Comparison of Haploidentical Hematopoietic Stem Cell Transplantation and Immunosuppressive Therapy for the Treatment of Acquired Severe Aplastic Anemia in Pediatric Patients.

This study aimed to compare the efficacy and safety between haploidentical hematopoietic stem cell transplantation (HHCT) and immunosuppressive therapy (IST) for the treatment of pediatric acquired severe aplastic anemia (SAA). The clinical data of 28 children with SAA treated from June 2010 to October 2014 at our hospital were retrospectively reviewed. Of these patients, 18 were treated with HHCT and 10 with IST. The median follow-up time was 23.5 months (range, 3-52 months). There was no significant difference in overall survival rate between the HHCT group and the IST group (66.7% vs. 70%, P > 0.05). Graft-versus-host disease occurred in 83.3% (15/18) of the HHCT group, including 5 cases with grade III or higher. In comparison with IST, HHCT has similar efficacy and safety profiles in the treatment of pediatric SAA.

[The effects of donor peripheral blood stem cell infusion in the prevention of relapse in high risk leukemia after haplotype hematopoietic stem cell transplantation].

OBJECTIVE: To explore the preventative effect of donor peripheral blood stem cell (PBSC) infusion mobilized by granulocyte colony-stimulating factor (G-CSF) for the relapsing patients with leukemia after haplotype hematopoietic stem cell transplantation (HSCT), as well as its therapeutic effect and safety. METHODS: G-CSF was given at 5 - 10 µg×kg(-1)×d(-1) to donor and PBSCs were obtained on day 5 and frozen and allotted for storing. PBSC infusion was given to all the 20 patients on day 90 after HSCT, and the second treatment was given to 4 patients on day 30 after the first infusion. The occurrence of graft-versus-host disease (GVHD), relapse rate of high risk leukemia and long-term survival were evaluate after PBSC infusion. RESULTS: A total of 19 patients had acute GVHD after PBSC infusion for a median of 25 (12 - 60) months, 4 of them were ≥ degree III. The cumulative incidence rate was 22.9%, and all of them accepted PBSC infusion twice. Thirteen patients had assessable chronic GVHD, 10 of them were restricted, and no one died of it. Nine patients died of relapse of leukemia. The remaining 11 patients survived leukemia free, including 4 with chronic myelogenous leukemia, 4 with acute myeloid leukemia (AML), 1 with lymphoma-leukemia and 2 with myelodysplastic syndrome-AML (MDS-AML). Kaplan-Meier analysis showed the disease free survival rate of 2-year was 52.5%. CONCLUSION: The prophylactic donor PBSC infusion mobilizing with G-CSF is effective, safe and feasible for the relapse of leukemia.

[Application of imatinib plus Hyper-CVAD chemotherapy regimen in patients with Ph chromosome positive acute lymphocytic leukemia].

OBJECTIVE: To report the efficacy and safety of imatinib plus Hyper-CVAD chemotherapy regimen in the treatment of patients with ph chromosome positive acute lymphocytic leukemia (Ph(+)ALL). METHODS: A sequential 2-year and 8-cycle treatment of imatinib plus Hyper-CVAD (A/B program) was administrated in 18 Ph(+)ALL patients treated at our hospital since January 2005 to January 2010. And another 18 Ph(+)ALL patients undergoing no allogeneic hematopoietic stem cell transplantation were selected as controls. RESULTS: Among 18 patients on chemotherapy, their average age was 33.1 years, the total response rate 100% following induction chemotherapy and the median survival 28.8 (8.0 - 60.0) months. And the 1-, 2- and 3-year overall survival rates were 77.8%, 72.2% and 66.7% respectively. In control patients, the response rate following induction chemotherapy was 100% and the median survival 22.5 (4.0 - 58.0) months. And the 1-, 2- and 3-year overall survival rates were 66.7%, 55.6% and 50.0% respectively. A recent follow-up showed that 5 patients (27.8%) died from relapse in the chemotherapy group and 4 (22.2%) in the control group. The overall deaths were 6 (33.3%) in the chemotherapy group and 9 (50.0%) in the control group. CONCLUSION: Imatinib plus Hyper-CVAD chemotherapy regimen is associated with significantly improved survival rates. Superior to allogeneic hematopoietic stem cell transplantation, it offers a prolonged median response time and survival time in Ph(+)ALL patients.

