Clinical Study on Dynamic Detection of Minimal Residual Disease in Acute Myeloid Leukemia by Multiparameter Flow Cytometry / 中国实验血液学杂志

OBJECTIVE@#To investigate the prognostic significance of dynamic detection of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) by 8-color flow cytometry.@*METHODS@#MRD of 282 AML patients who achieved remission after initial therapy was detected by 8-color flow cytometry. MRD threshold for predicting recurrence was determined by receiver operating characteristic (ROC) curve, and time from MRD-positive to clinical recurrence was analyzed. The differences in overall survival (OS) time and relapse-free survival (RFS) time of patients with different MRD-changes were compared, and the related factors of recurrence in patients with MRD-negative were analyzed by univariate and logistic regression analysis.@*RESULTS@#ROC curve determined that the MFC-MRD threshold for predicting the recurrence of AML was 0.105%, and the recurrence rate of MRD-positive patients was significantly higher than that of MRD-negative patients [52.45% (75/143 cases) vs 35.97% (50/139 cases), P=0.005]. The patients in MRD persistent positive group and negative to positive group recurred earlier than those in positive to negative group and negative-positive fluctuation group (P<0.005). Survival analysis showed that OS and RFS time of patients with MRD persistent positive were significantly shorter than those of patients with MRD persistent negative, positive to negative, and negative-positive fluctuation (P<0.005). There was no significant difference in OS and RFS between MRD negative to positive group and MRD persistent positive group (P>0.005), either between MRD persistent negative group and MRD positive to negative group (P>0.005). Among 139 MRD-negative patients, 50 recurred. Univariate and logistic regression analysis showed that the risk of recurrence increased with the increase of white blood cells level (95%CI: 1.000-1.013, P=0.045). The risk of recurrence in patients without hematopoietic stem cell transplantation (HSCT) was 9.694 times higher than that in patients who received HSCT (95%CI: 1.720-54.651, P=0.010), and in the high-risk group was 5.848 times higher than that in the low-risk group (95%CI: 1.418-24.121, P=0.015).@*CONCLUSION@#The prognosis of AML patients with different MRD changes is significantly different. No matter MRD-positive or MRD-negative at the initial remission, dynamic detection of MRD after treatment is more helpful to accurately guide treatment.

Effect of Chidamide on the Killing Acitivity of NK Cells Targeting K562 Cells and Its Related Mechanism In Vitro / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of chidamide on the killing activity of NK (Natural killer cell, NK) cells targeting K562 cells and its related mechanism.@*METHODS@#K562 cells were pretreated with chidamide at different concentrations and cocultured with NK cells at different effect-target ratios. The killing effect of chidamide on K562 cells by NK cells, the expression of natural killer group 2 member D (NKG2D) ligands and apoptosis rate of K562 cells were detected by flow cytometry.@*RESULTS@#The killing sensitivity of NK cells to K562 cells could be enhanced by chidamide. The expression of ULBP2 on K562 cell surface could be up-regulate, however, the expression of ULBP1 and MICA/MICB showed no statistically difference as compared with control group. Chidamide showed no obvious cytotoxicity to K562 cells.@*CONCLUSION@#Chidamide can significantly improve killing efficiency of NK cells on K562 cells, which may be related to the up-regulation of ULBP2 expression.

Killing Effect of A CD7 Chimeric Antigen Receptor-Modified NK-92MI Cell Line on CD7-Positive Hematological Malignant Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the killing effect of NK-92MI cells modified by chimeric antigen receptor (CD7-CAR) and specifically targeting CD7 to CD7 hematological malignant cells.@*METHODS@#Three types of hematological malignant tumor cells, including 5 cases of CD7 acute T-lymphoblastic leukemia (T-ALL), 10 cases of acute myeloid leukemia (AML) and 6 cases of T-cell lymphoma were collected, centrifuged, cultured and used to detect the expression levels of tumor cell surface targets; 7-AAD, CD56-APC, CD3-FITC, IgG Fc-PE flow cytometry were used to detected the transfection efficiency of NK-92MI and CD7-CAR-NK-92MI cells, killing efficiencies of CD7-CAR-NK-92MI cells to CD7 hematological tumor cells in vitro were determined by flow cytometry using PE Annexin V Apoptosis Detection Kit. Secretion differences of NK-92MI and CD7-CAR-NK-92MI cytokines interleukin (IL)-2, interferon (IFN)-γ, and granzyme B detection were estimated by using CBA kit.@*RESULTS@#The killing efficiencies of CD7-CAR-modified NK-92MI cells to CD7 T-ALL, AML, T-cell lymphoma tumor cells were significantly higher than those of NK-92MI cells without genetical modification. The difference showed statistically significant (P＜0.05). The level of IFN-γ and granzyme B were significantly increased among cytokines secreted by CD7-CAR-modified NK-92MI cells as compared with those of NK-92MI cells without genetical modification (P＜0.05) .@*CONCLUSION@#CD7-CAR-modified NK-92MI cells have significantly improved killing efficiency against CD7 T-ALL, AML and T lymphoma cells, and shows specific targeting effects, which provides a clinical basis for the treatment of CD7 hematological malignancies.

