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Objective@#To investigate the safety and efficacy of haploidentical hematopoietic stem cell transplantation with different intensity conditioning regimen in the treatment of childhood aplastic anemia (AA) .@*Methods@#Thirty-seven AA patients who underwent haploidentical transplantation in BaYi Children's Hospital Affiliated to PLA Army General Hospital from January 2013 to January 2017 were enrolled. According to the dosage of conditioning regimen, 34 patients excluding 3 other conditioning regimens were divided into high-dosage group (regimen 2, 22 cases) and low-dosage group (regimen 3, 12 cases). The data of Engraftment, graft-vs-host disease (GVHD), hematopoietic reconstitution, relapse, infection, overall survival (OS) were analyzed. The comparison between the two groups was tested by χ2 test.@*Results@#A total of 35 of 37 patients achieved primary engraftment; 2 cases died of regimen-related toxicity and severe infection before the infusing of the grafts. The activation rate of CMV and EBV was 60% (21/35) . Post-transplant lymphocyte disease (PTLD) of lung occurred in one case. The cumulative incidences of acute GVHD grade Ⅰ-Ⅳ and chronic GVHD were 29% (10/35) and 34% (12/35) respectively and the incidence of extensive chronic GVHD was 6% (2/35) . The median follow-up time was 18.8 (2.9-44.1) months, the OS was 92% (34/37) .All survived patients were no longer dependent on blood transfusion and none of them had recurrence. Comparing the rates of overall survival(86%(19/22) vs.100%(12/12)) and rates of chronic GVHD(40%(8/20) vs. 17%(2/12)) in regimen 2 and regimen 3 group, there were no significant difference (χ2=1.742, 1.841, all P>0.05) . Significant difference was found at the incidence of Ⅰ-Ⅳ acute GVHD (10% (2/20) vs. 50% (6/12) ,χ2=6.200, P=0.013).@*Conclusions@#Haploidentical hematopoietic stem cell transplantation is effective and safe. It is suitable for patients who are not eligible for matched donor transplantation. Application of reduced dose preconditioning in haploid transplantation is worth exploring.
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Objective To explore the efficiency and prognosis of hematological malignancies treated by haploidentical hematopoietic stem cell transplantation.Methods 70 patients who received haploidentical hematopoietic stem cell transplantation were analyzed retrospectively.According to tumor burden before transplantation,the patients were divided into three groups,the low tumor burden group,the mediate tumor burden group and the high tumor burden group.And then the effection of the tumor burden to survival was analyzed,and the engraftment,GVHD,infection,conditioning related toxicity,relapse and survival rate were also observed.Results The follow-up was terminated on January 1,2014.Follow-ups were performed for a median of 34.05 (7.4-83.6) months after transplantation.All patients achieved engraftments.The cumulative incidence of GVHD of grades 2-4 was 47.14 ％ (33/70) and that of grades 3-4 was 21.4 ％ (15/70).The chronic extensive GVHD was 20.0 ％ (14/70).The overall survival was 68.6 ％.Transplant-related mortality was 12.8 ％ and the relapse was 18.6 ％.The overall survivals in low tumor burden group,mediate tumor burden group,high tumor burden group were 91.67 ％,72.7 ％,33.3 ％ respectively.By SPSS 20.0,tumor burden was the high risk factor affecting the survival (low tumor group vs high tumor group,mediate tumor group vs high tumor group,low tumor group vs high tumor group,P =0.000,P =0.038,P =0.016).Conclusions Haploidentical hematopoietic stem cell transplantation in hematological malignancies is safe and effective.And for hematological malignancies with poor prognosis disease,it should be accepted the HSCT as soon as possible after remission in order to reduce the recurrence rate of malignancy.
