Biomedical subjects

Xian-hui He

Publications and source records attributed to Xian-hui He.

10 recordsLinked to original sources

PubMed2006

Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif.

AIM: To investigate the effect of incorporation of an isoleucine zipper (IZ) motif into CD40 on binding activity of CD40 for the CD40 ligand (CD40L). METHODS: Prokaryotic expression vectors for 2 soluble CD40 derivatives, shCD40His and shCD40IZ containing an IZ domain, were constructed and expressed in Escherichia coli. The recombinant proteins were purified to homogeneity after refolding from inclusion bodies. Their molecular weights in solution of shCD40His and shCD40IZ were compared by size-exclusion chromatography, and their binding activity for CD40L on Jurkat T cells was determined by flow cytometry. RESULTS: shCD40His and shCD40IZ were generated. Both of them possessed significant binding activity for the cognate ligand CD40L expressed on the cell surface. shCD40IZ had much higher binding activity to its ligand (CD40L) than did shCD40His. Furthermore, size-exclusion chromatography demonstrated that shCD40IZ existed in high molecular mass forms that were most likely to be trimers in solution. CONCLUSION: Incorporation of an IZ motif into CD40 enhances its binding activity for CD40L through trimerization of the CD40 derivative.

Binding, Competitive↗

PubMed2006

[Real-time quantitative PCR for evaluating murine thymic function].

OBJECTIVE: To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function. METHODS: The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR. RESULTS: The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains. CONCLUSIONS: Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.

Animals↗

PubMed2006

[Quantification and phenotypic analysis of hCMV specific CTL in peripheral blood from HLA-A2+ donors].

AIM: To optimize tetramer staining condition using HLA-A*0201 tetramer (A2-NLV tetramer) loaded with NLV peptide (pp65(495-503)) derived from structural protein pp65 of human cytomegalovirus and to investigate its application in phenotyping of specific cytotoxic T lymphocytes (CTL). METHODS: Peripheral blood from HLA-A2(+) donors was first stained with A2-NLV tetramer/PE under different conditions and then labeled with anti-CD3-FITC and anti-CD8-APC. The stained samples were analyzed with flow cytometry to find out the optimized staining condition. Meanwhile, the phenotype and activation antigen expression were determined. RESULTS: Tetramer staining with whole blood was superior to peripheral blood mononuclear cells. The optimized condition for tetramer staining was incubating 100 muL of whole blood with 0.3 mug of A2-NLV tetramer for 1 h at 4 degrees Celsius. Under this condition the specific staining was strong while unspecific staining of CD8(-) T cells was quite weak. Phenotypic analysis under this condition showed that the ratio of CD28 positive A2-NLV tetramer specific CTL was lower than that of nonspecific CTL, whereas the ratio of CD57 positive specific CTL was higher than that of nonspecific CTL. CD25 molecules were only expressed on the activated specific CTL. CONCLUSION: The optimized tetramer staining condition can increase the specificity of tetramer staining and decrease unspecific binding, therefore it is applicable for phenotyping and functional analysis of antigen-specific CTL.

Adult↗

PubMed2006

[Application of vital dye CFDA-SE and SNARF-1 to evaluate mixed lymphocyte reaction].

AIM: To establish a new method for more comprehensive evaluation of responsive intensity in one-way mixed lymphocyte reaction (MLR). METHODS: CFDA-SE labeled lymphocytes from lymph nodes of BALB/cJ mice were applied as responder cells, and SNARF-1 labeled splenocytes from BALB/cJ or C57BL/6 mice which had been pretreated with mitomycin C were applied as stimulator cells to establish mixed lymphocyte culture (MLC). After 96 h of culture, cells were harvested and analyzed by flow cytometry to acquire the proliferative information of SNARF-1 negative and CFSE positive staining responder cells. ModFit software was then used to get more proliferation related index. RESULTS: With increasing division cycles, the CFSE fluorescence was diluted to the extent that the responder cells could not be discriminated from stimulator cells, which was resolved by gating SNARF-1 positive cells out to define responder cells. Several important proliferation related indexes were obtained by ModFit software, such as precursor frequency and proliferation index, etc. CONCLUSION: Our results show that application of vital dye CFDA-SE and SNARF-1, combined with flow cytometry is a powerful tool for evaluation of responsive intensity in one-way MLR.

