Reaserch Advance on Langerhans Cell Histiocytosis Review / 中国实验血液学杂志

Abstract　　Langerhans cell histiocytosis (LCH) is a disease originated from bone marrow dendritic cells, and classified as a tumor by the discovery of a recurrent somatic BRAF-V600E point mutation in the RAS-RAF-MEK-ERK signaling pathway. The clinical manifestations of LCH are mainly granulomatous lesions composed of clonal pathological tissue cells. According to the lesions and invasive risk organs, it is divided into single system diseases, multi-system diseases with risk-free organ infiltration and multi-system diseases with risk organ infiltration. The diagnosis was based on immunohistochemical pathological dendritic cell-specific markers CD1α+and/or CD207,therefore, according to risk stratification, the regiment and intensity of combination chemotherapy and targeted therapy are drawn up. Prognosis is associates with risk organ infiltration, initial treatment response, and BRAF mutations. Due to the low incidence and lack of systematic knowledge, the clinical understanding of this disease is insufficient, thus the rates of misdiagnosis and therapeutic error are high. In this review, the pathogenesis, clinical manifestations, diagnostic and treatment are summarized. So on to provide a theroretical basis for clinical diagnosis and treatment of the diseases.

Effect of Pomalidomide on Activity of Myeloma Cell Line MM1.S and Expression of CRBN / 中国实验血液学杂志

OBJECTIVE@#To explore the effects of different concentration of pomalidomide on human multiple myeloma cell line MM1.S and the expression of CRBN.@*METHODS@#CCK-8 method was used for detecting inhibition effect of promalidomide on proliferation of MM1.S cells. Apoptosis rate of MM1.S cells was detected by flow cytometry with Annexin V-FITC/PI double staining. Real-time quantitative PCR was used to determine CRBN gene expression level. Western blot was used to detect the effect of pomalidomide on the protein expression of CRBN in MM1.S cells.@*RESULTS@#Pomalidomide has an inhibitory effect on MM1.S cells with time-and dose-dependent manners. Pomalidomide induced apoptosis in MM1.S cells. When the concentration of pomalidomide was 0, 40 and 80 μmol/L, the expression of CRBN gene after the treatment of MM1.S cells for 72 hours was 1.487±0.340, 0.211±0.054 and 0.055±0.005, by using actin as internal refereme. Pomalidomide significantly reduced CRBN protein expression in MM1.S cells.@*CONCLUSION@#Pomalidomide can inhibit the proliferation of MM1.S cells and promote its apoptosis. A certain concentration of pomalidomide can reduce the expression of CRBN gene and down-regulate its protein expression in MM1.S cells.

Changes of IL-21 and Its Mediated JAK/STAT Signaling Pathway in Patients with Immune Thrombocytopenia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the correlation between JAK/STAT signaling pathways and pathogenesis of immune thrombocytopenia(ITP).</p><p><b>METHODS</b>Twenty-six newly-diagnosed ITP patients was included in this study. They all meet the clinical and hematological criteria for the diagnosis of ITP, and patients with coronary heart disease, severe refractory hypertension, diabetes or with severe liver or kidney function incompetence were ruled out. 24 healthy control without autoimmune diseases, viral infectious diseases and with normal liver and kidney functions were also included. The expressions of Jak3, p-Jak3 mRNA, Stat3, and p-Stat3 were tested and the changes in levels of IL-21 mRNA, IL-21 cell secretion after DEX intervention and AG490 blockade were measured.</p><p><b>RESULTS</b>Compared with the healthy control, patients with ITP had significantly high expressions of Jak3, p-Jak3 mRNA, Stat3 and p-Stat3 protein, which significantly reduced after AG490 blocking (P<0.01). The expression of IL-21 mRNA and the secretion of IL-21 obviously decreased after DEX intervention, but increased after AG490 blocking(P<0.01).</p><p><b>CONCLUSION</b>The pathogenesis of ITP associates with the activation of JAK/STAT signaling pathways, and IL-21-mediated JAK/STAT signaling pathways play regulatory role in ITP.</p>