Expression of Lysosomal Membrane Proteins LAMP1, TPC1 and TPC2 in Acute Myeloid Leukemia Cells and Its Clinical Significance / 中国实验血液学杂志

OBJECTIVE@#To investigate the relationship between the expression of lysosomal membrane proteins LAMP1, TPC1 and TPC2 in acute myeloid leukemia (AML) cells and clinical indications of AML and to explore the possible role in the genesis and development of AML and clinical significance.@*METHODS@#Real-time quantitative PCR was used to detect the mRNA expression of LAMP1, TPC1 and TPC2 in AML cell lines (HL-60, NB4) and 57 patients with acute myeloid leukemia (including 44 initially treated patients and 13 relapsed and refractory patients). The relationship of mRNA expression levels with clinical indicators and post-chemotherapy remission was analyzed.@*RESULTS@#Compared with CD34 hematopoietic stem cells (HSC), the expression levels of LAMP1 and TPC1 in AML cell lines HL-60 and NB4 significantly increased, while the expression level of TPC2 was not significantly different. The expression levels of LAMP1, TPC1 and TPC2 in bone marrow mononuclear cells (BMMNC) of AML patients were higher than those in normal human BMMNC (P90%) were also high. There was no significant difference in the expression of LAMP1, TPC1 and TPC2 between CD34HSC of patients with AML and relapsed/refractory patients (P>0.05). No correlation was found between age, sex and genotype and expression of membrane proteins (P>0.05). The expression levels of LAMP1 and TPC1 positively correlated with the number of white blood cells in peripheral blood of patients (P<0.01). LAMP1 and TPC2 were found to be associated with remission after a course of chemotherapy in newly diagnosed patients. Initially treated patients with high expression of LAMP1 in the bone marrow not easily relieved after one course of chemotherapy. Patients with high expression of TPC2 in the bone marrow more likely to be relieved after one course of chemotherapy.@*CONCLUSION@#The mRNA of the three membrane proteins are highly expressed in AML patients, and LAMP1 and TPC1 are risk factors for AML disease progression. High expression of TPC2 is beneficial for chemotherapy of patients with newly diagnosed AML.

Effect of MiR-99a-5p on the Differential Ability of Human Bone Marrow Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of microRNA-99a-5p (miR-99a-5p) on differentiation ability of human bone marrow mesenchymal stem cells (BM-MSC).</p><p><b>METHODS</b>BM-MSC was cultured and then transfected with miR-99a-5p mimics or inhibitors. The transfection efficiency was detected by real-time quantitative PCR. The effects of miR-99a-5p on the adipogenic and osteogenic differentiation ability of BM-MSC were detected by differentiation experiment.</p><p><b>RESULTS</b>As compared with the negative control group, the expression of miR-99a-5p was significantly up-regulated after transfection with miR-99a-5p mimics(P<0.001), the expression of miR-99a-5p was down-regulated after transfection with miR-99a-5p inhibitor (P<0.001). In osteogenic differentiation experiments, the miR-99a-5p overexpression could promote the osteogenic differentiation, while the downregulation of miR-99a-5p expression inhibited the osteogenic differentiation. The same results were obtained by semi-quantitative detection through spectrophotometry. In the adipogenic differentiation test, transfection of miR-99a-5p mimics or inhibitors had no significant effect on the adipogenic differentiation of BM-MSC.</p><p><b>CONCLUSION</b>Overexpression of miR-99a-5p can promote the osteogenic differentiation of BM-MSC, but no significant effects are observed in the adipogenic differentiation.</p>

