| Part I: Observation on antitumor effect of mobilizing activated related HLA-haploidentical allogenic mononuclear cells in treatment of the metastatic or chemoresistant solid tumor patientsobjective To study the feasibility of activated related HLA-haploidentical allogenic monocytes in treatment of the metastatic or chemoresistant solid tumor patients. Methods 11 malignant solid tumor patients were enrolled with haploidentical relatives as donors. After mobilization with 10μg/kg-d of G-CSF for 3 days, haplo-HSCs were collected and activated by high dose of rhIL-2. Phenotypes and lysis activity of haplo-HSCs were compared by flow cytometry and LDH method. Multiple cytokines secreted in serum were examined by ELISA. Clinical effects were evaluated by CT scan. Results In 11 patients, 2.5-4.3 × 10~10 activated haplo-HSCs each was transfused. After mobilization, the proportion of T cells, especially CD4~+ T cells decreased and the proportion of NK cells increased which showed statistical significance. But after activation with high dose of rhIL-2, except a dramatic elevation in the proportion of activated cells, which highly expressed CD69 and CD25, no changes were found in the proportions of the other subsets. In LDH test, potent LAK-liked non-specific tumor lysis activity was observed against four solid tumor cell lines. After treatment, the concentration of Thl type cytokines, such as IL-2, IL-12,IFN-γ and TNF-α, rose significantly while the contents of Th2 type cytokines TGF-β dropped Eight of 11 patients showed regression in symptoms, tumor markers or CT scan after treatment, one of which reached PR 4 months post treatment Progression was observed in 2cases. Followed up to 1 year, six of 11 patients remained stable and survived, 5 cases diedo Conclusion: IL-2 activated haplo-HSCs possessed considerable antitumor effect, which could give a new light on treatment of the metastatic or chemoresistant solid tumor patients.Part II: Feto-Maternal Microchimerism between Donors and Recipients improves antitumor effect of activated related HLA-haploidentical allogenic monocytes Objective: To study the possible influences of the feto-matemal microchimerism in donors and recipients on antitumor effects of activated HLA-haploidentical allogenic monocytes. Methods: Genomic DNA samples were extracted from 30 pairs of patients and related donors who received activated haplo-HSCs treatment The feto-matemal microchimerism was analyzed by nested PCR-SSP typing. Then the survival time of donor cells transfused into patients who are feto-matemal microchimerism(+) were compared with those feto-matemal microchimerism(-) by FISH and STR-PCR method. And differences in cytokines secreted in serum were identified by ELISA method. Results: The purity of extracted genomic DNA was (1.843+0.136) with concentration of (213.7+116)ng/ul. A distinct 272 bp product was obtained after fist cycle PCR amplification with the general primers of HLA-DRB. The sensitivity and the specificity of nested PCR-SSP typing is relatively higher that tradional PCR-SSP typing that feto-matemal microchimerism could be identified even in the four times diluted genomic DNA sample. The positive rate of feto-matemal microchimerism in maternal/children donation is 40% but none inpaternal/children donatioa The survival time of donor cells transfused could last about 3 months detected by FISH in a feto-matemal microchimerism(+) patient, and the Thl-type of cytokines remained at die relative high level for more days. Conclusion: The feto-Matemal Microchimerism in Donors and Recipients improves antitumor effect of activated related HLA-haploidentical monocytes.Part HI: Mixed-chimerism in Recipients favors the antitumor effect of activated MHC-haploidentical donor lymphocytesObjective: To study the possible mechanism of mix-chimerism effecting on donor lymphocytes infusion (DLI) through a mice model of MHC-haploidentical allogeneic stem cell transplantation. Methods: Mice were treated with different doses of radiotherapy (9Gy and 6.5Gy) as meyloablative or non-meyloablative conditioning regimens respectively, followed by MHC-haploidentical allogeneic stem cell transplant The kinetics of allogeneic transplanted cell tagged with P32 in vivo, the activity of DLI anti-tumor by LDH and the effects of different chimerism status to DLI by LDH were measured. Results: A myeloablative response was reached under the 9Gy conditioning regimens and there was non-myeloablative response under the 6.5Gy regimens. The complete and stable chimerism was revealed in the myeloablative group. However, there was just mix-chimerism in the non-myeloablative group, with the ratio of chimerism decreasing overtime, resulting in less than 5% within one month.MCH-haploidentical allogeneic stem cell transplantation could just inhibit the tumor growth and prolong survival. But the tumor volume was almost reduced to 50% in the group which was treated with activated DLL There was no significant diflference in tumor growth between the 22% chimerism group and 10% group, and there was no effect to the allogeneic cell kinetics in the myeloablative or non-myeloablative group. Conclusion: It is necessary to get a mix-chimerism for the effect of graft-versus-tumor of MHC-haploidentical allogeneic stem cell transplantatioa... |
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