Anti-CD19 and anti-BCMA CAR T cell therapy followed by lenalidomide maintenance after autologous stem-cell transplantation for high-risk newly diagnosed multiple myeloma.

Few prospective studies have examined posttransplant chimeric antigen receptor (CAR) T cell infusion as candidates for front-line consolidation therapy for high-risk multiple myeloma (MM) patients. This single-arm exploratory clinical trial is the first to evaluate the safety and efficacy of sequential anti-CD19 and anti-BCMA CAR-T cell infusion, followed by lenalidomide maintenance after autologous stem cell transplantation (ASCT), in 10 high-risk newly diagnosed multiple myeloma (NDMM) patients. The treatment was generally well tolerated, with hematologic toxicities being the most common grade 3 or higher adverse events. All patients had cytokine release syndrome (CRS), which was grade 1 in 5 patients (50%) and grade 2 in 5 patients (50%). No neurotoxicity was observed after CAR-T cell infusion. The overall response rate was 100%, with the best response being 90% for a stringent complete response (sCR), and 10% for a complete response (CR). At a median follow-up of 42 (36-49) months, seven (70%) of 10 patients showed sustained minimal residual disease (MRD) negativity for more than 2 years. The median progression-free survival (PFS) and overall survival (OS) were not reached. Although the sample size was small and there was a lack of control in this single-arm study, the clinical benefits observed warrant ongoing randomized controlled trials.

Mesenchymal stromal cells as an adjuvant treatment for severe late-onset hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

The management of severe late-onset hemorrhagic cystitis (LO-HC) after allogeneic hematopoietic stem cell transplantation (HSCT) is still challenging. Because mesenchymal stromal cells (MSCs) possess anti-inflammatory and tissue repair-promoting properties, we retrospectively analyzed the efficacy and safety of MSC infusions in 7 of 33 patients with severe LO-HC after allogeneic HSCT. During treatment, each patient received at least one MSC infusion of Wharton's jelly derived from the umbilical cord of a third-party donor. In 6 patients, MSC treatment was initiated within 3 days of gross hematuria onset, while the 7th patient received an infusion 40 days later. The median dose was 1.0 (0.8-1.6) × 10(6)/kg. Five of 7 patients responded to treatment. Notably, gross hematuria promptly disappeared in 3 patients after 1 infusion, with a time to remission not seen in patients without MSC infusion. Two patients showed no response even after several infusions. No acute or late complications were recorded. Our findings indicate that MSC transfusion might be a feasible and safe supplemental therapy for patients with severe LO-HC after allogeneic HSCT.

Three times spontaneous remission of severe aplastic anemia following granulocyte transfusion from related donors: a case report and literature review.

Aplastic anemia (AA) is a bone marrow failure disease caused by abnormal activation of T lymphocytes, resulting in the apoptosis of hematopoietic cells and bone marrow failure. Currently, hematopoietic stem cell transplantation (HSCT), immunosuppressive - therapy (IST), and supportive care (e.g. transfusion adjuvant therapy, hematopoietic growth factors, and prevention of infection) are the main treatments of AA. Granulocyte transfusion has recently been accepted as an useful adjuvant therapy of HSCT and intensive IST. This article reported a severe AA patient who failed to respond to IST, but achieved spontaneous remission three times after granulocyte transfusions from related donors. Such cases have rarely been reported. Existence of human leukocyte antigen (HLA) cross between the patient and his relatives may influence the T cell-mediated immunity, which might explain this patient's recovery.

[Effects of Leukapheresis on Hemostatic Function in Patients with Hyperleukocytic Leukemia].