Hypobaric Hypoxia Induces Autophagy of Umbilical Cord Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the autophagy activity changes of umbilical cord mesenchymal cells (MSC) under hypobaric hypoxia and the effect of hypobaric hypoxia on cell viability.</p><p><b>METHODS</b>Umbilical cord mesenchymal cells were cultured in the chamber of hypobaric hypoxia with an air pressure of 41.1 kPa and an oxygen density of 1%. At 0, 4, 8, 16, 24 and 48 hours, the cells were harvested for Western blot and real-time PCR to observe the expression level of the autophagy marker protein LC3B. And the cell viability under hypobaric hypoxia was evaluated after treatment with autophagy inhibitors HCQ (8 μg/ml) and 3MA (5 mmol/L).</p><p><b>RESULTS</b>LC3B expression in MSC at protein and mRNA levels were up-regulated significantly after being cultured under hypobaric hypoxia condition for 8 hours. And compared with the control group, inhibition of autophagy reduced cell viability while increased Caspase-3 expression and the incidence of apoptosis.</p><p><b>CONCLUSION</b>Hypobaric hypoxia activates autophagy in MSC, and the activation of autophagy might play a protective role for cell survival.</p>

Molecular Mechanism of CRBN in the Activity of Lenalidomid eagainst Myeloma--Review / 中国实验血液学杂志

Cereblon（CRBN) is a brain-associated protein with ionic protease activity, which interacts with DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B), and regulator of Cullins 1 (RoC1) to form the functional E3 ubiquitin ligase complex(CRBN-CRL4) that performs proteolysis via the ubiquitin-proteasome pathway. And CRBN is a necessary target protein for the anti-myeloma effect of immunomodulators. The combination of lenalidomide and CRBN recruited a new substrate that binds to the CRBN-CRL4 complex, leading to increased ubiquitination and proteasome-dependent degradation, thus resulting in anti-myeloma activity. The substrates binding to this complex are IKZF1, IKZF3 proteins and GS, etc. The CRBN-dependent degradation of IKZF1 and IKZF3 after lenalidomide treatment is also the result of HO-mediated oxidative stress. In addition to ubiquitination, lenalidomide also mediates ubiquitin-independent pathways that prevent CRBN from binding to CD147-MCT1 in a competitive manner to regulate its antitumor activity. Lenalidomide can also play a role in multiple myeloma(MM) cells by modulating miRNA levels and CRBN binding to downstream protein AGO2 expression. Thus, there are many molecular mechanisms of lenalidomide anti-myeloma activity. This review summarizes the molecular mechanisms of CRBN in lenalidomide against myeloma activity in terms of ubiquitin-dependent and ubiquitin-independent pathways.