[Treatment of severe intestinal acute graft-versus-host disease with CD25 monoclonal antibody in haploidentical hematopoietic stem cell transplantation].

OBJECTIVE: To study the feasibility of CD25 monoclonal antibody (basiliximab) in the treatment of severe III-IV intestinal acute graft-versus-host disease (GVHD) following haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Twenty patients, 13 males and 7 females, who developed III-IV intestinal acute GVHD after haplotypic HSCT between October 2004 and September 2009, were treated with basiliximab (20 mg/d, d1, 4) and prednisolone (1 mg×kg(-1)×d(-1)) from the day of diagnosis. The therapy was repeated in the second week if the intestinal symptoms showed no improvement. The therapeutic effect was analyzed and the adverse reaction and cytomegalovirus (CMV) infection were observed. RESULTS: Ten patients had a complete remission and 5 were in a partial remission. The total effective rate was 75.0%. The clinical symptoms started to lessen in 1-12 days after using basiliximab (average: 7 days). During the 6-64 month follow-up (average: 25 months), 8/10 cases with a complete remission had no acute GVHD relapse, and the other 2 relapsing patients experienced a remission after a re-administration of basiliximab. Nine patients survived with a longest period of 64 months. Four withdrew corticosteroids and the other 5 stayed on a low-dose maintenance corticosteroid regimen. The 2-year disease-free survival was 47.5% by Kaplan-Meier calculation. CONCLUSIONS: Basiliximab is feasible in the treatment of III-IV intestinal acute GVHD after haplotype HSCT. It does not increase the relapse of leukemia or the incidence of infections.

Overexpression of miR­21 is involved in acute monocytic leukemia­associated angiogenesis by targeting IL­12.

Angiogenesis is important in pathophysiological processes, including the pathogenesis of acute monocytic leukemia (AML). MicroRNA­21 (miR­21) is overexpressed and exhibits oncogenic activity in cancer. However, the biological mechanism underlying the effect of miR­21 in AML remains to be fully elucidated. In the present study, the expression levels of miR­21 and vascular endothelial growth factor (VEGF) were determined in 26 patients with AML and 28 healthy individuals. The secretion of VEGF was also measured following the transfection of THP­1 cells with miR­21 mimic or inhibitor. The supernatants of the THP­1 cells, which were transfected with miR­21 mimic, inhibitor or small interfering RNA (si)VEGF, respectively, were used to incubate human umbilical vein endothelial cells (HUVECs), following which tube formation of the HUVECs was measured. miR­21 targets were predicted using a biological target prediction website and confirmed using a luciferase assay. The effects of interleukin (IL)­12 were investigated by examining the tube formation of HUVECs and the secretion of VEGF following recombinant human (rh) IL­12 pretreatment. The results revealed that miR­21 and VEGF expression was significantly increased in the peripheral blood monocytes of the patients, compared with the healthy controls. There was negative correlation between the expression of IL­12 and miR­21 in the serum of patients with AML. Furthermore, supernatant VEGF levels from the miR­21 mimic­transfected THP­1 cells were increased, whereas a decreasing trend was observed in the miR­21 inhibitor group. The angiogenic ability of the HUVECs pretreated with supernatant from the THP­1 cells transfected with miR­21 mimic was higher, and was lower in THP­1 cells co­transfected with miR­21 mimic and siVEGF, compared with the miR­21 mimic only group. A luciferase assay demonstrated that IL­12 was the direct target of miR­21, and the level of IL­12 in the supernatant of THP­1 cells transfected with miR­21 mimic was increased. IL­12 pretreatment increased VEGF expression and angiogenic ability in HUVECs. The inactivation of miR­21 or activation of its target gene may be a potential therapeutic strategy in human AML.