OBJECTIVE: To analyze and compare the effects of leukapheresis on hemostatic function in patients with hyperleukocytic leukemia. METHODS: A total of 139 patients with AML, ALL and CML who underwent leukapheresis from June 2009 to February 2020 and did coagulation test before and after operation were included in this study. The clearance efficiency of each group and the difference among three groups were evaluated, as well as hemostatic function including platelet counts, coagulation indicators, CDSS score and incidence of adverse events. The difference of hemostatic function caused by leukapheresis in different leukemia patients were compared. RESULTS: After leukapheresis, the WBC counts were decreased significantly in the three groups of patients (P<0.001), and the clearance efficiency was highest in ALL patients. However, the platelet counts also were decreased significantly (AML:P＜0.001, ALL: P＜0.001, CML: P＜0.01) in the three groups of patients, particularly for acute leukemia patients with a positive correlation with WBC clearance efficiency(r=0.284). After leukapheresis, fibrinogen decreased, PT and APTT prolonged. For acute leukemia patients, higher CDSS score was related to an elevated incidence of bleeding events (P<0.05). CONCLUSION: Leukapheresis is an effective method to decrease the leukemic burden, but it is necessary to monitor the impact on hemostatic function. It is recommended to assess the CDSS socre for acute leukemia patients, in order to identify the predictive value for bleedings.

Sequential CD19 and BCMA-specific CAR T-cell treatment elicits sustained remission of relapsed and/or refractory myeloma.

The low rate of durable response against relapsed and/or refractory multiple myeloma (RRMM) in recent studies indicates that chimeric antigen receptor T-cell (CART) treatment is yet to be optimized. This study aims to investigate the safety and efficacy of sequential infusion of CD19-CART and B-cell maturation antigen (BCMA)-CARTs for RRMM with a similar 3 + 3 dose escalation combined with a toxicity sentinel design. We enrolled 10 patients, among whom 7 received autologous infusion and 3 received allogeneic infusion. The median follow-up time was 20 months. The most common grade 3/4 treatment-emergent toxicities were hematological toxicities. Cytokine-release syndrome (CRS) adverse reactions were grade 1/2 in 9 out of 10 subjects. No dose-limited toxicity (DLT) was observed for BCMA-CAR-positive T cells ≤5 × 107 /kg), while two patients with dose-levels of 5-6.5 × 107 /kg experienced DLTs. The overall response rate was 90% (five partial responses and four stringent complete responses). Three out of four patients with stringent complete responses to autologous CART had progression-free survival for over 2 years. The three patients with allogeneic CART experienced disease progression within 2 months. These results evidence the sequential infusion's preliminarily tolerability and efficacy in RRMM, and present a simple and safe design applicable for the establishment of multiple CART therapy.

Combination of DNA methylation inhibitor 5-azacytidine and arsenic trioxide has synergistic activity in myeloma.

XAF1 is a newly identified candidate tumour-suppressor gene that can antagonise XIAP and sensitise cells to cell death triggers. This study was undertaken to study the effect of 5-azacytidine (AZA) on XAF1 expression in myeloma cells and efficacy of 5-AZA and arsenic trioxide (ATO) combination treatment in myeloma in vivo and in vitro. XAF1 expression was analysed by semi-quantitative PCR and western blotting. Methylation specific PCR was used to detect methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with various concentrations of 5-AZA and ATO. Expression of XAF1 mRNA variants were confirmed by gel electrophoresis and sequencing. Untreated RPMI 8226 cell expresses two variants of XAF1 mRNA. Untreated XG-7 cell has no expression of XAF1. Hypermethylation of XAF1 promoter CpG islands was detected in both cell lines. Both cell lines express full-length XAF1 transcript after treated with 2.5 mumol/L 5-AZA for 72 h. Our studies demonstrated that 5-AZA exhibits anti-myeloma synergy with ATO. In addition, ATO alone, 5-AZA alone, or combination of 5-AZA and ATO was effective in slowing myeloma growth and prolonging survival of myeloma-loaded nude mice. The findings suggested that 5-AZA and ATO may be an effective combination in the therapy of myeloma patients.

[Differentiating effect of PPARgamma ligand rosiglitazone and all trans-retinoic acid on myeloma cells and its possible mechanism].