Effect of MicroRNA-214 on Migration of Cord Blood CD34Cells Induced by Bone Marrow Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the possible effect of MicroRNA-214 (miR-214) on migration of umbilical cord blood CD34hematopoietic stem/progenitor cells induced by BM-MSC.</p><p><b>METHODS</b>After transfection of the inhibitor or mimic of miR-214, the cultured supernatant of BM-MSC were collected respectively. The expression of miR-214 in BM-MSC was detected by real time quantitative PCR, the effect of supernatant on CD34cell migration was evaluated by chemotaxis assays. The levels of chemokine(SDF-1) secreted by BM-MSC in the supernatant were detected by ELISA.</p><p><b>RESULTS</b>The cultured supernatant of BM-MSC could promote the migration of CD34cells. Compared with the group without transfection or negative control(NC) group, the transfection with miR-214 mimic could promote the migration of CD34cells (P<0.01), while the migration rate in miR-214 inhibitor groups decreased significantly (P<0.01). Further study found that the concentration of SDF-1 was not changed notably in all groups (P>0.05), as compared with the control group.</p><p><b>CONCLUSION</b>miR-214 signalings may indirectly increase the migration of CD34hematopoietic stem/progenitor cells by modulating BM-MSC functions, which may not significantly correlate with the chemokine SDF-1 secreted by BM-MSC.</p>

Effect of MicroRNA-146a on Differentiation Potential of Human Bone Marrow Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of MicroRNA-146a (miR-146a) on the ability of BM-MSC to differentiate into adipocytes and osteoblasts.</p><p><b>METHODS</b>BM-MSC were isolated from the bone marrow of healthy donors. The differentiation of BM-MSC into adipocytes and osteoblasts cells were done in vitro. After transfection with miR-146a inhibitor or mimics, the expression of miR-146a in BM-MSC was detected by real time quantitative PCR. The effect of MicroRNA-146a on the differentiation potential of BM-MSC was evaluated after transfection.</p><p><b>RESULTS</b>BM-MSC possessed the ability to differentiate into adipocytes and osteoblasts cells when cultured in the induction medium. The expression of miR-146a was correspondingly down-regulated and up-regulated in BM-MSC after transfection. Compared with the control group, the expression of miR-146a was down-regulated (P < 0.01) after transfection with miR-146a inhibitor, while after transfection with miR-146a mimics it was significantly up-regulated. This study proved that the transfection with miR-146a inhibitor can inhibit BM-MSC differentiate into adipocytes (P < 0.01), while transfection with miR-146a mimics can promote differentiation of BM-MSC into adipocytes (P < 0.01). No effect of miR-146a inhibitor or miR-146a mimics on osteogenic differentiation of BM-MSC was observed (P > 0.05).</p><p><b>CONCLUSION</b>BM-MSC possess the ability to differentiate into adipocytes and osteoblasts. The miR-146a can promote BM-MSC to differentiate into adipocytes.</p>

Influence of TLR2- and TLR4-activated Mesenchymal Stem Cells on Migration of Cord Blood CD34+ Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to investigate the possible effect of Toll-like receptors 2 (TLR2) and Toll-like receptors 4 (TLR4) on the migration function of umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells induced by bone marrow-derived mesenchymal stem cells (MSCs) and to explore the underlying mechanism.</p><p><b>METHODS</b>The expression of TLR2 and TLR4 on MSC was detected with flow cytometry. After the MSC were pretreated with TLR2 agonist (PAM3CSK4) and/or TLR4 agonist (LPS), the supernatants were collected. The effect of the supernatants on the migration of CD34+ cells was evaluated with chemotaxis assays. Alterations of chemokine (SDF-1) secreted by MSC in the supernatants were assayed by ELISA.</p><p><b>RESULTS</b>The expression levels of TLR2 and TLR4 were (31.5±4.6)% and (85.6±6.7)% respectively. Compared with the blank group, the migration ability of CD34+ cells increased significantly in control, LPS and/or PAM3CSK4 groups (P<0.01). Further study found that LPS and/or PAM3CSK4 enhanced the chemotactic ability of CD34+ cells (P<0.05), but the concentration of SDF-1 was not changed significantly in all of LPS and/or PAM3CSK4 groups (P>0.05) in comparison with the control group.</p><p><b>CONCLUSION</b>TLR2 and TLR4 signalings may indirectly increase the migration of CD34+ hematopoietic stem/progenitor cells by modulating BM-MSC functions, which may not significantly correlate with the production of chemokine SDF-1 by MSCs.</p>