Association of VEGF and VDR gene- gene and gene- smoking interaction on risk of multiple myeloma in Chinese Han population.

AIMS: To investigate the association of several single nucleotide polymorphisms (SNPs) within vascular endothelial growth factor (VEGF) and vitamin D receptor (VDR) gene polymorphisms and additional gene- gene and gene- smoking interaction with multiple myeloma (MM) risk in Chinese population. METHODS: Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction combination among SNPs and smoking. Logistic regression was performed to investigate association between 6 SNPs within VEGF and VDR gene, additional gene- gene and gene- smoking interaction on MM risk. RESULTS: MM risk is significantly higher in carriers with the rs699947- A allele within VEGF gene than those with CC genotype (CA+ AA versus CC), adjusted OR (95%CI) =1.72 (1.19-2.33), and higher in carriers with rs2228570- T allele within VDR gene than those with CC genotype (CT+ TT versus CC), adjusted OR (95%CI) = 1.68 (1.26-2.17). We also found a significant two-locus model (p=0.0010) involving rs699947 and rs2228570, and a significant two-locus model (p=0.0107) involving rs2228570 andsmoking. Participants with rs699947- CA+AA and rs2228570- CT+TT genotype had the highest MM risk, compared to participants with rs699947- CC and rs2228570- CC genotype, OR (95%CI) = 3.12 (1.82 -4.61). Smokers with rs2228570- CT+TT genotype had the highest MM risk, compared to never- smokers with rs2228570- CC genotype, OR (95%CI) = 3.27 (1.74-4.86). CONCLUSIONS: We found that the A allele of rs699947 within VEGF and T allele of rs2228570 within VDR gene, interaction between rs699947 and rs2228570, rs2228570 andsmoking were all associated with increased MM risk.

Analysis of allogeneic hematopoietic stem cell transplantation with high-dose cyclophosphamide-induced immune tolerance for severe aplastic anemia.

The study was aimed to explore the efficacy and safety of allo-HSCT with high-dose cyclophosphamide-induced immune tolerance for SAA. In the present study, 20 cases (12 male, 8 female; average age = 17.8 years) received reduced-intensity conditioning allo-HSCT from August 2012 to August 2014 in the Beijing Military Region General Hospital. All were HLA mismatched and received CSA; 11 received ATG-intensive immune therapy. Donors underwent mobilization with cell colony-stimulating factor. The modified preconditioning regimen included reduced-strength fludarabine combined with Busulfex and cytarabine, cyclophosphamide. Cyclophosphamide (50 mg/kg/d) induced immune tolerance 3 days after transplantation and was combined with immunosuppressive agents, including CSA, MTX, and FK506, for GVHD prophylaxis and the management of observed toxicity, GVHD and DFS. Hematopoietic reconstitution was achieved in 17 cases and engraftment after a second transplantation in an additional three cases. The average times to engraftment were 17.4 and 21.3 days, respectively, with neutrophils ≥0.5 × 109/L and platelets ≥20 × 109/L. Engraftment was confirmed by the evidence of 100 % donor hematopoiesis; T lymphocyte subset counts also increased significantly after transplantation. During follow-up monitoring to April 2015 (median duration = 17.7 months), three patients died of complications, while the other 17 showed disease-free survival (DFS rate = 85 %; longest DFS period = 32 months). Reduced-intensity allo-HSCT with high-dose cyclophosphamide-induced immune tolerance treatment is effective for SAA and can be the key technology extensively used in clinic, but its efficacy needs to be confirmed further with prospective randomized study with increased sample size.

[Clinical Efficacy of Haploidentical Allo-HSCT of Reduced Intensity Preconditioning Combined with Induced Immune Tolerance after Transplantation for Severe Aplastic Anemia].