OBJECTIVE: To investigate the effects of PPARgamma ligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to explore the possible mechanism. METHODS: Human myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [(3)H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27(Kip1) and p21(Waf1) expression was detected by Western blotting. RESULTS: The exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 micromol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G(0)/G(1) was (45.2 +/- 6.7)% and (40.3 +/- 7.3)%, respectively (P < 0.05). The proportion of the cells in phase G(2)/M and S was (52.2 +/- 7.4)% and (57.4 +/- 9.5)%, respectively (P < 0.05). These changes were more evident when the RGZ concentration was increased to 10 micromol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27(Kip1) and p21(Waf1) in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA. CONCLUSION: RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27(Kip1) and p21(Waf1) expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.

[Diagnostic Value of G and GM Tests in Acute Leukemia Patients with Invasive Fungal Disease].

OBJECTIVE: To analyze the diagnostic value of (1, 3) -ß-D-glucan and galactomannan (GM) tests in the patients with acute leukemia complicated by invasive fungal disease, and explore the application of serological detection (G/GM) and lung CT for early diagnosis of invasive fungal disease (IFD). METHODS: A total of 493 patients with acute leukemia complicated by high risk invasive fungal infection, also receival G and GM tests, in Department of hematology of our hospital from June 2016 to December 2016 were selected and were divided into IFD-confirmed group (62 cases) including confirmed and clinical diagnesed IFD, and IFD-unconfirmed group (431 cases) including suspected IFD and non-IFD according to the diagnostic criteria of IFD. The results of G and GM tests in patients of 2 groups were analyzed, then the diagnostic efficacy of G and GM done and combination evaluated. In addition, 26 patients whose lung CT negative at hospitalization, moreover, presentation of changes in lung by CT during hospitalization and serological G and GM test positive were selected, and the difference of time between serological that postive and presentation of changes in lung by CT were compared for the estimation of early diagnotic value. RESULTS: The positive rate of (1, 3) -ß-D-glucan in IFD-confirmed group and IFD-unconfirmed group was 56.5% and 10.4%, respectively. Meanwhile, that of galactomannan test was 41.9% and 9.0%, respectively. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of (1, 3) -ß-D-glucan was 56%, 90%, 44% and 92%, and that of galactomannan was 42%、91%、40% and 93%, respectively. Moreover, the combination of (1, 3) -ß-D-glucan and galactomannan could raise the sensitivity to 69% and specificity to 98%. The positive results of serological detection (G/GM) come earlier about five days than CT changes. CONCLUSION: Both (1, 3) -ß-D-glucan and galactomannan test have high sensitivity and specificity, and the combination of them can improve the diagnostic efficacy, and make the clinical antifungal therapy more precisely. In the early clinical diagnosis of IFD, the positive results of serological detection coming earlier than lung CT.

Expanded donor natural killer cell and IL-2, IL-15 treatment efficacy in allogeneic hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD), leukemia relapse, and immune deficiency remain the major limitations of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor natural killer cells (NK) and cytokines have good potential in GVHD prevention and immune reconstitution enhancement. Improved survival after allo-HSCT therefore requires effective prophylaxis to reduce GVHD and strategy to mediate graft-versus-leukemia (GVL) effect. We studied the administration of expanded donor NK cell infusion and interleukin-2 (IL-2) and IL-15 mixture treatment in a murine allo-HSCT model for its effects on GVHD, immune reconstitution and leukemia relapse. In the GVHD model, recipient mice were reconstituted with bone marrow (BM) cells and splenocytes via vein. In the leukemia model, recipient mice were inoculated with EL9611 leukemia cells via vein 8 d prior to transplant. NK cell infusion mice group had lower clinical GVHD scores and suffered less severe GVHD-associated weight loss than control mice group. 90% of control mice died of leukemia relapse within 52 d post-transplant. NK cell infusion and IL-2 and IL-15 treatment recipient mice had improved survival compared with control mice. NK cell infusion and IL-2 and IL-15 treatment recipient mice had also accelerated lymphoid immune reconstitution compared with control mice group. Expanded donor NK cell infusion and IL-2 and IL-15 treatment could promote lymphoid immune reconstitution, mitigate GVHD, and reduce leukemia relapse in allo-HSCT recipients. The findings may have important significance for complication prevention in clinical allo-HSCT.

