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Currently, biological immunotherapy is the main treatment for removing residual leukemic cells^[1]. The variations of precursor cells, cytokines, combined application of cytokines and culture conditions lead to different culture results, such as quantity of dendritic cells (DCs), maturation and phenotypic expression^[2-3]. How to obtain enough functional DCs faces many difficulties, especially the reports regarding immunomodulator with low toxicity are few. Mycobacterium phlei F.U.36 (Utilins) is one kind of inactivated mycobacterium suspension injection^[4]. In addition, inactivated Mycobacterium phlei DNA can directly inhibit cell proliferation and induce apoptosis in cancer cells. It is not yet reported whether Utilins could affect the proliferation of DCs. In this study, we evaluated the effect of Utilins on proliferation of DCs derived from human umbilical cord blood in vitro.

DCs are not only the most potent antigen presenting cells in the body, but also act as the main initiators of the antigen-specific immune response, which makes it play special role in the process of anti-tumor and anti-infection immunity^[8-9]. DCs vaccine and combination of DCs with other cytokines (such as interleukin-2, interleukin-12) are under extensive research in recent years^[10-11]. In particular, some research groups have begun phase Ⅰ of clinical trial. Yamanaka et al^[12] selected the glioma cell lysates from 7 patients with multiform glioblastoma and combined them with DCs to form DCs vaccines. After vaccinating and doing operation again, a number of immunoresponse cells, such as, CD8⁺ T cells, CD4⁺ T cells and CD56⁺ cells, were observed amid the tumor tissue specimens. DCs represent less than 1% of the peripheral blood circulating cells, so different origins to obtain and culture DCs in vitro have been described, such as, culture of umbilical cord blood^[13], bone marrow, purified peripheral blood monocytes or CD34⁺ progenitors cells in the presence of appropriate cytokines (GM-CSF, IL-6, FLT-3 and TNF-α)^[14-16]. Mature DCs high expressed MHC-Ⅱ, specific marker CD1a and CD83^[17]. Though some scholars achieved good results by importing cytokines into DCs by gene modification method, such as lentivirus trasfection^[18-19], the obviously diversity existed among different researches^[20]. High price of cytokines, few quantities of obtained DCs, and complex of MACS sorting technique limited the clinical application of DCs. Therefore, how to explore a simple and inexpensive way to acquire enough DCs in vitro plays a key role in widely application of DCs. Here, the cord blood mononuclear cells were cultured by Utilins in vitro. The effect of Utilins on amplification and maturity on DCs were observed. The smears Wright-Giemsa staining and inverted microscope observation showed with typical morphology can be detected in all the experimental groups, in particular in the Utilins group, the cell number and CD1a⁺, HLA-DR⁺ ratios of which were obviously greater than that of the control group, suggesting that Utilins can not only promote the proliferation of DCs, but also induce the maturity of DCs. Simultaneously, the CD1a⁺, HLA-DR⁺ ratios of the Utilins group was smaller than the cytokine group, which was less than the combination group, but there was no significant difference between the cytokine group and the combination group. It is suggested that the effect of Utilins on the proliferation of DCs is weaker than the combined effects of GM-CSF, TNF-α and IL-4, while Utilins has the ability to promote the mature effect induced by GM-CSF, TNF-α and IL-4. The mechanism by which Utilins promotes the proliferation and maturity of DCs is partially referred to stimulate the production of IL-4, TNF, INF and CSF, while the mechanisms in detail remain to be further studied. Compared to some cytokines, such as IL-4, GM-CSF and TNF-α, Utilins is characterized by safe and low cost, so this experiment may provide an experimental basis to explore more simple and inexpensive program for DCs proliferation.

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