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Objective:To evaluate the expression of CXCR4 and the migration rate of bone marrow stromal CD34+cells in differ-ent risk groups with myelodysplastic syndromes (MDS) using correlation analysis. Methods: Forty MDS patients were divided into low-and high-risk groups based on the International Prognosis Scoring System (IPSS). The former was composed of 20 patients with IPSS0.05). The migration rate of CD34+cells on the ef-fects of SDF-1αand marrow stromal cells were significantly increased in the high-risk MDS group compared with those in the low-risk and control groups (P<0.000 1). Migration rate of CD34+cells on the effect of marrow stromal cells was positively correlated with CX-CR4 expression (P=0.000 1). Conclusion:The CXCR4 expression and migration rates of CD34+cells on the effect of marrow stromal cells are significantly higher in the high-risk MDS group than in the low-risk group. Migration rate has a positive correlation with the CXCR4 expression, which further indicates that MDS is a heterogeneous group of hematopoietic stem cell malignancies. The expres-sion and function of SDF-1 and its receptor CXCR4 differ within each group with various risks. SDF-1 and CXCR4 may be involved in MDS pathogenesis.
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Objective To investigate early Epstein-Barr virus (EBV) reactivation and the outcome of preemptive therapy after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods From January 2007 to January 2009, totally 277 patients after allo-HSCT were studied (haploidentical 116,unrelated 75, matched sibling 86). Conditioning regimens were mainly busulfan (BU) + cyclophosphamide ( CY)/fludarabine(Flu) or total body irradiation (TBI) + CY/Flu. Antihuman thymocyte globulin (ATG)was added in haploidentical and unrelated transplants. Plasma EBV DNA was monitored once to twice weekly in the first 3 months after allo-HSCT with real time quantitative polymerase chain reaction (RQ-PCR). EBV viremia was diagnosed when EBV DNA was more than 5 × 102 copies/ml but without symptoms. Acyclovir (10 mg/kg, intravenous drip, 8 h) was used for preemptive therapy and immnuo-suppressants were decreased if possible. Results Totally 33 patients ( 11.9% ) developed EBV viremia with a median time at day 44 (day 19 to day 84). The incidences of EBV viremia in the transplants from matched sibling,haploidentical, unrelated donors were 0, 15.5%, 20. 0%, respectively. There was no significant difference between haploidentical and unrelated transplants ( P = 0. 09 ), but much less EBV viremia was seen in matched sibling transplant ( P = 0. 001 ). Twenty of 33 patients ( 60. 6% ) had complete response to preemptive therapy. The median time to reach EBV DNA negative in plasma was 11 (4-56) d. The median duration of preemptive therapy was 21 (14-60) d. Both univariate and multivariate analysis indicated that haploidentical and unrelated transplants, acute graft versus host disease (GVHD) were the risk factors for EBV viremia. Two-year overall survival in the patients with EBV viremia was significantly lower than that without EBV viremia (54. 2% vs 72. 1%, P = 0. 006 ). Conclusions Our large clinical study has demonstrated that preemptive therapy with acyclovir that is guided by EBV viremia is effective in majority of the patients with high-risk for EBV reactivation after allo-HSCT, which may further decrease the risk for developing life-threatening EBV disease or post-transplantation lymphoproliferative disorder. Haploidentical and unrelated transplants, acute GVHD are the risk factors for EBV viremia which has negative impact on survival.