OBJECTIVE: To investigate the efficacy and safety of haploidentical allo-HSCT in combination of reduced intensity preconditioning combined with cyclophosphamid (CTX)-induced immune tolerance after transplanitation for treatment of severe aplastic anemia (SAA). METHODS: A total of 15 patients with SAA received the haploidentical allo-HSCT of reduced intensity preconditioning combined with CTX-induced immune tolerance after transplartation in the General hospital of Beijing military command of chinese PLA from June 2012 to December 2014. The reduced intensity preconditioning regimen consisted of CTX, fludarabine, busulfex and amti-lymphocyte immunoglobin; the immune tolerance was induced with CTX (50 mg/kg·d) on day 3 after transplantation; the HSC donors were father and mother of patients. The GVHD was prevented by inmunosuppression consisted of cyclosporine A(CsA), methotrexate and tacrolimus. The aduvese reaction and disease-free survival (DFS) were observed in all the patients. RESULTS: All the SAA patients achieved hematopoietic reconstitution with 100% donor hematopoiesis, and all the T lymphocyte subsets increased. Out of 15 patients, 3 cases died of complication, and the DFS rate was 80% with a median follow-up of 19.8 month (6-36 months). CONCLUSION: The haploidentical allo-HSCT of reduced intensity preconditioning combined with CTX-induced immune tolerance after transplantation is safet and effective for SAA patients, that may be applied to clinical therapy.

[Clinical Significance of Monitoring T Lymphocyte Subsets after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To evaluate the relationship between T lymphocyte subsets and the incidence of graft-versus-host disease (GVHD) and its clinical significance of monitoring the changes of T lymphocyte subsets dynamicly on 1, 3, 6, 12 month after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Twenty cases received allo-HSCT in Department of Hematology of General Hospital of Beijing Military Command from January 2013 to January 2014, including 10 males and 10 females with average age of 20.3 years (3-46 years old), among them 4 cases rectived HLA matched transplantation and 16 cases rectived HLA mismatched transplantation. The levels of T lymphocyte subsets including CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+), CD4(+)CD25(high) FOXP3(+) in the peripheral blood were manitored with flow cytometry (FCM) on +1, +3, +6, +12 month after transplantation dynamicly. RESULTS: (1) Follow up to March 2015, the levers of CD3(+), CD4(+), CD8(+), CD4(+)/CD8(+), CD4(+) CD25(high) FOXP3(+) showed a different degree of recovery after transplantation for all cases and returned to the lever of pre-transplantation on 12 month basically, and CD8(+) T cells recovered earlier than CD4(+) T cells, while the decrease of CD4(+) T cells lasted more than 1 year; The proportion inversion of CD4(+)/CD8(+) also lasted for more than 1 year;(2) The level of CD4(+) CD25(high) FOXP3(+) in patients with acute GVHD was lower than that in patients without acute GVHD. CONCLUSION: The dynamic monitoring of the T lymphocyte subsets, especially CD4(+) CD25(high) FOXP3(+) after transplantation has importent clinical significance, it can forecast the incidence of acute GVHD before symptoms appeared; the dynamic monitoring of the T-lymphocyte subsets also can be used as reference indicator for prediction of GVHD, theraby it can reduce mortality of patients after transplantation.

Adenovirus co-expressing CD40 ligand and interleukin (IL)-2 contributes to maturation of dendritic cells and production of IL-12.

