Effect of endothelial cell-targeted soluble Notch ligand hD1R protein on the proliferation of acute myeloid leukemia cells / 中华血液学杂志

Objective: To evaluate the effects of endothelial cell-targeted soluble Notch ligand hD1R protein on the proliferation of acute myeloid leukemia (AML) cells. Methods: The expression levels of Notch1, Notch2, Notch3, Notch4, Hes1 in bone marrow CD34(+) cells from 24 cases of untreated AML (AML group) and 9 healthy controls (control group) were determined by real time quantitative polymerase chain reaction (PCR) . Recombinant hD1R protein was first induced and purified. Bone marrow CD34(+) cells were co-cultured on human umbilical vein endothelial cells (HUVEC) supplemented with a cocktail containing 5 types of human cytokines (5GF) and soluble hD1R. The cultured cells were tested under different culture conditions including PBS group (PBS replaces HUVEC) , hD1R group, 5GF group, GSI group (hD1R plus GSI) . Proliferation and apoptosis of cultured cells were also analyzed. Real time quantitative PCR was used to test the expression levels of Hes1 and Bcl-2 in cultured cells. Results: The expression levels of Notch1 and Hes1 in primary AML patients were significantly lower, and Notch4 expression was higher compared to the control group (P<0.05) . Cell counting showed a remarkable decrease of AML cells number in the culture with hD1R compared with that in the PBS group[ (0.74±0.13) ×10(5) vs (2.16±0.21) ×10(5), P<0.01]. FACS analysis showed that the percentage of AML cells was (18.48±2.51) % in apoptosis, which was higher than that of control cells (3.19±0.58) % after co-culture with hD1R. AML cells in the hD1R group underwent significantly increased apoptosis compared with those in the PBS one (P<0.05) . Moreover, apoptosis of AML cells in the GSI group was lower than that in the hD1R one (P<0.05) . Apoptosis in the PBS group also decreased compared with that in the 5GF one (P<0.05) . Finally, hD1R up-regulated Hes1 expression and inhibited Bcl-2 one in the AML cells. Conclusion: hD1R effectively activated Notch signaling and down-regulated Bcl-2 mRNA in AML cells, which lead to cell apoptosis.

Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage.</p><p><b>METHODS</b>PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni-beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The LinScal-1c-Kit cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10.</p><p><b>RESULTS</b>The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury.</p><p><b>CONCLUSIONS</b>A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.</p>

Three-year Follow-up on the Safety and Effectiveness of Rituximab Plus Chemotherapy as First-Line Treatment of Diffuse Large B-Cell Lymphoma and Follicular Lymphoma in Real-World Clinical Settings in China: A Prospective, Multicenter, Noninterventional Study / 中华医学杂志(英文版)

Background@#Prospective real-life data on the safety and effectiveness of rituximab in Chinese patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL) are limited. This real-world study aimed to evaluate long-term safety and effectiveness outcomes of rituximab plus chemotherapy (R-chemo) as first-line treatment in Chinese patients with DLBCL or FL. Hepatitis B virus (HBV) reactivation management was also investigated.@*Methods@#A prospective, multicenter, single-arm, noninterventional study of previously untreated CD20-positive DLBCL or FL patients receiving first-line R-chemo treatment at 24 centers in China was conducted between January 17, 2011 and October 31, 2016. Enrolled patients underwent safety and effectiveness assessments after the last rituximab dose and were followed up for 3 years. Effectiveness endpoints included progression-free survival (PFS) and overall survival (OS). Safety endpoints were adverse events (AEs), serious AEs, drug-related AEs, and AEs of special interest. We also reported data on the incidence of HBV reactivation.@*Results@#In total, 283 previously untreated CD20-positive DLBCL and 31 FL patients from 24 centers were enrolled. Three-year PFS was 59% (95% confidence interval [CI]: 50-67%) for DLBCL patients and 46% (95% CI: 20-69%) for FL patients. For DLBCL patients, multivariate analyses showed that PFS was not associated with international prognostic index, tumor maximum diameter, HBV infection status, or number of rituximab treatment cycles, and OS was only associated with age >60 years (P < 0.05). R-chemo was well tolerated. The incidence of HBV reactivation in hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative/hepatitis B core antibody-positive patients was 13% (3/24) and 4% (3/69), respectively.@*Conclusions@#R-chemo is effective and safe in real-world clinical practice as first-line treatment for DLBCL and FL in China, and that HBV reactivation during R-chemo is manageable with preventive measures and treatment.@*Trial Registration@#ClinicalTrials.gov, NCT01340443; https://clinicaltrials.gov/ct2/show/NCT01340443.

