Objective To provide experimental evidences for developing a safe and effective re- combinant fowlpox virus which can prevent the infection of HIV-2.Methods A fowlpox virus(FPV) transferring vector was constructed by inserting HIV-2 gag gene to the downstream of a synthetic complex promoter ATI-p7.5?20 of vector pUTA2.Transfection was then carried out,and recombi- nant FPV(rFPV)was screened by 5'-bromo-deoxyuridine(BrdU),genome PCR and western blot detection.Balb/c mice were immunized with rFPV by muscular injection.Anti-HIV-2 antibody, CD4~+ and CD8~+ T-cell count and specific target-killing activity of spleen CTL in immunized mice were analyzed by ELISA,FACS and LDH release assay,respectively.Results A transferring vector pA- gag was constructed and confirmed by amplifying a fragment of 766 bp from the rFPV genome.Mean- while,HIV-2 multi-antibody-specific protein blot(55 000)was detected from the recombinant virus and the HIV-2 specific antibody was detected from the immunized Balb/c mice.HIV-2 specific target- killing activity of spleen CTL was observed in immunized mice.Conclusion A recombinant fowlpox virus expressing HIV-2 structural protein Gag has been obtained,and it can stimulate HIV-2-specific eelluar and humoral immune reactions in mice.

To investigate the chemical constituents of the whole plants of Bidens bipinnata, the separation and purification of constituents were performed by chromatography on macroporous resin, silica gel, MCI and Sephadex LH-20. Their structures were elucidated by spectroscopic data as quercetin (1), quercetin-3-0-alpha-L-rhamnoside (2), keampferol-3-O-beta-D-glucopyranoside (3), keampferol-3-O-alpha-L-rhamnoside (4), 3', 5-dyhydroxy-3, 6, 4'-trimethoxyl -7-O-beta-D-glucopyranoside flavonoid (5), 7, 8, 3', 4'-tetraflavanone(6), (2S)- and (2R)-isookanin-7-O-beta-D- glucopyranoside (7a/7b), (2S)- and (2R)-3'-methoxy-isookanin-8-O-beta-D-glucopyranoside (8a/8b), 6, 7, 3', 4'-tetrahydroxyaurone(9), maritimetin (10), esculetin (11), 3-O-caffeoyl-2-methyl-d-erythrono-1, 4-lactone (12), (7S, 8R) balanophonin-4-O-beta-D-glucopyranoside (13), eugenyl-O-beta-apiofuranosyl-( 1"-6') -O-beta-glucopyranoside (14), and (+)-syringaresinol-4'-O-beta-D-glucopyranoside (15). Compounds 8, 13, 14, and 15 were isolated from this genus for the first time. Compounds 1 and 6 were potent inhibitors against HSC-T6 cells in vitro and compounds 1, 2, 6, and 7 were capable of decreasing the inflammatory cytokine production of macrophage cells in vitro.
Bidens ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.
AIDS Vaccines ; immunology ; Adjuvants, Immunologic ; Animals ; Antibodies, Viral ; immunology ; Fowlpox virus ; genetics ; immunology ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Humans ; Immunization ; Interleukin-18 ; biosynthesis ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; immunology

OBJECTIVETo discuss mass spectrum characterization of five valepotriates including 'monoene' type (didrovaltrate), 'diene' type (valtrate, acevaltrate) and 'four-olefinic' type (baldrinal and homobaldrinal) by electrospray ionization tandem mass spectrometry (ESI-MS(n)).

METHODThis study was carried out on the basis of electrospray ionization tandem mass spectrometric method and analysis of multistage fragments.

RESULTThe fragmentation patterns and structural assignment of 'monoene' type, 'diene' type and 'four-olefinic' type valepotriates in ESI-MSn under positive mode were summarized.

CONCLUSIONThe compounds have a strong pyrolysis rules and it can provide reference date for valepotriates in rapid structural identification, quantitative analysis and pharmacokinetic study.