Analysis of Curative Efficacy of Different Chemotherapy Regimen Combined with Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation on Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To evaluate the efficacy of autologous peripheral blood hematopoietic stem cell transplantation (auto-PBHSCT) on patients with multiple myeloma( MM) after Sequential different chemotherapy.@*METHODS@#Seven cases of patients with MM were included in the A group, and 14 cases of patients received 4-6 courses of chemotherapy with VAD and MP before transplantation were included in the B group and received 4-6 courses of chemotherapy with VTD and VD before transplantation. Auto-peripheral blood hematopoietic stem cell were mobilized by G-CSF. Condition regimen were melphalan(A group) or bortezomib combined melphalan(B group). IFN-α(A group) or Thalidomide(B group) was used as maintenance treatment after auto-PBHSCT.@*RESULTS@#Two cases of patients reached to complete remission (CR)(2/7,28.6%),1 case got very good partial remission (VGPR) (1/7,14.3%), 4 cases got partial remission(PR) (4/7,57.1%) in A group, and 9 cases got CR (9/14,64.3%), 3 cases got VGPR(3/14,21.4%), and 2 cases got PR(2/14,14.3%) in the B group before auto-PBHSCT. The CR and VGPR were significant difference between 2 groups (P<0.05). All the patients got hematopoietic recovery. In 2 groups, the median time of ANC recovery≥0.5×10/L was 13 (11-16) and 14（11-18）days, that of WBC recovery ≥4.0×10/L were 16(15-19) and 18(16-20)days, Plt recovery ≥ 50 ×10/L was 21 (18-25) and 21(17-25) days. Bone marrow showed CR in 21 to 28 days after transplantation. All of 7 cases of patients remised in 6 to 47 months after transplantation, and 4 cases died lastly and 3 cases failed to be followed up in A group. The median time of progression-free survival(PFS) was 36(6-47) months, and that of overall survival(OS) was 37(7-50) months. In B group, 2 cases of patients remissed in 5 and 17 months after transplantation, and did lastly, 1 case relieved in 12 months after transplantation and failed to be followed up. 1 case of patient relived in 46 months after transplantation, and then received the second auto-PBHSCT, and got CR for 105 months. Other 10 cases got CR, their median time of PFS was 45.5(4-105) months, the median time of overall survival(OS) was 45.5(4-105) months. The PFS and OS were very significant different between 2 groups (P<0.01).@*CONCLUSION@#Bortezomib-based chemotherapy, Auto-PBHSCT and maintenance treatment with thalidomide were favorable to the patients of MM for survival prolongation.

Retrospective Analysis of Genetics Abnormalities in Patients with Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To explore the characteristics of cytogenetics and molecular genetics in patients with multiple myeloma(MM).@*METHODS@#Fluorescence in situ hybridization(FISH) was used for molecular genetics analysis in 86 cases of newly diagnosed MM, at the same time the chromosome karyotype analysis was performed in 20 cases. Specimen were bone marrow cells.@*RESULTS@#FISH detection showed that 68 cases of MM (79.07%) had at least one type of the molecular genetic abnormalities. The positive rates of IgH rearrangement, 1q21 amplification, D13S319 deletion, RB1 deletion and.P53 deletion were 62.79%, 26.74%, 24.42% ,13.95% and 1.16%, respectively. The positive rate of IgH was significantly higher than that of any other probes(P<0.01). The positive rate of IgH was 79.41% in 68 cases. Out of which the positive rate of IgH single and combined with 1, 2, 3, 4 probes was 59.26%, 24.07%, 11.11%, 5.56% and 0 respectively. The positive rate of IgH only was very signficantly higher than that of combined with any other probes(P<0.01).The positive rate of 1q21 was 33.82% in 68 cases, Out of which the positive rates of 1q21 or combined with 1,2,3,4 probes was 21.74%, 43.48%, 21.74%,13.04% and 0 respectively, the 1q21 probe showed positive as combined with other probes(P<0.01), especially with IgH(P<0.05). The positive rates of D13S319 were 30.88% in 68 cases of patients, out of which the positive rates of D13S319 single or combined with 1, 2, 3, 4 probes was 14.29%, 28.57%, 42.86%, 14.29% and 0 respectively, the D13S319 combined with other probes appeared more significant positive(P<0.01), especially with 1 or 2 probes (P< 0.01). The positive rate of RB1 was 17.65% in 68 cases, the positive rate of RB1 singl or combined with 1, 2, 3, 4 probes were 0, 25%, 50%, 25% and 0, the RB1 appeared positive always combined with other probes, especially with D13S319 probe (P<0.01). The positive rate of P53 was 1.47%, as combined with RB1 and D13S319 probes. The chromosomal karyotyping showed that 3 cases carried abnormal chromosomal and 17 cases carried normal chromosome, Out of which 17 cases showed positive by FISH. There was a significant difference of sensitivity between FISH combined with chromosome karvotyping and single chromosome karvotype (P< 0.01).@*CONCLUSION@#The genetic abnormalies display obvious heterogenicity in MM. The sensitivity of FISH is higher than that of chromosomal karvotyping. If FISH and chromosome karvotyping are combined, the positive rate of abnormality can be raised.