The aim of the present study was to construct a chimeric adenovirus (Ad)5/F35 co-expressing human CD4O ligand (CD4OL) and interleukin (IL)-2 (Ad5/F35 CD40L-IL-2). The infection efficiency to human monocyte-derived dendritic cells (Mo-DCs), expression of genes, phenotype changes and IL-12 production of Mo-DC by Ad5/F35 CD40L-IL-2 were investigated. CD40L and IL-2 from total RNA extracted from human peripheral blood mononuclear cells (PBMCs) were cloned by reverse transcription-polymerase chain reaction and used to construct Ad5/F35 CD40L-IL-2. The infection efficiency, expression of CD40L, and phenotype changes of Mo-DC infected with Ad5/F35 CD40L-IL-2 were analyzed using flow cytometry. The quantities of IL-2 and IL-12 in the supernatants of Mo-DC following infection of Ad5/F35 CD40L-IL-2 were measured by enzyme-linked immunosorbent assay. The CD40L and IL-2 genes were successfully cloned and the Ad5/F35 CD40L-IL-2 was constructed. Ad5/F35 CD40L-IL-2 efficiently infected Mo-DCs with an infection efficiency of >75%, and the infected Mo-DCs expressed CD40L and secreted IL-2. The expression levels of cluster of differentiation (CD)80, CD86, CD40, and human leukocyte antigen-antigen D related on Mo-DC were moderate; however, CD83 was low prior to infection of Ad5/F35 CD40L-IL-2. Those molecules, particularly CD83, were markedly upregulated 24 h after the infection. Increasing quantities of IL-12 in the supernatants were detected subsequent to infection at different time points in a time-dependent manner. Thus, Ad5/F35 CD40L-IL-2 efficiently infected human Mo-DCs and its products, CD40L and IL-2, were subsequently expressed. In addition, infection with Ad5/F35 CD40L-IL-2 stimulated the maturation of Mo-DC and high levels of IL-12 production.

[Retrospective analysis of therapeutic efficacy of haploidentical allogeneic hematopoietic stem cell transplantation for severe aplastic anemia].

This study was purposed to investigate the therapeutic efficacy of haploidentical allogeneic hemopoietic stem cell transplantation (allo-HSCT) for severe aplastic anemia (SAA), and evaluate the safety of this treatment by retrospective analysis. A total of 21 patients with SAA (13 cases of SAA-I, 8 cases of SAA-II) were treated with haploidentical allo-HSCT. Donors were the relatives of the patients (12 were the parents, 9 were the siblings). The conditioning regimen contained cyclophosphamide, fludarabine and antithymocyte globulin. Methylaminopterin, mycophenolate mofetil and cyclosporin A were used for preventing graft versus host disease (GVHD). The chimerism rate was monitored periodically after successful graft. The long survival rate, incidence and severity of complication, such as GVHD, infection, and so on were analyzed. The results showed that 15 out of 21 patients were survived for 16 (3-46) months, survival rate was 71.4%. Graft tailure happened in one case who died of mycetes septicemia at 43 days after allo-HSCT. Two patients died of pulmonary infection at 6 days and 10 days respectively after transplantation. Rejection happened in one case at 3 months who died of pulmonary infection at 17 days after the second transplantation with the same donor. Two patients died of IV grade intestinal GVHD at 35 days and 52 days. GVHD occurred in 14 of 21 patients, the accumulative incidence was 66.7%, 5 cases of them were severe. It is concluded that the therapeutic efficacy of haploidentical allo-HSCT is effective for SAA and with slighter complications.

[Therapeutic efficacy of second allo-HSCT for aplastic anemia after failure of first allo-HSCT].

This study was purposed to evaluate the curative efficacy of second allogeneic hematopoietic stem cell transplantation (allo-HSCT) after failure of the first allo-HSCT in aplastic anemia patients, the cause of implant failure after allo-HSCT and clinical data of 10 severe aplastic anemia (SAA) patients in the second allo-HSCT were retrospectively analyszed. The second HSCT conditioning programs include: cyclophosphamide (CTX) + fludarabine (FLU)+ anti-thymocyte globulin (ATG) combination chemotherapy for 3 cases; CTX + FLU + white busulfan (Bu) + ATG combination chemotherapy for 7 cases. The prevention regimen of graft-versus-host disease (GVHD) include cyclosporine (CsA), mycophenolate mofetil (MMF) and methotrexate (MTX). The median count of mononuclear cell infusion was 12.17 (5.99-18.12)×10(8)/kg. The CD34(+) cell count was 5.2 (3.8-10.9)×10(6)/kg. The results showed that 10 evaluable patients achieved hematopoietic reconstitution with absolute neutrophil >0.5×10(9)/L, platelets >20×10(9)/L at 15d (8-21d) and 17d (11-27d) after transplantation. The grade I aGVHD occurred in 2 case, grade II in 1 case, chronic GVHD in 3 cases. Transplant-related deaths occurred in 4 cases. The disease-free survival rate, transplant-related mortality, GVHD after transplantation were 60%, 40% and 50% respectively. It is concluded that the second allo-HSCT is an effective therapy for aplastic anemia after allo-HSCT implant failure.

