Biomedical subjects

Xiao-Yan Che

Publications and source records attributed to Xiao-Yan Che.

15 recordsLinked to original sources

PubMed2006

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells.

AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector. METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured. RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P < 0.001). CONCLUSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Adenoviridae↗

PubMed2005

Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells.

AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli (E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDR-CDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured. RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.

Adenoviridae↗

PubMed2005

Comprehensive antibody epitope mapping of the nucleocapsid protein of severe acute respiratory syndrome (SARS) coronavirus: insight into the humoral immunity of SARS.

BACKGROUND: The epidemic outbreak of severe acute respiratory syndrome (SARS) posed a worldwide threat to public health and economic stability. Although the pandemic has been contained, concerns over its recurrence remain. It is essential to identify specific diagnostic agents and antiviral vaccine candidates to fight this highly contagious disease. METHODS: We generated 14 monoclonal antibodies (mAbs) specific to the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein and used these to thoroughly map the N protein antigenic determinants. We identified the immunodominant antigenic sites responsible for the antibodies in sera from SARS patients and antisera from small animals and differentiated the linear from the conformational antibody-combining sites comprising the natural epitopes by use of yeast surface display. RESULTS: We identified 5 conformational and 3 linear epitopes within the entire N protein; 3 conformational and 3 linear epitopes were immunodominant. The antibody responses to the N protein fragments in mammalian sera revealed that 3 regions of the N protein are strong antigenic domains. We expanded the specificity of the N protein epitope and identified 4 novel conformational epitopes (amino acids 1-69, 68-213, 212-341, and 337-422). CONCLUSION: The antigenic structures identified for the SARS-CoV N protein, the epitope-specific mAbs, and the serum antibody profile in SARS patients have potential use in the clinical diagnosis and understanding of the protective immunity to SARS-CoV.

Adult↗

PubMed2005

Antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229E and OC43.

Cross-reactivity between antibodies to different human coronaviruses (HCoVs) has not been systematically studied. By use of Western blot analysis, indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA), antigenic cross-reactivity between severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and 2 HCoVs (229E and OC43) was demonstrated in immunized animals and human serum. In 5 of 11 and 10 of 11 patients with SARS, paired serum samples showed a > or =4-fold increase in antibody titers against HCoV-229E and HCoV-OC43, respectively, by IFA. Overall, serum samples from convalescent patients who had SARS had a 1-way cross-reactivity with the 2 known HCoVs. Antigens of SARS-CoV and HCoV-OC43 were more cross-reactive than were those of SARS-CoV and HCoV-229E.

Adult↗

PubMed2005

SARS coronavirus detection methods.

Using clinical samples from patients with severe acute respiratory syndrome, we showed that the sensitivities of a quantitative reverse transcription-polymerase chain reaction (80% for fecal samples and 25% for urine samples) were higher than those of the polyclonal (50% and 5%) and monoclonal (35% and 8%) antibody-based nucleocapsid antigen capture enzyme-linked immunosorbent assays.

Enzyme-Linked Immunosorbent Assay↗

PubMed2004

Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome.

A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.

Animals↗

PubMed2004

Nucleocapsid protein as early diagnostic marker for SARS.

Serum samples from 317 patients with patients with severe acute respiratory syndrome (SARS) were tested for the nucleocapsid (N) protein of SARS-associated coronavirus, with sensitivities of 94% and 78% for the first 5 days and 6-10 days after onset, respectively. The specificity was 99.9%. N protein can be used as an early diagnostic maker for SARS.

Antibodies, Viral↗

PubMed2004

[Construction of recombinant adenoviruses encoding TK suicide gene driven by VEGF promoter using efficient AdEasier-1 system].