Drugs, Chinese Herbal ; chemistry ; Iridoids ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

BACKGROUNDThe results of clinical trials of rapamycin-eluting stents reduce restenosis have been quite promising. The main purpose of this study was to characterize the in vivo pharmacokinetics of high dose rapamycin (Rapa)-eluting stents in a miniswine coronary model.

METHODSTen miniswines underwent placement of 18 high dose Rapa-eluting stents in the left anterior descending and right coronary arteries. At the planned times of the 1.5th, 12th, 24th hour, 3th, 7th and 28th day, the animals (n = 1, 1, 2, 2, 2, and 2, respectively) were euthanized after completion of coronary angiography. Blood samples were obtained at 0, 10, 20, 30 minutes; 1, 2, 6, 24 hours; and 3, 7, 28 days to determine systemic Rapa levels. Rapa levels in whole blood, arterial wall, heart, renal and liver tissues were determined by high-performance liquid chromatography/mass spectroscopy.

RESULTSPeak whole blood concentration (Cmax), time to peak concentration (tmax), elimination half-life (t1/2beta), area under the curve (AUC), and apparent systemic clearance (Cl/F) were (10.91 +/- 1.28) ng/ml, (2.0 +/- 0.2) hours, (7.25 +/- 0.63) hours, (1.15 +/- 0.11) ng x h x ml(-1), and (180 +/- 12) ml x h(-1) x kg(-1), respectively. More than 95% Rapa detected is localized in the coronary artery surrounding the stent and heart.

CONCLUSIONStent-based delivery of Rapa via a copolymer stent is feasible and safe. This strategy holds promise for the prevention of stent restenosis.

Animals ; Chromatography, High Pressure Liquid ; Coronary Restenosis ; prevention & control ; Male ; Mass Spectrometry ; Sirolimus ; administration & dosage ; pharmacokinetics ; Stents ; Swine ; Swine, Miniature ; Tissue Distribution

Anti-HIV-1 gp120 single chain antibody(scFv) gene and staphylococcus extoxin A(SEA) gene were inserted into vector pPIC9K. The recombinant plasmid was integrated into Pichia pastoris by electroporation. High level expression was performed by determining the Muts phenotype and screening muti-copy integrants. The recombinant protein was about 57kD and the production was 50.1 mg/L. It was shown that the two kinds of protein affected the conformation of each other by antibody affinity assay, but the recombinant targeting toxins could highly mediate CTLs to kill HIV-1 target cells.
Enterotoxins ; genetics ; Genetic Vectors ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Breast Neoplasms/surgery* ; Carcinoma in Situ ; Carcinoma, Ductal, Breast ; Carcinoma, Intraductal, Noninfiltrating/surgery* ; China ; Female ; Humans ; Mastectomy

The objective of this study was to explore the possible effects of leukocyte elimination by filteration before storage on the quality of red blood cell concentrations (RCC) that prepared through two procedures. Eight units of red blood cell concentrations derived from whole blood after plasma separated (RCC1) and eight units of red blood cell concentrations derived from whole blood after platelet-rich plasma separated (RCC2) were divided randomly into filtered group and control group respectively. The RCC of filtered group were filtered by leukocyte deplete filter before storage. The control group didn't have any other treatments. These two groups were stored for five weeks at 4 degrees C according to AABB standard. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and plasma concentration of K(+) and lactate dehydrogenase (LDH), free hemoglobin (FHb), adenosine triphosphate (ATP) of red blood cell of all RCC were evaluated weekly, and bacteria contamination of all RCC was also detected after five weeks of storage. The results showed that there was no difference of MCV, MCH and MCHC and ATP level of red blood cell in all RCC of two groups, the ATP of red blood cell was lower than the control group on week 4 and 5. The average concentration of K(+) of the filtered group was less than the control group. The differences are significant except that of RCC1 stored till the third week. The plasma LDH concentration of filtered group was less than the control group, and the differences were exacerbate during the storing time prolonged. FHb release in the filtered group of RCC2 was significant less than that of control, but no significant difference was found between the two groups of RCC1. It was concluded that leukocyte elimination by filter before storage could be benefit to RCC preservation.
Adenosine Triphosphate ; metabolism ; Blood Component Removal ; Blood Preservation ; Erythrocytes ; physiology ; Filtration ; Humans ; L-Lactate Dehydrogenase ; blood ; Leukocytes ; Potassium ; blood