Amplification Ex Vivo and Cytocidal Activity of Leukemia Tumor-Associated Antigen-Specific Cytotoxic T Lymphocytes / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the feasibility of amplifying the leukemia tumor associated antigens-specific cytotoxic T lymphocytes (TAA-CTL) ex vivo and to evaluate the cytotoxicity of TAA-CTL.</p><p><b>METHODS</b>The peripheral blood mononuclear cells were enriched by density gradient centrifugtion; TAA-CTL were generated by stimulation of PBMNC with peptide-pulsed DC at an effector-to-target ratio of 10:1; immunophenotype of TAA-CTL was analyzed by flow cytometry; cytotoxicity assay was used to evaluate the cytotoxic activity of TAA-CTL against peptide-pulsed autologous target cells (PHA-Blasts).</p><p><b>RESULTS</b>TAA-CTL expanded from volunteer showed a mean expansion of 3.81±1.61, the phenotyping of the TAA-CTL was predominantly CD3+ (97.22±0.71)% with varying content of CD4+ (41.47±27.08)% and CD8+ (56.40±11.15)% T cells, it also contained few nature killer cells (0.50±0.31)% and rare residual B cells (0.14±0.20)%; the subpopulations of TAA-CTL and CTL were not statisticaly significantly different in the proportion (P>0.05); the detection of intracellular cytokines after stimulation with peptide showed that the secretion rates of IFN-γ and TNF-α in CD8+ TAA-CTL were (27.67±2.21)% and (34.2±0.71)%, while the secretion rates were (21.6±2.55)% and (9.97±3.44)% in CD4+ TAA-CTL. Compared with the CD8+ TAA-CTL group, the secretion rates of IFN-γ and TNF-α were (1.36±0.04)% and (5.58±0.03)% in CD8+ CTL, the rates of IFN-γ and TNF-α were (0.91±0.06)% and (1.60±0.07)% in CD4+ CTL. The secretions of IFN-γ and TNF-α in CTL were both significantly lower than those in TAA-CTL (P<0.01); the specific killing efficiency of the TAA-CTL against TAA-pulesd target cells were (77.00±1.00)%, (67.40±3.60)%, (60.55±2.45)% and (26.85±5.25)%, when the effecto-target ratios were 40:1, 20:1, 10:1 and 5:1, and there was negligible lysis of TAA-CTL for PHA-blast (P<0.01).</p><p><b>CONCLUSION</b>TAA-CTL can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood, and the TAA-CTL possess a specific killing activity.</p>

Extracellular HMGB1 promotes the migration of cord Blood CD34⁺ cells via SDF-1/CXCR-4 axis / 中国实验血液学杂志

This study was aimed to investigate the effect of high mobility group box1(HMGB1) and/or stromal cell derived factor-1(SDF-1) on the migration of cord blood CD34⁺ cells, and to explore whether HMGB1 promotes cord blood CD34⁺ cell migration via SDF-1/CXCR4 axis. Cord blood mononuclear cells were isolated by Ficoll-Paque density centrifugation, CD34⁺ cells were collected by a positive immunoselection procedure (CD34 MicroBeads) according to the manufacturer's instructions, the purity of the isolated CD34⁺ cells was detected by flow cytometry. In vitro chemotaxis assays were performed using Transwell cell chambers to detect cells migration. 1 × 10⁵ cells/well cord blood CD34⁺ cells were added into the upper chambers. Different concentrations of HMGB1 and/or SDF-1 (0, 10, 25, 50, 100, 200 ng/ml) were used to detect the optimal concentrations of HMGB1 and/or SDF-1 for inducing migration of cord blood CD34⁺ cells. Freshly isolated cord blood CD34⁺ cells express CXCR4 (SDF-1 receptor), and HMGB1 receptor TLR-2,TLR-4 and RAGE. To explore which receptors were required for the synergy of HGMB1 and/or SDF-1 on cells migration, the anti-SDF-1, anti-CXCR4 and anti-RAGE antibodies were used to detect the effect of HGMB1 alone or with SDF-1 on cord blood CD34⁺ cells migration. The results showed that the purity of CD34⁺ cells isolated from cord blood mononuclear cells by magnetic cell sorting was 97.40 ± 1.26%, the 25 ng/ml SDF-1 did not induce migration of cord blood CD34⁺ cells, whereas the optimal migration was observed at 100 ng/ml. HMGB1 alone did not induce migration up to 100 ng/ml. The dose test found that the the best synergistic concentrations for cells migration were 100 ng/ml HMGB1 combined with 50 ng/ml SDF-1. The blocking test showed that both the anti-SDF-1 (4 µg/ml) and anti-CXCR4 (5 µg/ml) antibodies could block cell migration induced by HMGB1 or combined with SDF-1, but the cord blood CD34⁺ cells in the presence of anti-RAGE, anti-TLR-2 and anti-TLR-4 antibodies did not modify the response to SDF-1 in the presence of HMGB1. It is concluded that both HMGB1 and SDF-1 can induce cord blood CD34⁺ cells migration, HMGB1 enhances SDF-1-induced migration exclusively via CXCR4 and in a RAGE and TLR receptors-independent manner, the exact mechanism needs to be further explored.