BACKGROUND & OBJECTIVE: One of the bottlenecks of suicide gene therapy is the low specific expression of suicide genes in tumor cells. Using tumor specific promoter to modulate suicide genes resulting in high specific expression of genes in tumor cells has became a hot research topic of tumor suicide gene therapy. It has showed that vascular endothelial growth factor (VEGF) over-expresses in almost all solid tumors, but not in normal tissues, which is highly relevant to the up-regulation of VEGF promoter (VEGFP) activity. This study was to use the simplified and efficient AdEasier-1 system to generate recombinant adenoviruses encoding TK gene driven by VEGFP, and determine whether VEGFP could increase the expression of TK suicide gene in tumor cells. METHODS: A fragment containing VEGFP was isolated from pEGFP-1-SV-VEGFP (Not I /Xho I), and cloned into the shuttle plasmid pAdTrack resulting in pAdTrack-VEGFP. TK gene by polyadenylation site was cut from pREP8-TK by HindIII,and XbaI, and subcloned into pAdTrack-VEGFP resulting in pAdtrack-VEGFP-TK, which was linearized by PmeI and transformed into AdEasier-1 Cells. Transformants were selected on LB agar plates containing 25 microg/mL kanamycin, and positive pAdEasy-VEGFP-TK was identified by electrophoretic analysis and enzymatic digestion, then digested with PacI, and transfected into 293 cells to produce the recombinant adenovirus Ad-VEGFP-TK. Ad-VEGFP-TK was finally confirmed by polymerase chain reaction (PCR) procedure, and DNA sequence analysis. RESULTS: The pAdTrack-VEGFP-TK (about 12 kb)and pAdEasy-VEGFP-TK(larger than 33 kb), both selectable by kanamycin resistance, could be clearly identified by electrophoretic analysis. Digesting pAdEasy-VEGFP-TK with PacI resulted in 1 specific small fragment (about 3.0 kb), and 1 large fragment (larger than 33 kb). The pAdEasy-VEGFP-TK was successfully constructed at a frequency of 60% (6/10). After being packaged in 293 cells, and purified by CsCl banding, the recombinant adenovirus Ad-VEGFP-TK, whose titer was as high as 5.6x1012 viral particle/ml, were produced, which were further proved to be correct by enzyme digestion, PCR analysis, and DNA sequence analysis. CONCLUSION: AdEasier-1 system is an efficient way to construct the recombinant adenoviruses encoding TK gene under control of VEGFP.

Adenoviridae↗

PubMed2003

[Development of a rapid quantitative double mAb sandwich ELISA for detecting galactomannan antigen of aspergillus fumigatus].

AIM: To develop a double mAb sandwich ELISA for rapidly and quantitatively detecting galactomannan (GM) antigen of aspergillus fumigatus. METHODS: Four monoclonal antibodies (mAb) against GM of aspergillus fumigatus were used as coating or enzyme conjugated antibodies respectively. Capture and sandwich mAbs were selected by sandwich ELISA paired one by one. RESULTS: Optimal capture and sandwich mAbs were selected and a highly sensitive double sandwich ELISA established. The sensitivity of detecting GM reached 0.1 microg/L, the detectable range was 0.1-10 microg/L. Coefficient of variation (C.V) obtained from detecting the same sample for 6 times by ELISA was (7.2+/-3.8)%. CONCLUSION: A sensitive, repeatable and rapid double mAb sandwich ELISA was established for quantitation of aspergillus fumigatus GM, which might apply to early diagnosis and treatment of patients with aspergillosis.

Antibodies, Monoclonal↗

PubMed2002

Detection of cell wall galactomannoprotein Afmp1p in culture supernatants of Aspergillus fumigatus and in sera of aspergillosis patients.

Mannoproteins are important and abundant structural components of fungal cell walls. The AFMP1 gene encodes a cell wall galactomannoprotein of Aspergillus fumigatus. In the present study, we show that Afmp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmp1p was capable of detecting this protein from the cell culture supernatant of A. fumigatus. The anti-Afmp1p antibody is specific since it fails to react with any protein from lysates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium marneffei, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum by Western blotting. In addition, this Afmp1p antigen-based ELISA is also specific for A. fumigatus since the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that 8 of 15 (53%) patients are Afmp1p antigen test positive. Furthermore, an Afmp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for invasive aspergillosis carry a sensitivity of 86.7% (13 of 15). The specificities of the tests are high since none of the 138 control sera, including 100 from normal blood donors, 20 from patients with penicilliosis marneffei, 6 from patients with candidemia, 8 from patients with typhoid fever, and 4 from patients with melioidosis, was positive by either test. In conclusion, the combined Afmp1p antibody and antigen tests are highly sensitive and specific for A. fumigatus invasive aspergillosis.

Antibodies, Fungal↗

PubMed2002

Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis.

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.

Animals↗

PubMed2002

Preparation and identification of monoclonal antibodies against human brain-derived neurotrophic factor.

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against human brain-derived neurotrophic factor (BDNF). METHODS: BALB/C mice were immunized with purified BDNF protein in conjunction with acid-treated Salmonella (antigen:thallus=1:5), and mAbs were subsequently derived by hybridoma technique. RESULTS: Three hybridoma cell lines secreting anti-BDNF mAbs were obtained, designated as B1, B2 and 4D1 respectively and all categorized into IgG1 subtype. The titers of the mAbs in the ascitic fluid of the rats ranged from 1x10(6) to 1x10(5), and their relative affinities were B2>B1>4D1. CONCLUSION: mAbs against BDNF are successfully prepared, which can be instrumental for further study of the expression and distribution of BDNF in vivo, and the detection of BDNF produced by genetic engineering.

