Efficacy and Safety of Allogeneic Hematopoietic Stem Cell Transplantation with Decitabine-containing Regimen in Myelodysplastic Syndromes and Myelodysplastic Syndromes Transformed Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To evaluate the efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) with decitabine (Dec)-conditioning regimen in the treatment of myelodysplastic syndrome (MDS) and MDS transformed acute myeloid leukemia (MDS-AML).@*METHODS@#The characteristics and efficacy data of 93 patients with MDS and MDS-AML who received allo-HSCT in our center from April 2013 to November 2021 were retrospectively analyzed. All patients were administered by myeloablative conditioning regimen containing Dec (25 mg/m2 /d×3 d).@*RESULTS@#Among the 93 patients, 63 males and 30 females, were diagnosed as MDS（n ＝77）, MDS-AML（n ＝16）. The incidence of I/II grade regimen-related toxicity (RRT) was 39.8%, and III grade RRT was only found in 1 patient (1%). Neutrophil engraftment was successful in 91 (97.8%) patients after a median neutrophil engraftment time of 14 (9-27) days; Successful platelet engraftment was achieved in 87 (93.5%) patients, with a median engraftment time of 18 (9-290) days. The incidence of acute graft versus host disease（aGVHD） and grade III-IV aGVHD was 44.2% and 16.2%, respectively. The incidence of chronic graft versus host disease（cGVHD） and moderate-to-severe cGVHD was 59.5% and 37.1%, respectively. Of the 93 patients, 54 (58%) developed posttransplant infections, among which lung infection (32.3%) and bloodstream infection (12.9%) were the most common. The median follow-up after transplantation was 45 (0.1-108) months. The 5-year overall survival (OS) rate, disease-free survival (DFS) rate, treatment-related mortality, and cumulative incidence of relapse were 72.7%, 68.4%, 25.1%, and 6.5%, respectively. And the 1-year graft-versus-host disease/relapse-free survival rate was 49.3%. The patients in different group of relative high-risk prognostic scoring or low-risk prognostic scoring, with or without poor-risk mutation(s), with mutations number ≥3 or <3 had similar 5-year OS rate (more than 70%). Multivariate analysis showed that the incidence of grade III-IV aGVHD was the independent risk factor affecting OS（P =0.008）and DFS (P =0.019).@*CONCLUSION@#Allo-HSCT with Dec-conditioning regimen is feasible and effective in the treatment of patients with MDS and MDS-AML, especially those in high prognostic risk and with poor-risk mutations.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin / 中国实验血液学杂志

OBJECTIVE@#To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms.@*METHODS@#MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot.@*RESULTS@#The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group.@*CONCLUSION@#MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Effects of Bone Marrow Mesenchymal Stem Cells Derived from Patients with Newly Diagnosed Acute Myeloid Leukemia on the Cell Proliferation, Cell Cycle and Immunophenotypes of HL-60 Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the role of bone marrow microenvironment(niche) in the development of acute myeloid leukemia (AML) and the effect of AML patients-derived MSC on the proliferation, cell cycle and immuno-phenotypes of HL-60 cells.@*METHODS@#The MSC derived from bone marrow of patients with newly diagnosed AML were isolated and co-cultured with HL-60 cells. The effect of MSC on proliferation of HL-60 cells was detected by using 3H-TdR incorporation method, the cell cycle and immunophenotypes of HL-60 cells were detected by flow cytometry.@*RESULTS@#The results of 3H-TdR incorporation assay showed that both AML-MSCs and normal MSCs remarkably suppressed the HL-60 cell proliferation in a time- and dose-dependent manner. The results of cell cycle analysis demonstrated that AML MSCs and normal MSCs induced arrest of the HL-60 cells in G/G phase. The results of immunophenotyping revealed that MSCs suppressed the expression of CD11a and CD154 on the surface of HL-60 cells. Moreover, AML MSCs exhibited increased inhibitory effects than that of normal MSCs. However, no remarkable effect of MSCs on CD54 expressions of HL-60 cells was observed in the current study.@*CONCLUSION@#AML-MSCs possess effects on HL-60 cell proliferation, cell cycle and immunophenotypes similiar to normal MSCs, but exhibited increased suppressive capacity on the expression of CD11a and CD154.