Establishment and Identification of SCL-tTA/BCR-ABL Transgenic Mouse Model with Chronic Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish transgenic mouse model with chronic myeloid leukemia(CML) by tetracycline withdrawal, so as to provide the experimental tool for studying pathogenesis of CML and searching new therapeutic method for this disease.</p><p><b>METHODS</b>The BCR-ABL expression in stem and progenitor cells in SCL-tTA/BCR-ABL transgenic mice was induced by tetracycline withdrawal in drinking water; and the peripheral hemogram and spleen index were examined after identifying genotypes in mice; the distribution of hematopoietic lineage in bone marrow, spleen and peripheral blood was detected by flow cytometry.</p><p><b>RESULTS</b>As compared with the control mice, the peripheral hemogram showed the obvious increase of neutrophile granulocyte count and spleen index in BCR-ABL transgenic mice. All induced double transgenic mice showed a severe myeloid cell expansion (Gr-1Mac-1) and reduction in T (CD3) and B(B220) cell lineages in bone marrow, spleen and peripheral blood.</p><p><b>CONCLUSION</b>A mouse model to induce BCR-ABL expression is successfully set up by tetracycline withdrawal, which resembles chronic phase CML.</p>

Correlation between Plasma microRNA Expression and Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the microRNA (miRNA) expression in plasma of patients with aGVHD and without aGVHD after allo-hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The miRNAs (miR-423, mirR199a-3p, miR93*, miR377) expression levels in peripheral blood plasma of 25 patients before and after allo-HSCT were detected by real-time PCR.</p><p><b>RESULTS</b>miR-423, miR199a-3p and miR-93* in aGVHD group were significantly upregulated (P<0.05); miR-377 expression was not significantly different between aGVHD and non-aGVHD (P>0.05).</p><p><b>CONCLUSION</b>The expression of miR-423, miR-199a-3p, miR-93* are upregulated in aGVHD group, which can be used as biomarkes to monitor and to diagnose aGVHD.</p>

Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21 / 中国实验血液学杂志

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Prognostic power of abnormal cytogenetics for multiple myeloma: a multicenter study in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China.</p><p><b>METHODS</b>All 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications.</p><p><b>RESULTS</b>The analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of β2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS.</p><p><b>CONCLUSIONS</b>Chinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.</p>

Clinical analysis of peripheral blood stem cell mobilization regimens in autologous transplantation for treating non-Hodgkin's lymphoma / 中国实验血液学杂志

The purpose of this study was to compare the efficacy of CEP plus G-CSF and CVP plus G-CSF regimens in the mobilization and collection of peripheral blood hematopoietic stem cells (PBHSC), and in the hematopoietic recovery. 57 patients with non-Hodgkin's lymphoma (NHL) underwent autologous PBHSC transplantation were analyzed retrospectively. The PBHSC were mobilized and collected by using CEP plus G-CSF and CVP plus G-CSF respectively, and were retransfused into these NHL patients after preconditioning, then the mobilization efficacy, adverse reactions and hematopoietic recovery were analyzed. The results showed that the WBC count decreased to ≤ 1.0 × 10(9)/L, platelet amount dropped to ≤ 40 × 10(9)/L during peripheral blood stem cell mobilization of all patients, which indicated successful collection of PBHSC. The mean value of (4.38 ± 3.40) × 10(8)/kg mononuclear cells (MNC) containing (2.79 ± 2.53) × 10(6)/kg CD34(+) cells were collected in CEP plus G-CSF group, while the mean value of (3.31 ± 1.23) × 10(8)/kg MNC containing (2.02 ± 0.87) × 10(6)/kg CD34(+) cells were collected in CVP plus G-CSF group. The efficacy of mobilization in CEP plus G-CSF group was significantly higher than that in CVP plus G-CSF group (p < 0.05). After preconditioning, bone marrow was suppressed in all patients. The average time of WBC count recovery to ≥ 1.0 × 10(9)/L was 11.4 days in CEP plus G-CSF group and 12.3 days in CVP plus G-CSF group; the average time of platelet amount recovery to ≥ 50 × 10(9)/L was 18.6 days in CEP plus G-CSF group and 19.3 days in CVP plus G-CSF group. The statistical analysis showed no significant difference in the average time of hematopoietic recovery between 2 groups. It is concluded that autologous PBHSC transplantation shows significant effect for treatment of patients with NHL. Either modified CEP or CVP plus G-CSF regimen is safe and effective in PBHSC mobilization. The CEP plus G-CSF regimen is better than CVP plus G-CSF regimen.