[A study of cytomegalovirus (CMV) glycoprotein B genotypes in patients with CMV infection after hematopoietic stem cell transplantation].

OBJECTIVE: To study the different cytomegalovirus (CMV) glycoprotein (G) B genotypes in the patients with CMV infection after hematopoietic stem cell transplantation (HSCT) and the correlation between genotype and pathogenesis of CMV disease. METHODS: Thirty-eight patients received allogenetic from March 2001 to December 2003. CMV GB DNA was detected with nested-PCR in all the patients and the products were digested with RsaI and HinfI. RESULTS: The distribution of GB types were as follows: GB1, 19/38 (50.0%); GB2, 3/38 (7.9%), GB3, 14/38 (36.8%). GB1 was associated with favorable clinical outcome, while GB3 might contribute to fatal interstitial pneumonia (IP). The incidences of graft versus host disease (GVHD) were not different significantly between the patients with GB1 and those with GB3. CONCLUSION: CMV GB genotypes might play an important role in the pathogenesis of CMV-IP, but not of GVHD.

Comparison of SPE, IFE, and FLC in Monitoring Patients with Multiple Myeloma After Autologous Stem Cell Transplantation.

Conventionally, serum protein electrophoresis (SPE) and serum immunofixation electrophoresis (IFE) are used as primary methods to diagnose and monitor multiple myeloma (MM). Recently, serum-free light chain (FLC) assay has been incorporated into hematological screening programs for myeloma. The purpose of this study is to compare the performance of the three methods in monitoring MM patients after autologous stem cell transplantation (ASCT). SPE, serum IFE and serum FLC assay were performed on 38 MM patients who underwent ASCT. In total, four patients had unexpected protein bands (UPBs) and 13 patients had relapsed after ASCT. Our results indicate that IFE is more sensitive than SPE and FLC assay in detection of UPBs and relapse. The results of IFE may provide useful information in advance of patient relapse.

[Study on the correlation between CMV reactivation and bronchiolitis obliteans after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To investigate the correlation between CMV reactivation and obliterative bronchiolitis (BO) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: From January 2011 to December 2013, 769 patients underwent allo-HSCT. The CMV infection was diagnosed by fluorescence quantitative PCR method for detecting the level of CMV-DNA and immunofluorescence staining of PP65 antigen in white blood cell. The frequency of BO in patients with and without CMV infection was compared, and the correlation between CMV infection and BO was analyzed. The clinical data of CMV infection patients with and without BO were analyzed and compared. RESULTS: Of 259 diagnosed CMV infection patients, BO occurred in 32 cases, the incidence rate was 12.35%, while in 510 cases without CMV infection, BO occurred in 8 cases, the incidence was 1.56%. The incidence rate of BO is significantly higher in patients with CMV infection than that in patients without CMV infection (P<0.001). The CMV related clinical data between the 32 cases with BO and 227 cases without BO were analyzed among the 259 cases of diagnosed CMV infection patients. BO incidence is higher in patients with more than 105 copies/ml CMV-DNA than that in patients with less than 10² copies/ml CMV-DNA. CONCLUSION: Among the risk factors related to BO post allo-HSCT, CMV infection is one of them to be worthy of attention. CMV reactivation with high virus titer, multiple CMV reactivations and CMV pneumonia are the risk factors.

[Clinical study of unrelated umbilical cord blood transplantation for 25 patients with hematological diseases].

