10(8) U/mg, and for huIL-6, it reached 1.95 x 10(7) U/mg. CONCLUSION: huGM-CSF(9-127)-IL-6(29-184) fusion protein with high purity and both huGM-CSF and huIL-6 biologic activities was obtained.

[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.