[Establishment of A Novel Method to Activate and Expand Human NK Cells Ex Vivo].

OBJECTIVE: To develop a new method to activate and expand human NK cells ex vivo by using sodium hyaluronate as a major activating agent and to explore its related mechanism. METHODS: Mononuclear cells were isolated from 3 samples of peripheral blood from three healthy donors. New NK cell culture method and the control method were used to culture NK cells from each samples separately for 14 days. Flow cytometry was used to analyze the ratio of NK cells and CD69 expression. To measure the in vitro cytotoxicity of NK cells cultured by the two methods, the K562 cells were used as the targeting cells and flow cytometry combined with CFSE marker was used as the testing method. RESULTS: After culturing for 14 days, the number of NK cells obtained by new culture method for NK cells expanded by 188.63±3.83 times while the number of NK cells cultured by control method expanded by 152.77±5.77 times. The ratio of NK cells in new cell culture method was above 90%, while the ratio of NK cells in control method was about 70%. The ratio of CD69+ NK cells in new cell culture method was 32.37%±3.22%, while the ratio of CD69+ NK cells in control method was 17.29%±3.79%. The results of cytotoxicity experiment in vitro showed that NK cells cultured by the new method had a higher killing ability to the target cells as compared with NK cells cultured by the control method. CONCLUSION: New NK cell culture method using sodium hyaluronate as a major activating agent can expand NK cells more efficiently as compared with the cells cultured by control method, which may be related to the direct and/or indirect activation of sodium hyaluronate to NK cells, further causing the dominant expansion of the NK cells.

[The role of third-party tolerogenic dendritic cells in the prevention of acute graft-versus-host-disease following allogeneic bone marrow transplantation in mice].

OBJECTIVE: To explore the biological characteristic of third-party-derived tolerogenic DC(tDC) and the influence of third-party-derived tDC on acute graft-versus-host-disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) in mice. METHODS: tDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-ß1D1. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4(+)T cells proliferation in vitro. Different doses of D1-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Th1/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control. RESULTS: tDC expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86 and CD40, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-ß, Fas Ligand, indoleamine 2, 3-dioxygenase (IDO) and arginase. In the allogeneic MLR, third-party tDC suppressed allo-CD4(+)T cells proliferation, which was relative to the dose of tDC. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 10(4) third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 10(3) cells, only 20% of mice survived day 60, and when increased tDC to 10(5), all of the mice died within day 37 after allo-BMT. The cytokine levels in serum indicated that 10(4) tDC-treated mice secreted in vivo high level of IL-10 21d after BMT (P < 0.05), the levels of IL-10 in 10(3), 10(4) and 10(5) tDC-treated mice were (114.23 ± 7.78), (646.18 ± 212.02), (121.97 ± 10.47) ng/L, respectively. CONCLUSION: Third-party tDC could suppress allo-CD4(+)T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-ß1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.

[Using real-time PCR to evaluate the effect of viral inactivation by Methylene Blue with visible light].

To investigate the feasibility of using Real-Time PCR to evaluate the effectiveness of Sindbis virus inactivation by Methylene Blue with visible light. Sindbis virus was treated by Methylene Blue with different intensity of visible light and the transcribed cDNA was quantified by Real-Time PCR. Residual infectivity of treated virus was tested by cell infection method as parallel control at the same time. The residual infectivity of virus decreased from 6.50 lgTCID50/mL to under the limit of detection as light intensity increased. Meanwhile, the quantity of virus cDNA decreased significantly (P < 0.05), which correlated to the decline of virus infectivity (R2 > 0.98). Methylene Blue with visible light could cause lesion to nucleic acid of Sindbis virus, the extent of which was light intensity-dependent and correlated to the decrease of virus infectivity. The results demonstrated that Real-Time PCR can be a useful tool for evaluating effect of virus inactivation after Methylene Blue treatment with light.

HLA-A, -B, -DR haplotype frequencies from DNA typing data of 26,266 Chinese bone marrow donors.

HLA phenotypes of 26,266 Chinese individuals who were recruited as potential hematopoietic stem cell donors by the Shanghai Red Cross Marrow Donor Registry, part of the China Marrow Donor Program, were determined for HLA-A, -B, and -DRB1 alleles at low to intermediate resolution using DNA-based typing methods. The large sample size of the study allowed accurate calculation of the Chinese HLA haplotype frequencies. The observed alleles correspond to 19 HLA-A, 44 -B, and 13 -DR split antigens. The serologic equivalents of HLA-A36, -A80, -B78, and -DR18 alleles were not observed. A total of 2,241 distinct HLA-A, -B, -DRB1 haplotypes were identified. Three-locus haplotype frequency was estimated using the maximum likelihood method. The lowest haplotype frequency that can be reliably estimated at a 95% confidence level was 0.000057. Using this cutoff value, 1,220 haplotypes (54%) were statistically reliable and their cumulative haplotype frequency was 0.9730. The cumulative haplotype frequency of the remaining 1,021 haplotypes (46%) was 0.0270. A regression equation of p = 0.192 log N - 0.576 was derived to estimate the probability (p) of finding an HLA-A, -B, -DR split antigens-matched donor in a pool of N Chinese donors.

Impact of transfection with total RNA of K562 cells upon antigen presenting, maturation, and function of human dendritic cells from peripheral blood mononuclear cells.

BACKGROUND: Vaccination of dendritic cells (DCs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. The purpose of this study was to investigate whether human monocyte-derived DCs were able to present P210(Bcr-Ab1) protein and induce antigen-specific cytotoxic T lymphocyte (CTL) responses in vitro after transfected with total RNA of K562 cells (K562-RNA). STUDY DESIGN AND METHODS: DCs derived from human peripheral blood mononuclear cells were transfected with K562-RNA with electroporation or DOTAP lipofection. The successful transfection was determined by reverse transcription-polymerase chain reaction and Western blot. The phenotypes of the DCs were analyzed by flow cytometry (FCM), and cytotoxicity of CTL was assessed by propidium iodide staining followed by FCM analysis. The CD1a expression and purity of DCs were measured by FCM. RESULTS: The Bcr-Abl fusion gene was detected in the DCs with 24 hours after the transfection. The transfected cell expressed increased levels of CD80, CD83, CD86, and HLA-DR. Moreover, the transfected DCs strongly stimulated the T lymphocytes to gain cytotoxic activity against K562 cells. Culture medium containing 1 percent human plasma was the most effective for DC growth. CONCLUSION: Human DCs transfected with K562-RNA effectively induce specific immune responses. This method can be used to induce tumor-specific immune response and may have potential application in immunotherapy of tumors.