This study was purposed to investigate the engraftment, graft-versus-host disease (GVHD), transplantation related mortality (TRM), relapse and survival in hematologic patients received unrelated umbilical cord blood transplantation (UCBT). A total of 25 patients with hematological disease underwent UCBT, including 8 pediatric and 17 young adult patients. Among them 3 cases received single unit of UCBT and 22 cases received double units of UCBT. For donor/recipients human leukocyte antigen (HLA) matching: HLA 6/6 loci matched in 9 cases, HLA 4-5/6 loci matched in 16 cases. There were 19 patients with hematologic malignancies, including 3 cases in the period of disease progression and 6 cases of non-hematologic malignancies. Conditioning regimens were TBI/Cy ± Flu ± ATG or BuCy ± Flu ± ATG for 21 patients and Cy+Flu+ATG for 4 patients. For prophylaxis of acute graft-versus-host disease (aGVHD) the regimen of cyclosporine (CsA) as dominant drug was used. The results showed that among 16 patients (80.0%) achieved engraftment, 20 patients survived for more than 42 d after transplantation. The cumulative neutrophil recovery rate on day 42 after transplant was 64.0%, with a median time of 17.0 d;the cumulative platelet recovery rate on day 100 after transplant was 60.0 %, with a median time of 35.0 d. The cumulative rate of grade II-IV and III-IV aGVHD after transplantation 100 d was 44.0% and 30.7%, respectively. Until the end of the follow-up, the cumulative rate of TRM was 54.3%. For all the patients, overall survival rate was 42.7%. Out of 17 evaluable patients with hematologic malignancies 7 cases (41.2%) survived to date, and only 1 case relapsed, so event-free survival rate was 35.3%. Out of 5 evaluable patients with non-hematologic malignancies, 4 patients survived and 2 patients were in stable engraftment state, 2 cases with autologous hematopoietic recovery. Among 3 cases of hematologic malignancies at advanced stage, only 1 case survived to date. It is concluded that HLA-4-6/6 loci matched UCBT is an effective option to treat hematological diseases. Double cord blood transplantation (dUCBT) can overcome the disadvantage of insufficient cells of single cord blood UCBT to treat overweight children and adult.

[Quantitative monitoring of multi-donor chimerism after multi-donor allogeneic hematopoietic stem cell transplantation].

This study was aimed to establish a model for detecting the donor chimerism rate following the multi-donor hematopoietic stem cell transplantations, and simplify its calculation method. Patients with hematologic disease receiving allogeneic hematopoietic stem cell transplantation including single-donor and multi-donor were selected in this study and the donor cell chimerism rates were detected, using STR-PCR combined with capillary electrophoresis. The results indicated that the peaks of the sister alleles coming from the same individual were confirmed to have the approximate areas and can be replaced each other in the situation of mixed chimerism. In the calculation model, the value between reference chimerism and approximate chimerism have no significant difference using the hypothetical peak areas, and the result was confirmed to be accepted basing on typical measurement error between sister alleles (5% - 20%). It is concluded that the areas of share peaks can be replaced by non-share peaks and this conclusion can be used to calculate the double-donor CHM (DD-CHM)(%). Compared to the D alleles, R alleles show more strategic importance because it can lead to more accurate result and allowed simplifying the arithmetic calculations for DD-CHM(%).

[Establishment of murine allogeneic umbilical cord blood cell transplantation model and study on the mechanism of keratinocyte growth factor enhancing immune reconstitution after transplantation.].