Animals↗

PubMed2005

Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

AIM: To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). METHODS: The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. RESULTS: A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. CONCLUSION: PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

Adult↗

PubMed2005

[Development of a method for isolation, culture and proliferation of mouse NK cells].

AIM: To establish a method to isolate, purify, and culture mouse nature killer (NK)-cells in vitro. METHODS: NK-cells were isolated from splenic mononuclear cells (MNC) by a two-step adherence system and magnetic microbeads actived cell sorting (MACS), then these cells were cultivated with feeder cells and IL-2 in RPMI 1640 medium for 5, 10, 15, and 20 days. The enriched cells were counted and stained with anti-mouse CD3-FITC and anti-mouse NK1.1-PE. The purity of the NK-cells was determined by flow cytometry and the cytotoxicity to YAC-1 targets was detected by MTT assay. RESULTS: In the two-step adherence system, the enriched cells cultivated for 5, 10, 15, and 20 days were 0.5 x 10(7), 1.4 x 10(7), 2.6 x 10(7), and 3.0 x 10(7) respectively, and the percent of CD3(-) NK1.1(+) cells was 18.36%, 43.44%, 55.68%, and 60.03% respectively. The cytotoxicity to YAC-1 targets was significantly higher than that of splenic MNC(P<0.01), and increased from 41.93% up to 54.38%, 66.54%, 79.38%, and 83.86% respectively at 25:1 of effector:target ratio. After NK-cells were purified by MACS, the purity reached 93.60%ls, the enriched cells was 1.5 x 10(6), but proliferated to only 1.9 x 10(6) 20 days later. CONCLUSION: NK-cells isolated by using the two step adherence system proliferate abundantly, and the purity reaches 55%-60%. The cytotoxicity to YAC-1 targets is about 80% at 25:1 of effector:target ratio.

Animals↗

PubMed2005

[Effects of berberine on DNFB-induced delayed type hypersensitivity in mice].

AIM: To investigate the effect of berberine (Ber) on DNFB-induced delayed type hypersensitivity (DTH) and explore the mechanism of the immunosuppression effect of berberine. METHODS: BALB/c mice were divided into three groups: control group, DTH group, and Ber-treated DTH group. The DTH group was sensitized to DNFB by painting the shaved abdomen with DNFB dissolved in propanone/olive oil. The Ber-treated DTH group was injected (i.p.) with berberine daily for 7 consecutive days, with the total dose of 30 mg/kg. The control group was treated with the solvent without DNFB. All mice were then challenged by painting 10 microL of 2 g/L DNFB on the left ears. The weight of right and left ears was measured at 48 h after challenge. Histological changes of mouse auricles were observed under light microscopy. Violet crystal staining was used to determine the effect of Ber on adhesion of mouse splenic lymphocytes to extracellular matrix (ECM). Annexin-V staining was used to detect the apoptotic rate of the lymphocytes in the lymph nodes. RESULTS: The results of ear weighting showed that Ber significantly inhibited DTH reaction (P<0.05). Histological observation indicated Ber reduced markedly the infiltration of lymphocytes. The adhesion of T lymphocytes to ECM was also notably decreased by Ber treatment (P<0.05). There was no obvious difference in apoptotic rates of the lymphocytes among Ber-treated DTH group, DTH group and control group (P>0.05). CONCLUSION: Ber can significantly inhibit the DNFB-induced DTH in mice. The reduced adhesion between mouse T lymphocytes and ECM may be one of the mechanisms of suppression of DTH by Ber.

Animals↗

PubMed2004

[Expression of CTLA4-EGFP fusion protein in K562 cells and its subcellular localization].