The clinical value of F-18 FDG PET/CT in patients with secondary hemophagocytic syndrome / 中国实验血液学杂志

The aim of this study was to investigate the role of F-18 fluoro-2-deoxyglucose positron emission tomography/computed tomography (F-18 FDG PET/CT) in diagnosis and prognostic evaluation of secondary hemophagocytic syndrome (HPS). A total of 11 secondary HPS patients examined with 18F-FDG-PET/CT were retrospectively analyzed. The diagnostic value of F-18 FDG PET/CT for malignancy detection was assessed. The values of maximum standardized uptake value (SUV(max)) in spleen (SUVS(p)) and in bone marrow (SUVBM) were measured to analyze their relationship with various laboratorial parameters and clinical outcome of secondary HPS patients. The results showed that 4 out of the 11 patients had malignancies, the sensitivity, specificity and diagnostic accuracy of F-18 FDG PET/CT for malignancy detection were 100%, 66.7% and 75% respectively, the SUV(max) of spleen and bone marrow showed no significant correlation with laboratorial parameters, a maximum SUVS(p) of 3.10 and a maximum SUVBM of 3.47 were the optimal cutoffs for predicting patients' outcome, the increased uptake of F-18 FDG in the BM and spleen were significantly associated with shorter survival time according to univariate analysis. It is concluded that 18F-FDG PET/CT may especially play an important role in diagnosis and predicting outcome of secondary HPS for the small sample size.

Immunophenotyping characteristics of AML and their correlation with the curative effects / 中国实验血液学杂志

This study was aimed to explore the immunophenotyping characteristics of acute myeloid leukemia (AML) and their correlation with the curative efficacy. The bone marrow or blood samples were collected from 516 patients with newly diagnosed AML, and their immunophenotypes were analyzed by flow cytometry. The results showed that (1) In 516 cases, the ratios of myeloid antigen expression were higher, as follows: MPO 95.0%, CD33 93.0%, CD13 88.8%, CD117 69.4%; and the expressions of CD14, CD15, CD64 and CD71 were lower, meanwhile 145 cases were accompanied with lymphocyte antigen expression, the ratios were as follows: CD7 21.5%, CD19 6.0%, CD2 0.78%, CD10 0.58% and CD20 0.58%; the positive expression rate of CD71 in M6 was 100%, and that of CD64 in M5 was the highest (30.2%); the overall positive rate of CD34 was 57.8%. (2) After first chemotherapy, the complete remission (CR) rate was 64.7%, CR rate of CD34(+) patients was lower than that of CD34(-) in M3 group (P = 0.019). The CR rate of CD34(+) patients was significantly lower than that of CD34(-) in non-M3 group (P = 0.002). The CR rate of CD19(+) patients was higher than CD19(-) (P = 0.028); the CR rate of CD7(+) patients was significantly lower than that of CD7(-) (P = 0.002); the CR rate of CD71(+) patients was lower than that of CD71(-) (P = 0.013); the CR rate of MPO(+) patients was higher than that of MPO(-) (P = 0.015). Between the CR rate of CD11b, CD13, CD33-positive and-negative group, the difference was not statistically significant (P > 0.05). It is concluded that the phenotype is a prerequisite for the diagnosis of AML, and can help to guide the clinical typing, selection of treatment protocols and evaluation of prognosis.