Bone Marrow Mesenchymal Stem Cells from Newly Diagnosed Acute Myeloid Leukemia Patients Exhibit Enhanced Osteogenic Differentiation Capacity / 中国实验血液学杂志

OBJECTIVE@#To study the influence of acute myeloid leukemia (AML) microenvironment on mesenchymal stem cells (MSCs).@*METHODS@#MSCs were isolated from the bone marrow of newly diagnosed AML patients (AML-MSCs) and were cultured. The morphology of MSC was observed by inverted microscopy, the immunophenotypes of MSC were detected by flow cytometry, the proliferation ability of MSC was detected by using MTT method, the multi-differentation ability of MSC was assayed by osteogenic, lipogenic and chrondrogenic induction. The morphologic features, immunophenotypic characteristics, cell proliferation, and multipotential differentiation capability were compared between the MSC derived from normal healthy donors and AML patients.@*RESULTS@#AML-MSCs presented the morphological features similar to the normal MSCs. In addition, AML-MSCs highly expressed CD29, CD44, CD73, CD105 and HLA-ABC. Meanwhile, they were homogenously negative for CD14，CD31, CD34, CD45, CD80, CD86 and HLA-DR. Further-more, AML-MSCs showed cell proliferation ability similar to normal MSCs. Notably, AML-MSCs exerted increased osteogenic-differentiation capacity as compared with normal MSCs.@*CONCLUSION@#AML-MSCs possess typical MSC phenotypes but displayed enhanced osteogenic-differentiation capacity.

Clinical research of severe aplastic anemia with pulmonary invasive fungal infection after transplantation / 解放军医学杂志

Objective To study the clinical characteristics of pulmonary invasive fungal infection (IFI) in patients with severe aplastic anemia (SAA) after allogeneic hematopoietic stem cells transplantation (allo-HSCT).Methods The clinical data of 30 SAA patients received allo-HSCT in 307 Hospital of PLA from Jan.2012 to Jan.2015 were analyzed retrospectively.All the SAA patients accepted combined bone marrow with peripheral blood stem cells transplantation,and then the clinical features and outcome of pulmonary IFI after transplantation were analyzed.Results Six (20％) of the 30 SAA patients were complicated by IFI after transplantation,including 4 males and 2 females with average age of 18.3(6-42) years.All the 6 patients showed characteristic imaging changes in pulmonary,of which 3 cases were definitely diagnosed by bronchoscopy or sputum fungal culture,and the other 3 cases by imaging and clinical manifestation.Four cases were cured and 2 cases died after first-line antifungal therapy,the total mortality was 33.3％.Conclusion High incidence of complicated IFI in SAA patients after allo-HSCT may lead to the high mortality,it's necessary to give an empiric antifungal therapy as earlier as possible.

Relationship between NK Cell Reconstitution and aGVHD after Allo-HSCT / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between NK cell count/activity and acute graft-versus-host disease (aGVHD) in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>A total of 26 patients who had undergone allo-HSCT from January to July 2015 were enrolled in this study. The NK cell count/activity in the peripheral blood of recipients on day 30 after allo-HSCT were monitored by using 4-color flow cytometry. The incidence of aGVHD in patients was evaluated by clinical manifestation combinating with related pathologic indicators, and the relationship between NK cell count/activity and aGVHD were analyzed.</p><p><b>RESULTS</b>In the aGVHD group and the no-aGVHD group, the NK cell count and activity on days 30 after allo-HSCT were 655±216 cells/µl vs 1169±372 cells/µl(P=0.002) and 7.3±3.6% vs 9.0±3.6% (P=0.008). In the II-IV grade aGVHD group and the 0-I grade aGVHD group, the NK cell count/activity were 617±220 cells/µl vs 1081±399 cells/µl (P=0.001) and 4.2±1.7% vs 8.3±3.5%(P=0.001). As compared with the 0-I grade aGVHD group, patients in the II-IV grade aGVHD group had higher relapse rate (57% vs 5%)(P=0.010) , lower 1-year progression-free survival(PFS) rate (43% vs 84%)(P=0.010).</p><p><b>CONCLUSION</b>NK cell count/activity on day 30 after allo-HSCT were closely relates with aGVHD, which may be a potential marker for aGVHD and can provide a new target for aGVHD therapy.</p>