[Clinical analysis of reduced conditioning intensity allo-HSCT treatment for relapsed ETO-positive AML].

This study was aimed to explore the effect and feasibility of reduced conditioning intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of relapsed ETO positive acute myeloid leukemia (AML) patients. Fifteen cases of relapsed AML received the reducing conditioning intensity allo-HSCT from January 2011 to January 2013 in Beijing Military Command General Hospital. All patients were high-risk type of relapsed or refractory AML, including 10 males and 5 females, aged from 16 to 48 years old with mean age of 32.5 years. Ten cases are HLA-identical matching and other 5 cases are HLA-haploidentical.donors received granulocyte colony-stimulating factor (G-CSF) to mobilize the peripheral blood stem cell for transplantation. Conditioning regimen was fludarabine combined with busulfex, cytarabine and cyclophosphamide. The preventive donor's peripheral blood stem cell infusion were performed after 3 months of transplantation, and the toxicity, GVHD and disease-free survival were observed in patients after transplantation. The results showed that all patients achieved hematopoietic reconstitution, the average time of neutrophils ≥ 0.5 × 109/L and platelets ≥ 20 × 109/L were 15.5 d and 16.8 d respectively. Implantation was confirmed by the evidence of 100% donor hematopoiesis. Follow-up to June 2014, with a median follow-up duration of 27.5 months (18-54 months), GVHD occurred in 8 cases of all patients, one died of complication, the other 4 cases died of relapse and the other three patients remained in disease-free survival. The disease-free survival rate of 2-year was 66.7%,the longest disease-free survival time was up to 54 months. It is concluded that the reduced conditioning intensity allo-HSCT is the effective and safe method for relapsed AML with ETO-positive, and it may be chosen as a treatment method for relapsed ETO positive AML patients.

[Clinical research of reduced-intensity allogeneic hematopoietic stem cell transplantation for multiple myeloma].

This study was purposed to explore the efficacy and feasibility of reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of multiple myeloma (MM). Three patients with MM from January 2011 to January 2012 in General Hospital of Beijing Military Area were treated by reduced-intensity allo-HSCT. All donors are compatriots and affinity HLA identical. Donors were mobilized with granulocyte colony-stimulating factor (G-CSF), the MM patients were given combined transplantation of bone marrow and peripheral blood stem cells. Preconditioning regimen consisted of fludarabine combined with melphalan and anti-human thymocyte globulin, and the classic cyclosporin A (CsA) combined with methotrexate (MTX) was used to prevent graft-versus-host disease (GVHD). The preventive donor peripheral blood stem cell infusion in dose 0.2×10(8)/kg mononuclear cells (MNC) was applied after 3 months of transplantation, then the toxicity, GVHD and disease-free survival (DFS) in patients were observed after transplantation. The results showed that 3 patients got hematopoietic reconstitution, the average time of neutrophils ≥ 0.5×10(9)/L and platelets ≥ 20×10(9)/L was 14.3 d and 15.3 d respectively, the detection of implanting efficacy displayed 100% complete donor hematopoiesis. Follow-up to January 2013, the median follow-up time was 13 months (12 to 15 months), As a result, none of the patients got GVHD, infection and other serious complications, all patients are still in complete remission (CR), the longest DFS time has reached to 15 months. It is concluded that the reduced-intensity allogeneic hematopoietic stem cell transplantation is the effective method for MM, this method has the high safety and efficacy, as well as high complete remission rate in early transplantation, the MM patients may get a long-term survival. This method can be used as a key technology in clinic for treating MM.

[Clinical analysis of haploidentical or unrelated donor hematopoietic stem cell transplantation for patients with severe aplastic anemia].