Influence of TLR2 and TLR4 agonists on migration of human bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the influence of TLR2 and TLR4 agonists on the migration and adhesion activity of human bone marrow-derived mesenchymal stem cells (MSC) and to clarify the underlying mechanisms. The expression of TLR2 and TLR4 on MSC was detected by flow cytometry. The effects of TLR2 agonist (PAM3CSK4) and TLR2 agonist (LPS) on MSC migration and adhesion ability were evaluated with chemotaxis and adhesion test. The results indicated that expressive levels of TLR2 and TLR4 on surface of human bone marrow MSC were (24.5 ± 3.2)% and (91.3 ± 5.2)% respectively. Compared with the control group, the migration activity of MSC toward SDF-1 was decreased significantly in PAM3CSK4 group, while the adhesion activity of MSC was promoted by PAM3CSK4 exposure. However, both the migration activity toward SDF-1 and the adhesion activity of MSC were not changed significantly in LPS-treated group. Further, it was found that PAM3CSK4 did not affect the expressive level of CXCR4 on MSC, however, it could inhibit the spontaneous migration of MSC in dose dependent manner. It is concluded that activation of TLR2 can decrease the migration ability of MSC, which may associate with the decreased spontaneous migration ability and the increased adhesion activity of MSC.

Comparison of curative efficacy after G-CSF-mobilized sibling HLA-matched peripheral blood hematopoietic stem cell transplantation versus that combined with BMT for patients with hematologic malignancies in a single center / 中国实验血液学杂志

This study was aimed to retrospectively analyze and compare the clinical curative efficacy of patients with hematologic malignancies after G-CSF-mobilized sibling HLA-matched (sm) peripheral blood hematopoietic stem cell transplantation (sm-allo-PBHSCT) and sm-allo-PBHSCT combined with bone marrow transplantation (BMT). 100 patients received sm-allo-HSCT in a single center from October 2001 to October to 2010, included 38 patients received sm-allo-PBHSCT and 62 patients received sm-allo-PBHSCT combined with BMT. The myeloablative or reduced intensity conditioning regimens were chosen according to the condition of patients. All patients received standard cyclosporine (CsA) and mycophenolate mofetil (MMF) as prophylaxis for GVHD. The results showed that the rapid hematopoietic reconstitution was observed in all patients. The median time of ANC ≥ 0.5 × 10(9)/L in both groups were 12 days, the median time of platelet count ≥ 20 × 10(9)/L was 15 days in sm-allo-PBHSCT group and 16 days in sm-allo-PBHSCT + BMT group. The incidence of acute GVHD, acute GVHD of III-IV grade and chronic GVHD in sm-allo-PBHSCT and sm-allo-PBHSCT + BMT groups were 37.1% and 34.2%, 7.89% and 8.06%, 36.11% and 41.38% respectively, there were no statistical differences. The relapse rates were similar in two groups (sm-allo-PBHSCT 13.16% vs sm-allo-PBHSCT + BMT 12.9%). The 3-year disease-free survivals in sm-allo-PBHSC and sm-allo-PBHSCT + BMT groups were 57.1 ± 8.7% and 61.3 ± 6.4% respectively (p = 0.852). The 2-year overall survival of high-risk patients was 41.4 ± 12.8% in sm-allo-PBHSCT group, while 60.9 ± 9.6% in sm-allo-PBHSCT + BMT group (p = 0.071). It is concluded that the rhG-CSF mobilized sibling matched allo-PBHSCT + BMT is superior to the rhG-CSF mobilized sibling matched allo-PBHSCT in increasing the overall survival of high-risk hematologic malignancies.

Influence of TLR2 and TLR4 agonists on migration of cord blood CD34(+) cells / 中国实验血液学杂志

This study was aimed to investigate the influence of TLR2 and TLR4 agonists on the migration and adhesion activity of umbilical cord blood (UCB) CD34(+) cells and to explore the underlying mechanism. The expression of TLR2 and TLR4 on UCB CD34(+) cells was detected with flow cytometry. The effect of TLR2 agonist (PAM3CSK4) and TLR2 agonist (LPS) on the migration and adhesion ability of UCB CD34(+) cells was evaluated with chemotaxis and adhesion assays. The results indicated that expression levels of TLR2 and TLR4 were (14.2 ± 3.8)%, (19.6 ± 4.1)% respectively. Compared with the control group, the migration activity of UCB CD34(+) cells toward SDF-1 decreased significantly in LPS group (p < 0.01). The adhesion activity was not altered significantly in LPS group. However, both the migration activity towards SDF-1 and the adhesion activity of UCB CD34(+) cells were not changed significantly in PAM3CSK4 group. Further study found that LPS did not affect the expression level of CXCR4 on CD34(+) cells, but could inhibit the spontaneous migration ability of CD34(+) cells. It is concluded that TLR4 activation can decrease the chemotaxis function of CD34(+) cells towards SDF-1, which may associate with the decreased spontaneous migration ability of CD34(+) cells.