Construction and expression of recombinant adenovirus vector Ad5-hTRX-EGFP / 中国实验血液学杂志

This study was purposed to construct and prepare the recombinant adenovirus vector carrying human thioredoxin (hTRX) and enhanced green fluorescence protein (EGFP), and transfect it into HEK293 cells, so as to lay a foundation for further gene therapy. The PCR-amplified products of hTRX with a pair of primers containing Not I and EcoR V restriction sites were subcloned into shuttle plasmid pDC316-mCMV. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC316-hTRX-EGFP and large adenovirus-helper plasmid pBHGlox (delta) E1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells, purified by CsCl gradient centrifugation, counted for virus particles and determined for titer. The recombinant adenovirus was identified by PCR. The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry. The expression of hTRX was confirmed by Western blot. The results showed that according to PCR and restriction endonuclease assay, the target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of designed fragments. The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5.558×10(10) pfu/ml. A transfection efficiency of 92.25% could be achieved at MOI = 100. Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells. It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells, which laid a foundation for further investigation.

Effect of human bone marrow mesenchymal stem cell on allogeneic T lymphocyte phenotype in vitro / 中国实验血液学杂志

The purpose was to study the effect of mesenchymal stem cell (MSC) on immune function, and to explore the new strategy to prevent graft versus host disease (GVHD) and host versus graft reaction (HVGR) in allogeneic bone marrow transplantation. MSCs from human bone marrow cells were isolated and cultured. The purity of MSCs were identified with the spindle-fibroblastic morphological characterization by microphotography, and the phenotypes were tested by flow cytometry. MSCs were planted in 6-well plates (8 x 10(4)/well for group A, 4 x 10(4)/well for group B and 2 x 10(4)/well for group C), and cocultured for 7 days with T cell isolated from peripheral blood. Peripheral blood T cells non-cocultured with MSC acted as the control group. CD3, CD4, CD8, and CD25 expressed on T cells were analyzed by flow cytometry after coculture with MSCs for 0, 24, 72 hours and 7 days respectively. The results showed that CD4(+)CD25(+) T cells and CD8(+) T cells of allogeneic T lymphocytes cocultured with bone marrow MSCs (group A and group B) increased obviously as compared with control group (T lymphocytes uncocultured with MSCs), and there were no difference between groups A and B. CD3, CD4, CD8 and CD25 expressed on T cells had no significant difference between experimental groups and control group. The result suggested that CD4(+)CD25(+)-regulatory T cells and CD8(+) T cells were increased apparently when cocultured with allogeneic MSCs. It is concluded that MSCs pretreatment may be useful in the prevention of GVHD and HVGR in allogeneic bone marrow transplantation and provides a new strategy to induce transplantation tolerance.

The effect of MSC on cytokine production by naive T cell differentiated in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of mesenchymal stem cell (MSC) on naive T cell and to explore its mechanism of immunoregulation.</p><p><b>METHODS</b>After culturing for 3 passages, MSC was incubated with naive T cells differentiated from cord blood CD34(+) cells in vitro. Variance of cytokine produced by naive T cell in culture supernatant was analyzed by enzyme-linked immunoassays.</p><p><b>RESULTS</b>On the 7th day of co-culture, a mild proliferation of T cells in the co-culture group was observed: (9.15 +/- 0.68) x 10(5)/well in MSCs + naive T cells group versus (4.87 +/- 1.33) x 10(5)/well in naive T cells alone group (P < 0.05). IFN-gamma production was lower in MSCs + naive T cells group than that in naive T cells alone group: (1.147 +/- 0.181) pg/ml versus (4.897 +/- 0.189) pg/ml (P < 0.05), but IL-2 production was higher in the co-culture group: (16.141 +/- 2.729) pg/ml versus (2.551 +/- 0.460) pg/ml (P < 0.05). Neither IL-4 nor IL-10 were detected.</p><p><b>CONCLUSIONS</b>MSC have allogeneic effect on naive T cell, but may suppress alloreactive T lymphocyte and reduce the incidence of GVHD partly by decreased IFN-gamma production. The result may provide new clues for explaining immunoregulatory mechanism of MSC.</p>