The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Molecular Sequence Data ; Neutralization Tests ; Nucleocapsid Proteins ; chemistry ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests ; Vesicular stomatitis Indiana virus ; genetics ; Vesicular stomatitis New Jersey virus ; genetics

BACKGROUNDAt the end of 2005, 650,000 people lived with human immunodeficiency virus type-1 (HIV-1) in China, of whom 75 000 were AIDS patients. Many AIDS patients received highly active antiretroviral therapy (HAART) supported by the "China CARES" program but the immune responses of HAART were seldom reported. This study investigated the effect of HAART on the activation and coreceptor expression of T lymphocytes in Chinese HIV/AIDS patients and evaluated its effect on immune reconstitution.

METHODSSeventeen HIV/AIDS patients were enrolled and three-color-flow cytometry was used to detect the activation of HLA-DR CD38 and the coreceptor CCR5, CXCR4 expression on T lymphocytes in whole blood samples taken from the patients before and after 3- or 6-month HAART.

RESULTSThe activation percents of CD4(+), CD8(+) T lymphocytes were significantly higher before therapy than the normal controls (HLA-DR/CD4: 40.47 +/- 18.85 vs 11.54 +/- 4.10; CD38/CD4: 81.34 +/- 10.86 vs 53.34 +/- 11.44; HLA-DR/CD8: 63.94 +/- 12.71 vs 25.67 +/- 9.18; CD38/CD8: 86.56 +/- 11.41 vs 58.84 +/- 6.16, all P < 0.01). After 6-month combined antiretroviral treatment, the activation of T lymphocytes in HIV/AIDS patients was significantly decreased (HLA-DR/CD4: 28.31 +/- 13.48; CD38/CD4: 69.88 +/- 12.64; HLA-DR/CD8: 46.56 +/- 18.64; CD38/CD8: 70.17 +/- 14.54, all P < 0.01 compared with the pre-treatment values). Before the treatment, CCR5 expression on CD8(+) T lymphocytes was up-regulated while CXCR4 expression on CD8(+) T lymphocytes downregulated in HIV/AIDS patients compared with the normal controls (CD8/CCR5: 70.91 +/- 10.03 vs 52.70 +/- 7.68; CD8/CXCR4: 24.14 +/- 11.08 vs 50.05 +/- 11.68, all P < 0.01). After 6-month HAART, CCR5 expression on CD8(+) T lymphocytes significantly decreased (56.35 +/- 12.96, P < 0.01), while CXCR4 expression on CD8(+) T lymphocytes increased (36.95 +/- 9.96, P < 0.05) compared with the pre-treatment and the normal controls. A significant statistical relationship was observed between the expression of activation markers, CCR5 and the CD4(+) T lymphocyte counts after HAART (P < 0.05).

CONCLUSIONSReduced activation of T lymphocytes and a normalization of coreceptor expression were observed in Chinese HIV/AIDS patients after HAART. Immunity can be restored in HIV/AIDS patients receiving HAART.

Acquired Immunodeficiency Syndrome ; drug therapy ; immunology ; Adult ; Antiretroviral Therapy, Highly Active ; China ; Female ; HIV Infections ; drug therapy ; immunology ; Humans ; Lymphocyte Activation ; drug effects ; physiology ; Male ; Middle Aged ; Receptors, Chemokine ; analysis ; drug effects ; T-Lymphocytes ; immunology