Clinical analysis on the mobilization and collection of peripheral hematopoietic stem cells in 198 healthy donors / 白血病·淋巴瘤

ObjectiveTo investigate the methods and effects on the mobilization and collection of peripheral hematopoietic stem cells.MethodsPeripheral hematopoietic stem cells of 198 cases of healthy donors were selected and mobilized by subcutaneous recombinant human granulocyte colony stimulating factor (rhG-CSF) by (5-10) μg·kg-1 ·d-1 and collected on the 5th day. The effects of gender, height, age and WBC count of donors on mobilization and collection were analyzed. ResultsAll the peripheral hematopoietic stem cells of donors were successfully mobilized.The average counts of mononuclear cells(MNC)and CD34+ cells were (4.19±1.96)×108/kg and (2.98±1.40)×106/kg, respectively, which had no correlation with gender, height and age.The counts of MNC and CD34+ cells collected were positively correlated with the WBC count of peripheral blood (r =0.9201, P =0.0035; r =0.8420, P =0.0149). The donors with WBC ≥20.0×109/L had moresignificant effect than thoseWBC＜20.0×109/L (F =4.688, P =0.0013; F =4.622, P =0.0006). Conclusion The WBC count of peripheral blood from healthy donors is a simple and feasible indicator to predict the quantity of CD34+ cells.

Role of Delta-like 1 in differentiation and antigen presentation of mouse bone marrow-derived dendritic cells / 中国实验血液学杂志

The aim of this study was to investigate the role of Delta-like 1 (Dll1) in differentiation and antigen pre-sensation of mouse bone marrow-derived dendritic cells (DCs). In the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4), mouse bone marrow cells were co-cultured with OP9-Dll1 and OP9-GFP cell lines respectively. After 8 days, the immature DCs were stimulated with tumor antigen. The surface molecules of the activated DCs including MHC II, CD80 and CD86 were analyzed by flow cytometry. Levels of IL-12 and IL-10 in the culture supernatant were detected by ELISA. In addition, the proliferation of T-cells co-cultured with DCs was analyzed by FACS through mixed T-lymphocyte reaction. The results showed that compared with OP9-GFP, the bone marrow cells co-cultured with OP9-Dll1 produced significantly more CD11c(+) DCs (p < 0.05), and possessed higher levels of surface molecule expression including MHC II, CD80 and CD86 after tumor antigen stimulation. The DCs secreted higher level of IL-12 (p < 0.05) and less IL-10 (p < 0.01). They also resulted in significantly stronger T-cell proliferation response. It is concluded that Dll1 can promote the differentiation of DCs from mouse bone marrow cells and enhance their antigen presentation capacity.

Multicenter report of nonmyeloablative allogeneic stem cell transplantation for hematologic diseases / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the treatment effect and toxicity of nonmyeloablative allogeneic stem cell transplantation (NST) for hematologic diseases.</p><p><b>METHODS</b>A total of 243 hematologic diseases patients received HLA-identical NST were enrolled in this study from 9 transplant centers of NST Cooperative Group in China. Nonmyeloablative conditioning regimen was based on fludarabine (Flud), rabbit anti-human thymocyte globulin (ATG), cyclophosphamide (CTX) (FAC), and plus cytarabine or busulfan (BU) etc. Graft-versus-host disease (GVHD) prophylaxis included cyclosporin A (CsA) and mycophenolate mofetil (MMF).</p><p><b>RESULTS</b>Among the 243 patients, 219 (90.1%) achieved full donor chimerism (FDC), 2(0.8%) engraftment failure. 78 (32.1%) had mixture chimerism (MC) at 4 weeks after NST, out of which 56 switched to FDC, 16 remained MC and 6 (2.5%) developed graft rejection. The incidence of acute GVHD was 34.2%, including 6.6% of grade III-IV acute GVHD. Chronic GVHD developed in 78 (32.1%) patients. The follow-up durations were 3 - 99 months, 162 (66.7%) were still alive and the overall survival rates were 76.5%, 73.9%, 70.7%, and 27.8% for MDS/SAA, chronic myeloid leukemia, acute leukemia at first remission, and refractory or relapsed leukemia, respectively.</p><p><b>CONCLUSIONS</b>The nonmyeloablative allogeneic stem cell transplantation based on FAC conditioning results in sustained engraftment and mild aGVHD, providing a new feasible curative therapy for hematology diseases.</p>

Construction and expression of a fusion protein containing extracellular domain of human Jagged1 and Fc fragment of human IgG1 in Pichia Pastoris / 中国实验血液学杂志

In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jagged1 and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.