OBJECTIVE: To explore the impact and mechanism of keratinocyte growth factor (KGF) on immune reconstitution post murine allogeneic umbilical cord blood transplantation (UCBT). METHODS: Perpheral blood (PB) from 19.5-day embryos post-conception (E 19.5 d) mice was used as umbilical cord blood (UCB) graft. Thirty-two BALB/c mice were randomly assigned to 4 groups with 8 mice each in the first cohort UCBT. Mice were infused with PBS (control group) or 1 x 10(6) (group 1A), 2 x 10(6) (group 1B), 3 x 10(6) UCB mononuclear cells (MNCs) (group 1C), respectively. Twenty-four BALB/c mice were randomly assigned to 3 groups with 8 mice each in the second cohort UCBT. Mice were injected with 1 x 10(6) (group 2A), 2 x 10(6) (group 1B) or 3 x 10(6) (UCB) MNCs (group 2C). All mice received platelet transfusion on +8d. Sixteen BALB/c mice were randomly assigned to 2 groups with 8 mice each in the third cohort UCBT. Mice were injected s.c. with KGF (group 3) or PBS (control group) before TBI. All mice were injected with 2 x 10(6) UCB MNCs and were supported with platelet transfusion on +8 d. The survival time, splenic lymphoid cell subsets, sjTREC assay were observed after UCBT. RESULTS: The 100-day survival of mice were 2, 3 and 3 in group 1A, 1B, 1C and 7, 8, 8 in group 2A, 2B, 2C, respectively. The splenic T, NKT, NK and B cell counts on +35 d were (9.57 +/- 0.74) x 10(6), (0.64 +/- 0.06) x 10(6), (1.43 +/- 0.10) x 10(6) and (19.13 +/- 1.50) x 10(6) in control group, respectively; while were (13.47 +/- 0.74) x 10(6), (0.89 +/- 0.03) x 10(6), (1.79 +/- 0.04) x 10(6) and (20.50 +/- 0.91) x 10(6) in group 3, respectively, being significantly higher than in control group. The sjTREC level was 182.2 +/- 10.7copies per 10(5) cells in control group; while was 224.2 +/- 9.6 copied per 10(5) cells in group 3, being significantly higher than in control group (P = 0.019). CONCLUSIONS: Peripheral blood from E19.5d is rich in hematopoietic stem cells. A murine allogeneic UCBT model with platelet support on +8 d is established. KGF treatment can enhance thymic output and improve T cell immune reconstitution after UCBT.

Preliminary study on 5-azacytidine anti-myeloma activity in vitro.

This study was aimed to investigate the effect of 5-azacytidine (5-AZA) on XAF1 expression in myeloma cells and efficacy of 5-AZA treatment for myeloma in vitro. XAF1 expression was analyzed by semi-quantitative PCR. Methylation-specific PCR (MSP) was used to detect the methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with 0-5 micromol/L of 5-AZA. Expression of XAF1 mRNA variants was confirmed by gel electrophoresis. The results indicated that the untreated RPMI 8226 cell expressed XAF1 mRNA transcript 1 and transcript 2, untreated XG-7 cells did not express XAF1 mRNA. Hypermethylation of XAF1 promoter CpG islands could be detected in both cell lines. Both cell lines expressed full-length XAF1 transcript after being treated with 2.5 micromol/L of 5-AZA for 72 hours. 5-AZA treatment led XAF1 promoter CpG island to hypomethylation in both cell lines. 5-AZA exerted anti-myeloma activity in a time- and concentration-dependent manner. The IC(50) value of XG-7 cells treated with 5-AZA for 48 hours was 2.6 micromol/L. 1.0, 2.0, 2.5 and 5.0 micromol/L of 5-AZA treatment for 48 hours induced (34.3 +/- 8.0)%, (54.8 +/- 3.1)%, (64.1 +/- 3.4)%, (81.0 +/- 4.1)% apoptosis in XG-7 cell line respectively. The combination of 1.0 - 4.0 micromol/L of 5-AZA with 1.0 - 4.0 micromol/L of arsenic trioxide (ATO) exhibited synergistic toxicity in myeloma cells with all CI values less than 1.0. It is concluded that lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with the hypermethylation of XAF1 gene promoter CpG island. 5-AZA treatment can induce the expression of XAF1 mRNA and protein in myeloma. 5-AZA exerts anti-myeloma activity via apoptosis at clinically achievable concentrations. The findings suggested that 5-AZA and ATO may be an effective combination in the therapy of patients with multiple myeloma.

[Rosiglitazone and all-trans retinoic acid inhibit human myeloma cell proliferation via apoptosis signaling pathway modulation].