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Objective To determine the antigen-specific CTL in PBMC induced by a fusional family-gene vaccine of the immunoglobulin heavy chain variable gene framework region combined with the sequence of cytokine CM-CSF in vitro with MHC pentamer. Methods Peripheral blood samples were collected from two healthy donors and two patients. One was follicular lymphoma and another was hair cell leukemia. PBMC were isolated by density gradient centrifugalization with Ficoll and then subsequently differentiated into immature DCs (imDCs) induced by recombinant human GM-CSF and recombinant human IL-4. Gene gun was used to deliver the plasmids of the gene vaccine or the control plasmids into the imDCs. RT-PCR and ELISA assay were used to detect IgHVl-GM-CSF mRNA and GM-CSF in order to validate the transfection of the vaccine. After adding the cytokine cocktail, the imDCs became mature DCs. Then the mature DCs were co-cultured with lymphocytes from the blood samples for the induction of the antigen-specific CTL. The cultured cells were classified into vaccine group and control group and harvested at different time points of 0 d,7d, 17 d and 24 d after transfection. The subset of CD3+CD8+ T cells was analyzed by FCM assay. Finally, the CTL levels were detected with fluorescently labeled MHC pentamer antibody targeting vaccine epitopes. Results With the induction of cytokines, the imDCs with typical morphology were generated in PBMC. After delivering, the efficient expressions of the vaccine in the imDCs were determined by RT-PCR. And ELISA results also confirmed that GM-CSF was produced at a level of (28 ±6) ng/106 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group (10 ± 3) ng/106 cells (t = 5. 191, P <0.01). FCM assay result showed that the CD3+ CD8+ T cells increased in a stepwise pattern during the culture. For control group, the levels at 0 d,7d, 17d and 24 d were ( 34. 24 ± 2. 72 )% , (46.06 ± 3.08)%, ( 65. 34 ± 4. 26 )% and (73.86 ±4.85 )% , respectively. For vaccine group, the results were (32. 28 ± 2. 08 ) % , (45. 32 ± 3. 81)% , ( 63. 37 ± 4. 21)% and (75. 01 ±3. 20)%. The differences between each time point had statistical significance (F = 176. 966 ,P <0.01) ,but there was no statistical differences between vaccine group and control group ( F = 0.657,P>0.05). The MHC pentamer analysis showed that the DCs loaded with IgHV1-GM-CSF fusional vaccine could efficiently induce the antigen-specific CTL response and the CTL levels increased gradually with the culture time, with the highest level of 4. 36% in the lymphoma blood and 3. 89% in the hair cell leukemia blood. Conclusions MHC pentamer assay could efficiently determine the antigen-specific CTLs response induced by the gene vaccine of family IgHV frame region in vitro. It could be a useful method for monitoring of anti-tumor cell immunity and evaluating of diagnosis and prognosis of the tumors in clinical application.
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Objective To establish a method for detecting chimerism after allogeneic stem cell transplantation using microsatellite polymorphism. Methods DNA was extracted from bone marrow or peripheral blood of 10 recipients and their donors, DNA fragments including 5 microsatellite loci were amplified by PCR, the polymorphism of PCR products was analyzed by PAGE and sliver staining, and the quantitative analysis of chimerism was performed using image analysis software. Results [WTBZ]The five selected STR loci had high polymorphism, and were suitable for detecting chimerism. The sensitivity of sliver staining was 90%. Conclusion Microsatellite polymorphism analysis based on PCR is a sensitive and accurate method to detecting chimerism after allogeneic stem cell transplantation, but the sensitivity of sliver staining was not so good,so more sensitive methods should be used.
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Retrovirus (RV) is the most widely used vector for gene transfer, but its efficiency is very low for human cells. A Flow-through Transduction System made in General Hospital of PLA was used to deliver interleukin-12 RV vector into human leukemia cell line K562. Compared with static procedure, the efficiency could be significantly improved,and detected by clony forming units (CFU) for NeoR gene in semisolid culture medium with G418 (83.1%±3.9% vs 9.2%±2.3%, P＜0.01). No significant difference was found in the levels of IL-12 p70 protein expression detected by ELISA, by flow-through transducted cells without G418 selection and by statically-transducted cells with G418 selection (11.3±7.5ng/ml vs 12.0±6.4ng/ml, P>0.05), while statically transducted cells without G418 selection could express very low IL-12 (0.1±0.024ng/ml，P＜0.01)。The Flow-through Transduction System is efficient and easy for use, it can be widely used in research and clinic of gene therapy.