Objective of this study was to evaluate the efficacy and safety of haploidentical or unrelated donor hematopoietic stem cell transplantation (HSCT) for patients with severe aplastic anemia (SAA). Twenty patients with SAA received allogeneic HSCT from haploidentical or unrelated donors (14 from haploidentical donors and 6 from unrelated donors) from November 2005 to May 2011. Conditioning regimen consisted of fludarabine (FLU), cyclophosphamide (Cy) and anti-thymocyte immunoglobulin (ATG). The patients were administrated with G-CSF-primed bone marrow and mobilized peripheral blood as grafts from haploidentical donor or only mobilized peripheral blood from the unrelated donor. The results showed that the median time of neutrophil and platelet engraftment were 14 (11 - 20) d and 17 (13 - 31) d respectively. All patients who achieved engraftment had complete hematologic recovery with complete donor chimerism, except for two patients who developed graft failure in 2 months after transplantation. Four cases developed acute grade IIGVHD. The chronic GVHD occurred in 7 of the 16 evaluable cases (6 limited, 1 extensive). 14 patients got disease-free survival with follow-up to January 2012. The disease-free survival rate was 68.9%. It is concluded that the haploidentical or unrelated donor hematopoietic stem cell transplantation may become a viable therapeutic option for severe aplastic anemia patients who lack suitable human leukocyte antigen-matched donors and fail immunosuppressive therapy.

[Clinical analysis of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation].

The objective of this study was to explore the incidence and therapeutic efficacy of cytomegalovirus (CMV) infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical data of 140 patients undergoing allo-HSCT in our department of hematology from 2010-01 to 2012-01 were retrospectively analyzed. The results showed that the incidence of CMV infection was 4.3% (48/140), the time for the first detection of positive CMV-DNA was at day 45 (33 to 68) after allo-HSCT, and the CMV quantitative range was 1.25×10(3) - 5.5×10(6). There were 2 cases of CMV-related interstitial pneumonia and 5 cases of hemorrhagic bladder inflammation. A total of 65 patients suffered from graft versus host disease (GVHD), in which 32 cases (49.2%) were accompanied with CMV infection, CMV-DNA negative in patients treated with ganciclovir, foscarnet sodium anti-CMV was at day 45 (33 to 68) with the effective rate of 100%. 12 patients with CMV infection were accompanied with transient neutropenia and thrombocytopenia. It is concluded that after allo-HSCT the CMV infection occurs frequently. The patients with GVHD have a higher incidence of CMV infection. Ganciclovir and foscarnet sodium are reliable to be used for treatment of CMV infection with fewer adverse reactions.

A clinical study of cytokine-induced killer cells for the treatment of refractory lymphoma.

Cytokine-induced killer (CIK) cell therapy, an adoptive T-cell immunotherapy, has been reported to be a safe and effective mode of treatment for patients with metastatic diseases, lymphoma and acute leukaemia. To investigate the clinical efficacy of cytokine-induced killer cells for the treatment of refractory lymphoma, the present clinical study was conducted. A total of 8 male patients with a mean age of 41 years (range 22-65) who were pathologically diagnosed with malignant lymphoma (Hodgkin's disease, 2 and non-Hodgkin's lymphoma, 6) were enrolled. CIK cells were expanded by priming with IFN-γ, monoclonal antibody (mAb) to CD3 and IL-1α, followed by the addition of IL-2 the following day using peripheral blood mononuclear cells (PBMCs) of the 8 male patients. The CIK cells were then transfused back to the patients as treatment. On day 13, the CIK cell count reached 7-18×10(19) (mean, 12.7×10(9)), a 44- to 140-fold increase (mean, 98-fold). The average percentage of cells expressing CD3(+), CD4(+), CD8(+) and CD3(+)CD56(+) were also increased from 50.9±3.5, 29.9±1.7, 41.3±3.2, 1.6±0.2% to 90.2±1.6, 40.6±5.5, 52.8±4.9 and 33.1±4.0%, respectively. Patients showed measurable radiographic tumor reduction, increased T-cell subset levels, and relief of symptoms after treatment. No severe toxicity or side effects were reported. CIK cells developed by this culture method have a high in vitro proliferation rate and tumor-killing capacity. In conclusion, CIK cell treatment of patients with malignant lymphoma achieves effective clinical responses, causing few side effects.