OBJECTIVE: To investigate the effects of artificial ligand of peroxisome proliferators activated receptors (PPARs), rosiglitazone (RGZ) and all trans-retinoic acid (ATRA) on human myeloma cell line growth in vitro and in vivo. METHODS: U266 and RPMI 8226 cells were treated with different concentration of RGZ in the presence or absence of ATRA and the results were studied by 3H-TdR thymidine incorporation (for cells proliferation), Annexin V-PI staining and caspase-3 activity assay (for cells apoptosis), RT-PCR (for FLIP, XIAP and survivin mRNA expression), and tumor formation test in BALB/c nude mice. RESULTS: Exposure to RGZ induced proliferation inhibition in a dose-dependent manner in both U266 (r = 0.991, P < 0.01) and RPMI 8226 cells (r = 0.961, P < 0.01). A combination of RGZ with ATRA could enhance the inhibition effect (P < 0.001 in U266, P < 0.01 in RPMI8226). 10 micromol/L of RGZ induced apoptosis of (9.8 +/- 1.7)% in U266 cells and (10.7 +/- 3.3)% in RPMI8226 cells, in a time and dose dependent manner and combined with ATRA intensified the apoptosis induction effects (P < 0.01 in both cell lines). The FLIP, XIAP and survivin mRNAs were expressed in both cell lines and their levels decreased significantly after cultured with RGZ. The addition of RGZ + ATRA in the culture further decreased the levels. Caspase-3 activity increased substantially with the increase of RGZ concentration and the addition of RGZ + ATRA in the culture medium showed similar synergism effect on caspase-3 activation (P < 0.01). The xenograft of U266 cells in BALB/c nude mice were inhibited by RGZ and so did more by the combination of RGZ and ATRA (P < 0.01). CONCLUSION: The down-regulation of FLIP, XIAP and Survivin induced by RGZ can activate caspase-3, whereby induced apoptosis and proliferation inhibition in myeloma cells. ATRA can enhance these effects of RGZ.

[Relationship between CMV reactivation and KIR haplotype/HLA-Cw genotype in patients after unrelated-donor hematopoietic stem cell transplantation.].

OBJECTIVE: To explore the relationship between CMV reactivation and KIR haplotype or HLA-Cw genotype in patients after unrelated-donor hematopoietic stem cell transplantation (HSCT). METHODS: From January 2003 to December 2008 the HLA-Cw/KIR genotype of 48 patient-donor pairs were determined by polymerase chain reaction with sequence specific primers (PCR-SSP) and sequence specific nucleotide (PCR-SSOP). Posttransplant CMV reactivation was performed by immune histochemically assay. RESULTS: Of 48 patients, 15 were transplanted from unrelated donors with an antigen mismatch for HLA Cw and 33 patient-donor pairs were matched for HLA-Cw. The CMV reaction rate was 66.7% for HLA-Cw mismatch group and 48.5% for HLA-Cw match group (chi(2) = 1.39, P = 0.2375). Thirty-seven donor-patients pairs belonged to group C1 and 11 to group C2, and CMV reaction rate was 64.9% in group C1 and 18.2% in group C2 (chi(2) = 18.13, P < 0.0001). Twenty-six patients received graft from KIR haplotype A (group A donor) and 22 from KIR haplotype B donors (group B donor) and CMV reaction rate was 57.7% in group A donor and 50.0% in group B donor (chi(2) = 0.28, P = 0.5941). The number of donor activating KIRs (aKIRs) was less than that of recipient aKIRs in 34 patient-donor pairs in which the CMV reaction rate was 70.6%, and the number of donor aKIRs was more than that of recipient aKIRs in 14 patient-donor pairs in which the CMV reactivation was 14.3%. There was a significan difference between the two group (chi(2) = 12.44, P = 0.0004). CONCLUSION: KIR and HLA-Cw genotypes influence the rate of CMV reactivation following non-T cell deleted unrelated donor hematopoietic cell transplantation.

[Clinical study of allogeneic hematopoietic stem cell transplantation for relapsed/refractory acute lymphocytic leukemia].