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Objective To explore the difference of immunosuppresive effect between the expanded Stro-1+ and Stro-1-subgroups of human mesenchymal stem cells in vitro. Methods Mononuclear cells (MNCs) were isolated from bone marrow (BM) samples and seeded in a T-75 cm2 tissue culture flask contained with Dexter medium. When 50% confluence was obtained, adherent cells were collected and incubated with anti-stro-1 antibody, and the Stro-1+ and Stro-1-MSCs were further seeded for expansion. The total culture time (median) was 15 days. Cells were then analyzed by flow cytometry. One-way mix lymphocyte reaction (MLR) (1?105 responding cells and an equal number of stimulating cells/well were co-cultured in 96-well plates) and nonspecific mitogenic stimuli phytohemagglutinin (PHA) plus interleukin-2 (IL-2) induced lymphocytes proliferation (PBL 1?105/well were mixed with PHA 10?g/ml and IL-2 500U/ml in 96-well culture plates) were established in vitro. 1?103-3?104 irradiated Stro-1+ MSCs and Stro-1-MSCs were added into the two systems at the beginning of reaction. Immunosuppressive actions of both Stro-1+ or Stro-1-MSCs were compared. Results Adherent cells contained a median of 9% (range 5%-26%) Stro-1+ cells, which expressed higher immunophenotype of MSCs. In both reaction systems, suppressive actions occurred in a dose-dependent fashion when whatever Stro-1+ MSC or Stro-1-MSC were added. However, that the addition of 1?103 Stro-1-cells enhanced rather than inhibited the lymphocyte proliferation in one-way MLR. In the presence of various concentrations of MSCs, Stro-1+ MSCs always showed a significantly increased inhibitory effects in comparison to Stro-1-MSCs (P
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35), while the expression level of TGF-?1 in Stro-1-MSC was significant higher than that detected in Stro-1+MSC (2-??Ct=0.39, P
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Objective To study the therapeutic efficacy of HSV-TK/GCV in acute graft versus host disease (GVHD) produced by infusion of H-2 haplotype matched CD34 + cells in combination with TK +T lymphocytes transplantation. Methods CD34 + cells were separated from bone marrow of male donor mice, and T lymphocytes were acquired from the spleen and transfected with retroviral vectors carrying tk gene. Forty-eight female F1 recipient mice were divided into six groups. Group 1 received 1?10 5 CD34 + cells, groups 2 and 3 received 1?10 5 CD34 + cells and 1?10 7 T lymphocytes, groups 4～6 received 1?10 5 CD34 + cells and 1?10 7 TK +T lymphocytes. GCV was administered to group 5 and group 6 in the dosage of 50mg/(kg?d) for 7 days by intraperitoneal injection on day 0 and 7 posttransplant, respectively. Results All the recipient mice in group 1 survived more than 30 days and no acute GVHD symptoms emerged. Acute GVHD symptoms appeared in the recipient mice in groups 2～6 showing typical pathological manifestations. The recipient mice in groups 2～4 died of acute GVHD 3 weeks posttransplant. The survival time of recipient mice in groups 5 and 6 was 26.6?4.8 days and 40.5?8.4 days, respectively, which was significantly longer than that of groups 2～4 (P
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Objective Clinical research was made to observe the relationship between idiopathic thrombocytopenic purpura(ITP)and Helicobacter pylori(ITP)and to evaluate the effect of HP eradication in the treatment of chronic ITP.Methods ~(14)C urea breath test were made in 17 patients with chronic idiopathic thrombocytopenic purpura,including 6 males and 11 females,aging from 15-48,with course of disease from 4 months to 8 years.Omeprazole,clarithromycin and amoxicillin were used for Helicobacter pylori eradication,and the effect was evaluated by platelet count.Results Thirteen out of 17 patients were HP positive,and eleven turned negative after HP eradication,eight of whom were observed platelet recovery.Conclusion Platelet count can be raised in some patients with chronic ITP after eradication of Helicobacter pylori.