[Conditioning regimen containing fludarabine instead of cyclophosphamide for haploidentical hematopoietic stem cell transplantation].

OBJECTIVE: To explore the feasibility and safety of conditioning regimen containing fludarabine (Flud) for haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Preparative regimen containing Flud 40 mgxm(-2)xd(-1) on day -7 to -3 in place of cyclophosphamide (CTX) for haploidentical HSCT was given to 35 patients with hematologic malignancies (4 standard risk, 16 high risk, 15 relapse with no remission). All donors received rhG-CSF followed by HSC harvest. One patient received peripheral blood HSCT (PBSCT), one bone marrow transplantation (BMT), and the others BM combination with PBSCT. The regimen-associated side effect, engraftment, incidence of graft-versus-host disease (GVHD) and disease-free survival (DFS) probabilities were observed. RESULTS: All patients achieved sustained, full donor-type engraftment. Thirty-four patients obtained primary durable engraftment, and 1 who rejected graft from his mother obtained successful durable engraftment after the second graft from his father. The cumulative incidence of grade III-IV acute GVHD and chronic GVHD was 12.1% and 31.7%, respectively. With a follow-up duration of 8-25 months, 6 patients were dead, in which 3 died of relapse, 2 of acute GVHD, 1 of fungal infection, none died of regimen-associated side effect. The other 29 patients remained alive and DFS probability was 79.7%. CONCLUSION: Flud based conditioning regimens for haploidentical HSCT is safe and feasible, which reduces regimen-associated side effect, with no increasing the rate of relapse and infection, and decreases the incidence of aGVHD.

[Induction of efficient T-cell immunity against autologous leukemia cells by dendritic cells pulsed with the leukemia cell total RNA].

OBJECTIVE: To assess the feasibility and efficiency of eliciting leukemia-specific T cell responses in acute myeloid leukemia patients in complete remission (AML-CR) in vitro by dendritic cells (DC) pulsed with the leukemia cells total RNA. METHODS: The immature DCs were generated from the adherent bone marrow mononuclear cell in vitro in the presence of combined cytokines (GM-CSF 100 ng/ml, IL-4 500 U/ml), and pulsed with total RNA isolated from autologous leukemic cells by cationic lipid 1,2-dioleoyloxy-3-trimethyl ammonium propane (DOTAP) at day 5 of culture. Then the cells were incubated for another 24 h in a medium containing 10 ng/ml of TNF-alpha for maturation of DC. After the total 7 days culture, the cells were harvested as the mRNA-DC and the expression of mature DC markers were determined by FACS. The proliferative capacity of T cell activated by mRNA-DC was determined by MTT assay. Meanwhile, the mRNA-DC was co-cultured with T lymphocytes at a ratio of 1:3 for 7 days. The activated T lymphocytes were harvested, the secretion of IFN-gamma was determined by ELISPOT assay, and the cytotoxicity was analyzed in vitro by LDH release assay. RESULTS: After culture, the BMMNC from 14 AML-CR patients developed morphologic and phenotypic characteristics of mature DC. At a stimulator/reactor ratio of 1:16, auto-T lymphocytes primed with mRNA-DC exhibited significant proliferative activity compared with T lymphocyte primed with non-pulsed DC [(36.84 +/- 5.68)% vs (12.20 +/- 3.16)%, (P < 0.05)]. An expansion of mRNA reacted T cell secreting IFN-gamma could be observed on ELISPOT assay. At an effector/target ratio of 20:1, the auto-T lymphocytes primed with mRNA-DC exhibited significant killing activity to auto-AML cells (45.46 +/- 6.34 )% as compared with that stimulated by IL-2 alone (13.26 +/- 2.28)% or primed by non-pulsed DC (12.32 +/- 1.32)% (P < 0.05). CONCLUSION: Immunization with DC-leukemia cell RNA vaccines may be a simple, rapid and potent approach to elicitation of T cell-mediated anti-leukemia immunity.