| Objectives: The present study is aimed to explore whether the graft-versus-host disease (GVHD) could be decreased and graft-versus-leukemia effects could be retained by syngeneic mixed with granulocyte colony-stimulating factor- mobilized H-2 haploidentical marrow grafting. Part I Experimental study of G-CSF moderating GVHD after Mixed bone marrow transplantationMethods: Female BALB/c mice and neonatal BALB/c mice were recipients and male (BALB/cxC57BL/6) Fl (BCFi) mice were donor mice. Donor mice were injected subcutaneously with G-CSF daily at 0.01ug/g body weight or saline (control diluents) for 6 days, and splenocytes were harvested on day 6. By using of spleen enlargement of neonatal mice and mortality of lethally irradiated (60Co, 8.5Gy) adult mice after the injection of different mixed spleen cells or different mixed marrow cells. GVL effects were determined by observation of survival time, survival rate of the mice; whereas GVHD was assessed by observing signs of weight loss, ruffled fur, diarrhea and histological change of skin, liver and small intestines. Allogeneic chimerism was detected using cytogenetic analysis. Enzyme-linked immunosorbemt assay (ELISA) method was used to detect cytokines (IL-2, IL-4, IFN-y). Fluorescence-activated cell sorting (FACS) analysis was used to detect T cell phenotype.Results(1) The neonatal mice received 2:1 and 1:1 mixed spleen cells and H-2 haploidentical spleen cells all detected GVHD signs. The neonatal mice received 7:1-3:1 mixed groups didnt show signs of moderate GVHD .The severity of acute GVHD in mice receiving G-CSF-mobilized splenocytes was dramatically reduced.(2) The GVHD signs and histological change were observed in most mice of 4:1-1:1 MBMT groups. The mice of Syn-BMT group and 5:1MBMT group had no GVHD signs and histological changes. However, the survivalperiod of G-CSF- mobilized MBMT groups was longer than control diluents groups (P<0.01) and these mice did show signs of moderate GVHD.(3) The absolute number of Thyl.2+ and Iyt2+ cells was decreased and relative ratio in both subsets was come down in G-CSF-treated donor mice.(4) The supernatants from a primary MLR were collected at 48 hours for cytokine measurement. The results showed an increased production of IL-4 and a decreased production of IL-2 and IFN-y after stimulation with ConA for 48 hours.Part II Experimental study of G-CSF enhancing GVL effects afterMixed bone marrow transplantationMethods: Female L615 mice were recipients and male (615 x BALB/c) Fl (6BF1) mice were donor mice. Lethally irradiated L615 mice were given a lethal dose of L615 leukemia cells along with syngeneic marrow or a mixture of syngeneic plus G-CSF-treated (control diluents) H-2 haploidetical marrow cells.Results(1) The mice merely received L615 leukemia cells were all died of leukemia. The L615 mice of 3:1 and 4:1 MBMT groups survived longer than those post syngeneic BMT (P<0.01).(2) The survival time of mice in the G-CSF-treated MBMT groups was longer than that of the pure MBMT groups. MBMT of G-CSF-treated donors could markedly increase the survival rate of L615 leukemia mice of MBMT (P<0.01) without clinical evidence of GVHD.(3) As the post-transplanted time prolonged, the rate of allogeneic chimerism were decreased. As the mice died of leukemia relapse, the chimerism rate decreased to zero.(4) Splenocytes from recipients at day 14 post-MBMT after G-CSF pretreatment donors showed an increased production of IL-4 and a decreased production of IL-2 and IFN-y.Conclusions1.The GVHD could be reduced using syngeneic mixed with H-2 haploidentical marrow cells.2.The severity of acute GVHD in mice receiving G-CSF-mobilized splenocyte or marrow cells could be further moderated.3.Syngeneic mixed with H-2 haploidentical marrow transplantation had a potential way to increase GVL effects, and this GVL effects could be enhanced by G-CSF pretreatment in MBMT.4.Improved survival in recipients of G-CSF-treated donors is associated with increased IL-4 pr...
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