Research advance of notch signal in ex vivo expansion of hematopoietic progenitor cells - review / 中国实验血液学杂志

Ex vivo expansion of hematopoietic progenitor cells (HPCs) is valuable for clinical application, however, traditional ex vivo culture negatively affects long-term hematopoietic reconstitution ability. In the hematopoietic system, the expression of Notch receptors and their ligands has been widely reported. Active Notch signal inhibits the differentiation of HSCs while promotes their expansion, suggesting that ex vivo expansion of hematopoietic progenitor cells could be enhanced by manipulating Notch signal pathways. In this article the Notch signal pathways, Notch signal and maintenance of hematopoietic progenitor cells, Notch signal and expansion of hematopoietic progenitor cells and molecular mechanism of Notch signal maintaining undifferentiation of hematopoietic progenitor cells were reviewed.

Activating effects of protein transduction domain mediated BCR/ABL protein on CML T cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the activating effect of protein transduction domain (PTD) mediated BCR/ABL protein on T cells from CML patients.</p><p><b>METHODS</b>The plasmid containing PTD and b3a2 bcr/abl of CML was constructed by genetic engineering and expressed in E. coli. The peripheral blood mononuclear cells from CML patients were stimulated in vitro with purified PTD-BCR/ABL protein and the expression of the early activation antigen CD(69) on CD(8)(+) and CD(4)(+) T cells was detected by flow cytometry (FCM).</p><p><b>RESULTS</b>The optimal concentration of PTD-BCR/ABL protein for activating CD(8)(+) T cells in vitro was 100 micro g/ml, CD(69) expression peaked in three days stimulation. CD(8)(+) T cells were activated in 10 of 15 CML patients, the expression rate of CD(69) was (15.01 +/- 3.75)%. CD(4)(+) T cells were activated in 4 of 15 patients, the expression rate of CD(69) was (10.32 +/- 3.08)%. Both CD(8)(+) and CD(4)(+) T cells were activated simultaneously in 3 of them. However, neither CD(4)(+) nor CD(8)(+) T cells was activated by stimulation with BCR/ABL protein in all 15 specimens, the expression rate of CD(69) on CD(8)(+) and CD(4)(+) T cells was (1.36 +/- 0.31)% and (1.41 +/- 0.43)%, respectively. There was no difference compared with that of PBS control group (P > 0.05).</p><p><b>CONCLUSION</b>By using a PTD-mediated antigen delivering system, exogenous BCR/ABL protein can be delivered into APC, processed and presented onto surface of APC to activate Ag-specific CD(8)(+) and CD(4)(+) T cells in vitro.</p>

Alteration of the Cell Cycle during the Differentiation of HL-60 Cells by Induction of Retinoic Acid / 中国实验血液学杂志

To study the alteration of the cell cycle during the differentiation of human myeloid leukemia cell line HL-60 induced with all trans-retinoic acid (RA), the flow cytometry was used to assay the various phases of cell cycle in HL-60 cells treated with RA. The results showed: (1) S + G(2)/M phase proportion kept relative invariability during the 48 hours incubation of HL-60 cells with RA, however, the proportion alteration of S-phase cells was associated with the RA concentrations. At 10(-6) mol/L RA, the proportion of S-phase cells appeared a temporarily increasing peak followed by persistent decrease of S-phase proportion. At 10(-5) mol/L RA, S-phase cell proportion only appeared the persistent decreasing tendency. (2) Re-culture of HL-60 cells without RA showed the decrease of S + G(2)/M and S-phase cells was associated with the increase of differentiated cells, but not all HL-60 cells were triggered into differentiation at the same time. Once the cells start to differentiate, even if there is no RA presence, the HL-60 cells were still differentiated until maturation. In conclusion, HL-60 cells are able to differentate and maturate after exposure to RA for a period of time. S-phase proportion is related to the concentrations of RA. Once the cells start to differentiate, even if there is no RA presence, the HL-60 cells still differentiatiated until maturation.