Functional defect of partial homing receptor on human cord blood hematopoietic stem/progenitor cells / 中国实验血液学杂志

This study was aimed to investigate the function defect of partial homing receptor on cord blood hematopoietic stem cells (CBHSC) and explore efficacy and feasibility of intervention in vitro. The expression and activity of active groups in P, E-selectin ligands on CD34+ cells from cord blood, bone marrow and peripheral blood were detected by flow cytometry; meanwhile the expression of active groups in selectin ligands on CD34+ cells treated by fucosyl transferase in vitro was determined by flow cytometry. The results indicated that the expression levels of CD26 on the surface of stem/progenitor cells (CD34+) from cord blood, bone marrow and peripheral blood were (7.62+/-0.63)%, (6.35+/-0.89)% and (6.18+/-0.91)% (p>0.05) respectively. And the activities of CD26 of the three sources of stem cells were 67.15 U/1000 cells (1 U=1 pmol/min), 26.85 U/1000 cells and 20.95 U/1000 cells respectively, in which the activity of CD26 on surface of CD34+ from cord blood was significantly higher than that from other both sources (p<0.01). The expression levels of P-selectin ligand on the stem/progenitor cells three kinds were (83.46+/-6.33)%, (15.65+/-0.89)% and (80.17+/-6.85)%, and the expression levels of E-selectin ligand on stem/progenitor cells of three kinds were (25.31+/-1.03)%, (26.34+/-0.89)% and (29.79+/-1.78)% respectively. The expression of E-selectin ligand on the surface of cord blood stem/progenitor cell CD34+ increased from (25.31+/-1.03)% to (63.23+/-1.08)% after glycosylation engineering. It is concluded that there is no significant difference of the expression of CD26 between the three sources of stem/progenitor cells, but the activity of CD26 in cord blood was obviously higher than that in bone marrow and peripheral blood. The expression of P-selectin ligand on bone marrow stem/progenitor cell was lower than that on stem cells of cord blood and peripheral blood. Glycosylation engineering can promote and elevate the expression of E-selectin ligand on the surface of CD34+ cells from cord blood.

CD47 and leukemia stem cells / 中国实验血液学杂志

CD47, also known as integrin-associated protein (IAP), is an immunoglobulin-like protein. It can inhibit the phagocytosis of macrophages through binding with signal-regulatory protein alpha chain of inhibitory receptor on macrophage (SIRPα). The expression of CD47 on normal hematopoietic stem cells (HSCs) is useful for maintaining the stability of HSCs in body, but the high expression of CD47 existed on leukemia stem cell (LSCs) of AML patients which can reduce the macrophage-induced phagocytosis to LSCs and decrease the clearance of innate immune system of organism to LSCs. In this article, the expression and function of CD47 on HSCs and LSCs as well as the role of CD47 in the prognosis and target therapy of AML are reviewed.