Animals↗

PubMed2002

[High-level mIL-12 expression and replication of replicating adenovirus carrying mIL12 gene in naso-pharyngeal carcinoma cells].

OBJECTIVE: To enhance the therapeutic effects on nasopharyngeal carcinoma by combining treatment with selectively replicating adenovirus and IL12. METHODS: Replicating adenovirus with mouse IL12 gene insert (CNHK200-mIL12) was constructed to transfect nasopharyngeal carcinoma cell lines CNE3 and 915. Adenovirus hexon was detected by immunohistochemical staining and flow cytometry (FCM), and mIL12 expression examined by enzyme-linked immunosorbent assay (ELISA). The replication rates of CNHK200-mIL12 and dl1520 was determined by 50% tissue culture infectious dose (TCID50). RESULTS: Twenty-four hours after transfection with CNHK200-mIL12, most of cells were positive for adenovirus hexon and FCM demonstrated increased positivity rates of 39% and 4% among CNE3 and 915 cells respectively. It was observed that CNHK200-mIL12 replication increased by 1 000 folds with mIL12 expression level reaching as high as 84.5+/-4.6 ng in CNE3 cells and 75.6+/-3.4 ng in 915 cells as determined 72 h after transfection with 1x10(5) PFU CNHK200-mIL12 into 1x10(4) cells. CONCLUSION: CNHK200-mIL12 can replicate in vitro in nasopharyngeal carcinoma cells (dl1520 cells, for instance) with high mIL12 expression, which suggests that CNHK200-mIL12 may potentially be used to treat nasopharyngeal carcinoma.

Adenoviridae↗

PubMed2002

[Construction of specifically targeted chimeric hepatitis E virus DNA vaccine and its efficacy assessment].

OBJECTIVE: To design chimeric DNA vaccine targeted to antigen-presenting cells (APCs) with enhanced efficacy to induce immunization. METHODS: The plasmid containing the gene encoding cytotoxic T-lymphocyte antigen 4 (CTLA4), a surface molecule on T cells, was directly fused to the gene fragment HEVE2 coding for hepatitis E virus antigen by molecular engineering technology. The plasmid containing HEVE2 gene fragment alone was also constructed to serve as control and transfection of COS-7 cells with the 2 resultant plasmids was performed respectively, followed by assay of the expressions of CTLA4- HEVE2 fusion protein and HEVE2 protein in COS-7 cells by way of Western blotting. BALB/c mice were injected intracutaneously with the DNA vaccine (100 microgram) for 3 times at 2-week intervals, and the titer of anti-HEVE2 total IgG and IgG subclasses were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The mammalian expression plasmids of CTLA4-HEVE2 fusion protein or HEVE2 protein alone were cloned. The culture supernants of COS-7 cells transfected with the plasmids showed the production of CTLA4-HEVE2 and HEVE2 proteins, which were secreted in the form of dimers. Mice immunized with pCTLA2-HEVE2 produced high levels of specific anti-HEVE2 total IgG titers with IgG2a, IgG2b and IgG1 subclasses predominant in the serum, approximately 50- to 100-fold higher than those in mice immunized with pHEVE2, whose serum contained predominantly IgG1. CONCLUSION: CTLA4-HEVE2 chimeric vaccine stimulates strong immune responses in mice, making it possible for further exploration into chimeric DNA vaccines that target the antigen to APCs.

Animals↗

PubMed2001

Preparation of anfi-CD3 and anti-IgM&mgr; chain bispecific antibody by cell fusion.

OBJECTIVE: To prepare anti-CD3 and anti-Igm&mgr; chain bispecific antibody (BsAb) by means of cell fusion and assess the stability and activity of the hybrid hybridoma. METHODS: Mouse hybridoma cell line secreting anti-human CD3 monoclonal antibody (mAb) was trans formed into HAT-sensitive cells by 8-azaguanine, which was subsequently transfected by plasmid pCDaA3 containing neoR marker gene via FuGENETM6. The resulted mutant phenotype, named alphaCD3 HATs G418R, was fused with the hybridoma producing anti-IgM&mgr; chain mAb, and enzyme-linked immunosorbent assay and flow cytometry were employed to identify the fusion cells producing the target BsAb. RESULTS: Six rounds of cell fusion was performed and a total of l 080 wells inoculated, yielding 5 hybrid hybridoma cell lines. Two of the 5 cell lines were subcloned and continuously cultured in vitro for 2 months without losing their capability to secrete BsAb. CONCLUSION: Cell fusion technique can be utilized to prepare BsAb-producing hybrid hybridoma that has similar stability and activity to its parent hybridoma.

Journal Article↗