OBJECTIVE: To explore the efficacy and toxicity of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for relapsed/refractory acute lymphocytic leukemia (ALL). METHODS: Forty-seven patients with relapsed/refractory ALL received allo-HSCT, which containing 19/47 from HLA-identical sibling donors (sib-HSCT), 18/47 from HLA-identical unrelated donors (URD-HSCT) and 10/47 from haplo-identical donors (Hi-HSCT). Conditioning regimens included "TBI plus Cyclophosphamide (Cy) (42/ 47)" or "busulfan (Bu) plus Cy (5/47)". Cyclosporine (CsA) combined with a short-course Methotrexate (MTX) were used for graft versus host disease (GVHD) prophylaxis. In addition, patients receiving URD-HSCT or Hi-HSCT were given mycophenolate mofetil (MMF) and anti-thymocyte immunoglobulin (ATG). Patients with molecular or cytogenetic relapse tendency on minimal residual disease (MRD) monitoring received donor lymphocyte infusion (DLI). RESULTS: All patients tolerated the therapy well except for mucositis. Renal dysfunction occurred in 2 patients on CsA therapy. Epilepsy occurred in 1 patient, fatal infectious complications in 9 (including 3 interstitial pneumonia), grade III-IV acute GVHD (aGVHD) in 7, chronic GVHD (cGVHD) in 22 and hemorrhagic cystitis (HC) in 4 patients. Thirteen patients relapsed after transplantation. The median time of hematopoietic reconstitution was + 17 ds. Nineteen patients received DLI, and 6 of them had no disease progression. With a median follow-up duration of 43 (10-77) months, the estimated 5-year overall survival (OS) and disease free survival (DFS) rates were 49.65% and 46.55%, respectively. CONCLUSION: Allo-HSCT is an effective therapy for relapsed/refractory ALL. Relapse after transplantation, fatal infection, and severe acute GVHD are the main causes for failure. DLI might decrease the relapse rate after transplantation.

[Experimental study on IL-2- and IL-15 application in allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To explore the impact of IL-2- and IL-15-activated donor natural killer (NK) cell infusion on graft-versus-host-disease (GVHD) and graft-versus-leukemia (GVL) effect post allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The C57BL/6 mice splenic NK cells were selected by microbeads, and then expanded in the media containing IL-2 and IL-15. The killing activity of NK cells was detected. In the leukemia mouse model, recipients (BALB/c) were intravenously inoculated with EL9611 leukemia cells 8 days before transplantation. Lethally irradiated BALB/c recipient mice were transplanted with 5 x 10(6) bone marrow cells (BMCs), or 5 x 10(6) BMCs plus 1 x 10(7) splenocytes with or without 1 x 10(7) activated NK cells. Additionally, NK cell infusion group mice were intraperitoneally injected with a mixture of IL-2 and IL-15 post transplant. Survival time, GVHD occurrence, lineage chimerism, TRBV spectra-typing were observed post transplant. RESULTS: The purity of isolated splenic NK cells was 95.7% - 97.1%. The killing activity of NK cells after activation was increased by 3 times. GVHD did not occurred in allogeneic BMCs infusion group, whereas did from 1 week after transplant in allogeneic BMCs + splenocytes infusion group. The severity of GVHD in total body irradiation (TBI) experimental group was significantly lower than in splenocytes infusion group (P < 0.05). The survival time was 9.5 - 14.0 d in TBI alone conditioning group. In leukemia mouse model, 100 day survival rate was 10% the rest of them were died of leukemia while in experimental group, the more than 100 days survival rate was 80% (P < 0.01). PB NK cells at 2 week post-transplant were 4.8% in experimental group and 2.8% in control group. NK cells recovery in experimental group was earlier than that in control group (P < 0.05). TRBV reconstitution was faster in experimental group than in control group, moreover, the number of TRBV family expression was more in experimental group than in control group which mainly expressed monoclone or oligo-clone. CONCLUSIONS: Donor alloreactive NK cells can be efficiently expanded and activated with IL-2 and IL-15. Donor activated NK cell infusion and IL-2, IL-15 treatment can promote immune reconstitution, mitigate GVHD and reduce leukemia relapse.