AIM: To construct a eukaryotic expression vector for cytotoxic T lymphocyte antigen-4 (CTLA4) gene fused with enhanced green fluorescent protein(EGFP) gene and to analyze the expression and subcellular localization of the fusion protein in K562 cells. METHODS: The human CTLA4 gene was cloned by RT-PCR and was then inserted into plasmid pEGFP-N1 to construct the expression vector for CTLA4-EGFP fusion gene. The expression and subcellular localization of the fusion protein in transfected K562 cells were analyzed by flow cytometry and confocal microscopy, respectively. RESULTS: The cDNA of CTLA4 gene was cloned from human peripheral blood cells. The expression vector for CTLA4-EGFP fusion gene was constructed by PCR through introducing a Kozak sequence before the initiation site and deleting the stop codon. Flow cytometry analysis revealed that the proportion of both CTLA4+ and EGFP+ K562 cells was 16% at 24 h after the transfection. Confocal microscopy observation showed that the expressed fusion protein was mainly distributed in the intracellular compartments and had small part on the cell membrane. In contrast, only EGFP expression could be detected in K562 cells transfected with empty vector with diffuse distribution in the cells. Moreover, the expression level of the fusion protein on the cells increased to 29% after stimulation with phorbol ester and ionomycin. In addition, the fusion protein inside the cells tended to move towards and fused with the cell membrane. CONCLUSION: The expression vector for CTLA4-EGFP fusion gene has been constructed successfully and expressed in K562 cells. The expressed fusion protein has similar subcellular localization and transport characteristics to native CTLA4 in activated T cells.

Abatacept↗

PubMed2004

[Cloning of the cDNA of human PD-L1 gene and the expression of its extracellular domain in Escherichia coli].

AIM: To clone human PD-L1 cDNA and construct a prokaryotic expression vector for the extracellular domain of PD-L1 gene. METHODS: The human PD-L1 cDNA was cloned by RT-PCR from the total RNA of activated human peripheral lymphocytes. The prokaryotic expression vector for the extracellular domain of PD-L1 gene was constructed and protein expression in E.coli strain BL21(DE3) was determined. RESULTS: The full-length gene coding sequence of PD-L1 was cloned and confirmed by DNA sequencing. The prokaryotic expression vector for the extracellular domain of PD-L1 gene fused with His(6) tag at the C-terminus was then constructed. The recombinant protein was expressed in E.coli after IPTG induction and identified by Western blot. The expressed product had a relative molecular mass (M(r)) being 22 000, which was consistent with theoretical value. CONCLUSION: The PD-L1 gene was successfully cloned and the extracellular domain of the protein was expressed in E.coli, which lays the foundation for further study of the function of PD-L1.

Antigens, CD↗

PubMed2004

[Effect of natural killer cell on hematopoiesis and immunity recovery in mouse allogeneic bone marrow transplantation].

OBJECTIVE: To study the effect of natural killer (NK)-cell on reconstitution of hematopoiesis and immunity in mouse allogeneic bone marrow transplantation (allo-BMT). METHODS: Lethally irradiated BALB/c (H-2(d)) mice were transplanted with C57BL/6 (H-2(b)) bone marrow plus peripheral T cells and/or NK cells. Recipients CD34(+) cells and H-2K(b+), CD3(+) and CD19(+) cells were detected by flow cytometry, peripheral white blood cell (WBC) by auto-cytometry, and the survival rates, engraftment, hematopoietic and immune recovery were observed. RESULTS: In the transplantation with NK cell infusion group, the survival rates, the WBC and CD34(+) cell counts, and the H-2(b+) and CD19(+) cells were significantly higher than that in without NK cell infusion group (P < 0.01). Twenty-eight days after transplantation, the CD3(+) cells in the NK cell infusion group [(33.69 +/- 3.36)%] were lower than that in without [(50.4 +/- 5.06)%] (P < 0.01), and there was no longer difference between these groups 60 days after transplantation (P > 0.05). CONCLUSION: In mouse allo-BMT, alloreactive NK cell enhances engraftment, promotes reconstitution of hematopoiesis and immunity and increases survival rates.

Animals↗