Effects of HMGB1 on human cord blood CD34(+) hematopoietic stem cells proliferation and differentiation in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To study both the release of HMGB1 from irradiation-treated mesenchymal stem cells (MSCs) and the effects of HMGB1 on human cord blood CD34(+) hematopoietic progenitor cell proliferation and differentiation.</p><p><b>METHODS</b>MSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12 Gy gamma-ray irradiation was determined by enzyme linked immunosorbent assay (ELISA). CD34(+) cells were positively selected with a MACS CD34 isolation kit. The freshly isolated CD34(+) cells were cultured in the presence of HMGB1 for 6 days. Phenotype of cultured cells surface molecules (CD13, CD14, CD11c, CD41 and CD71) were analyzed by flow cytometry. The proliferation and differentiation capacities of cord blood HSCs were assayed by colony forming cell assay. The receptors of HMGB1 (RAGE, TLR2 and TLR4) on cord blood CD34(+) cells were detected by flow cytometry.</p><p><b>RESULTS</b>HMGB1 level in the supernatant \[(4.3 +/- 0.9) ng/ml\] of the irradiated MSC was significantly higher than that in control \[(0.4 +/- 0.2) ng/ml\] (P < 0.01). Human cord blood CD34(+) cells expressed the HMGB1 receptors RAGE, TLR2 and TLR4. The HMGB1-treated CD34(+) cells contained higher proportions of CD13(+) \[(32.6 +/- 5.9)% vs (18.4 +/- 3.8)%\], CD14(+)\[(25.4 +/- 4.4)% vs (12.6 +/- 2.7)%\], CD11c(+) \[(20.3 +/- 3.9)% vs (9.8 +/- 2.1)%\], CD71(+) \[(47.1 +/- 7.4)% vs (26.6 +/- 4.6)%\] cells compared with control group did. But HMGB1 did not induce the generation of CD41(+) cells \[(1.3 +/- 0.5)% vs (1.1 +/- 0.4)%\]. Furthermore, HMGB1 profoundly induced the growth of BFU-E, CFU-GM and total CFU in a dose-dependent manner, and this effect was partially inhibited by TLR2 and TLR4 antibodies.</p><p><b>CONCLUSION</b>Human MSC treated with gamma-ray irradiation can release HMGB1, which can induce the proliferation and differentiation of human cord CD34(+) cells.</p>

Early antigen diagnosis of invasive fungal infection in patients with hematological malignancies and patients receiving hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to evaluate the sensitivity and specificity of G/(1, 3-β-D glucan)/GM (galactomannan) combined detection for early diagnosis of invasive fungal disease (IFD), determine the GM positive cut-off value, and investigate the change of diagnostic level before and after G/GM test and the relation of GM value with therapeutic effect. The ELISA with double antibody sandwich was used to detect the serum levels of G and G/M. The results showed that according to determined GM positive cut-off value, the sensitivity, specificity, positive and negative predictive value of G/GM combined detection were 100%, 65.7%, 67.6% and 100%, respectively. The case number of the clinical diagnosis of IFD increased from 1 to 24 cases, the diagnosis of 12 non-infective patients was changed as suspected IFD with the help of G/GM combined detection; the GM cut-off value decreased in patients whose GM cut-off value was higher before therapy and antifungal therapy was effective, while the GM cut-off value increased in patients no-responded to therapy. It is concluded that G/GM combined detection can increased the sensitivity of the diagnosis and reduce false negative rate in the early diagnosis of IFD. The dynamically monitoring G/GM cut-off value can be used as evaluation indicator of therapeutic efficacy.

Unrelated cord blood transplantation in adult patients with hematologic malignancies / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyse the engraftment, transplant-related complications and survival after unrelated cord blood transplantation (UCBT) in patients with hematologic malignancies.</p><p><b>METHODS</b>Twenty eight consecutive adult patients with hematological malignancies were treated with UCBT and 20 of them were advanced-stage diseases. Double or multiple UCB grafts were used for 18 patients, while single UCB graft for 10 patients. Myeloablative conditioning regimens were given to 26 cases and nonmyeloablative regimens to 2 cases. All patients were given a combination of cyclosporine (CsA) and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis.</p><p><b>RESULTS</b>Median time to neutrophil engraftment (≥ 0.5 × 10(9)/L) in 26 patients was 18 (14 - 37) days and platelet engraftment (≥ 20 × 10(9)/L) in 22 patients was 30 (25 - 49) days. Chimerism was weekly assessed by PCR analysis of short tandem repeat (STR) sequences in whole blood or bone marrow and 22 cases were confirmed of fully donor chimeric from 7 to 21 days after transplantation. Eighteen cases developed acute GVHD, greater than grade II in 1, and 6 of 22 patients who survived more than 100 days developed limited chronic GVHD. Eighteen cases were alive in hematologic remission at a median follow-up of 9.5 (2.5 - 72.0) months. The probability of event-free survival at 3 years was 56.7%. Two cases relapsed and 8 of 10 cases died of transplant related complications.</p><p><b>CONCLUSIONS</b>UCBT could be safely and effectively used for adult patients with hematologic malignancies. Use of double UCB units is a strategy extending the feasibility of UCBT.</p>