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Objective To explore the application of particle-gun technique in transfecting dendritic cells(DCs),and investigate the expression of DNA vaccine particle IgHV1-GM-CSF in DCs induced by particle-gun.Methods Monocytes were isolated from donor peripheral blood collected by Ficoll density gradient method and adherent culture,and then differentiated into immature DCs(imDCs) by rhGM-CSF and rhlL-4 induction.The plasmids for transfection were IgHV1-GM-CSF/pcDNA3.0 and pEGFP,which were prepared with endofree plasmid purification kit.The DCs were transfected with plasmid pEGFP by particle-gun at different transfection conditions,the green fluorescence positive cell and the total cell numbers were counted respectively under fluorescence microscope,and the transfection efficiency was calculated;the viable cell count was performed after trypan blue staining;the transfection efficiencies of particle-gun,liposome-mediated transfection and electroporation method were compared.The plasmids IgHV1-GM-CSF/pcDNA3.0 and pEGFP were cotransfected into imDCs by particle-gun under the optimum conditions,and then DNA was extracted,the expression of IgHV1-GM-CSF was determined by RT-PCR,and of GM-CSF by ELISA.Results The optimum conditions of particle-gun transfection in imDCs were: gas pressure at 120psi,PVP concentration in 0.02mg/ml,microcarrier loading in 0.5mg gold/shot,and DNA loading ratio in 1.5?g plasmid/mg gold.The transfection efficiencies of particle-gun and electroporation were 10.5%?2.4%(n=3) and 45.2%?5.6%(n=3) respectively,while that of liposome-mediated transfection was very low,and significant difference existed among the three methods(P
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C57BL/6 synergistical mice were divided into 4 groups (10 mice each). The first group was negative control without any interference. Each mouse in the other 3 groups was subcutaneously inoculated with 1 10 6 wide type (wt) EL4 tumor cells. Then each group was treated either with interleukin 12 (mIL 12) in vivo vaccine or NeoR control vaccine or PBS(positive control group) on day 1, 7, 14, 21, on the same place where wt EL4 tumor was inoculated. mIL 12 in vivo vaccine and NeoR control vaccine were package cells which can produce retrovirus (RV) with mIL 12 or NeoR gene, the vaccine was 60 Co irradiated and injected (1?10 7 cells/mouse). All mice in positive group and NeoR control group died of tumors in a month, while 5/10 mice in mIL 12 in vivo vaccine groups survived without tumors for more than 60 days. The 5 survived mice were rechallenged with 5?10 5 wt EL4 cells, 3/5 mice even survived without tumors for another 60 days. Among the mice with tumors in vivo vaccine group mice, compared with the controls, the development of tumors was delayed ( P
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Objective To morphologically evaluate the bone marrow (BM) smear specimens collected from the patients with chronic myelogenous leukaemia (CML) in accelerated phase and blastic phase receiving a short term Gleevec therapy, and to determine its clinical significance in evaluating the changes in disease condition to guide the treatment. Methods Sequential BM smear specimens of 16 Ph positive CML patients, including 9 in accelerated-phase and 7 in blastic-phase, were examined before and 3,6 and 9 weeks after Gleevec treatment (0.4/d or 0.6g/d, PO) with routine method. Periodic acid-Schiff reagent (PAS) staining was performed for a proper identification of abnormal erythroid precursor cells. Results The treatment rapidly caused following conspicuous BM changes in the process: both cellular proliferation and neutrophil granulopoiesis decreased significantly, while the accumulation of erythroid precursor cells increased obviously, and megakaryocytes decreased markedly at 3rd week. A few patients showed no such changes, but an accumulations of erythroid precursor cells, leading to misdiagnosis in some one third of the patients. In 21 percent of patients, in whom no erythroid cells were found, were classified into accelerated phase and blast phase based on both WHO classification system and FAB system. In fact the increase in red system was a bone marrow response to anemia caused by Gleevec therapy. If a condition of severe cellular proliferation of BM with a lower WBC and platelets appeared, most patients could not tolerate the therapy, then it should be ceased. Conclusion Initial treatment with Gleevec therapy exerts pronounced changes in BM morphology, which can be used as a simple and effective method to evaluate the outcome of the patients.
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Retrovirus (RV) is the most widely used vector for gene transfer, but its efficiency is very low for human cells. A Flow-through Transduction System made in General Hospital of PLA was used to deliver interleukin-12 RV vector into human leukemia cell line K562. Compared with static procedure, the efficiency could be significantly improved,and detected by clony forming units (CFU) for NeoR gene in semisolid culture medium with G418 ( 83.1%?3.9% vs 9.2%?2.3%, P0.05), while statically transducted cells without G418 selection could express very low IL-12 (0.1?0.024ng/ml,P