Clinical Analysis of Adoptive Immunotherapy after Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation in B Lymphocyte Malignant Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy of autologous peripheral blood hematopoietic stem cell transplantation(auto-PBHSCT) combined with adoptive immunotherapy for patients with B lymphocyte malignant lymphoma(ML).</p><p><b>METHODS</b>A total of 110 cases of ML treated with adoptive immunotherapy after auto-PBHSCT from January 2000 to December 2009 were enrolled in adoptive immunotherapy group (treated group), while 74 cases of ML treated without adoptive immunotherapy after auto-PBHSCT from January 1995 to December 1999 were used as control group. The efficacy of 2 groups were analyzed and compared, 110 case of ML in treated group included 78 cases of non-Hodgkin's lymphoma(NHL), 32 cases of Hodgkin's lymphoma(HL),74 cases of ML in control group included 52 NHL and 22 HL. All of the patients were treated sequentially with chemotherapy regimens for 6 courses. After that, all the patients received auto-PBHSCT. After hematopoietic reconstruction, the patients in treated group were given 6 courses of adoptive immunotherapy(rhIL-2 100 WU/day for 10 days monthly for each course), while the patients in control group were not given immunotherapy. All the patients were followed-up for more than 5 years.</p><p><b>RESULTS</b>There was one patient in each group, who died of liver failure and cerebral hemorrhage respectively within 3 and 2 months, and all the other patients achieved hematopoietic reconstruction. Following-up for 1, 3, 5 years, the disease-free survival (DFS) rate in treated group was 97.3%,93.6%,87.3% while 91.9%, 73.0%, 64.9% in control group. Following-up for 3 and 5 years, there was very significant difference in DFS between 2 groups(P<0.01). The 1,3 and 5 year DFS rate of patients in stage I/II and III/IV in the treated group were 100%,100%,91.7% and 96.5%,91.9%,86.0% respectively while DFS of control group was 100%, 93.3%, 86.7% and 89.8%, 67.8%, 59.3%, there was a significant difference in 3 and 5 years DFS of III/IV stage patients between 2 groups (P<0.01). The 1,3 and 5 year DFS rate of HL patients were 100%, 93.8%,84.4% in treated group and 100%,72.7%,59.1% in control group respectively. There was significant difference in 3 and 5 years DFS of HL between 2 groups (P<0.05). The 1,3 and 5 year DFS rate of stage I/II HL patients were 100%,100%,88.9% in treated group and 100%,100%,80.0% in control group. The 1,3 and 5 year DFS of HL patients in stage III/IV was 100%,91.3%,82.6% and 94.1%,64.7%,52.9% respectively. There was significant difference in 3 and 5 years DFS of III/IV stage of HL patients between 2 groups (P<0.05). The 1,3 and 5 year DFS rate of NHL patients is 96.2%, 93.6%,88.5% in treated group and 90.4%,73.1%,65.4% in control group respectively. There was a significant difference in 3 and 5 years DFS of NHL between 2 groups(P<0.01). The 1,3 and 5 year DFS rate of stage I/II NHL patients was 100%, 100%, 93.3.9% in treated group and 100%, 90%, 90.0% in control group, respectively. The 1,3 and 5 year DFS of NHL patients in stage III/IV is 95.2%, 92.1%,87.3% and 88.1%,69.0%, 59.5% respectively. There was significant difference in 3 and 5 years DFS of III/IV stage NHL patients between 2 groups (P<0.05).</p><p><b>CONCLUSION</b>Therapeutic efficacy is satisfactory for the patients of B lymphocyte ML treated with adoptive immunotherapy after auto-PBHSCT, especially benefited the patients of stage III/IV significantly.</p>

Relationship between Gene Polymorphism of HLA-A(*)/-B(*)-DRB1(*) and Aplastic Anemia in Chinese Han Population of Northwestern Plateau / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the correlation between the HLA genes and pathogenesis of aplastic anemia (AA), so as to find the susceptible AA genes.</p><p><b>METHODS</b>Polymerase chain reaction with specific sequence primers (PCR-SSP) method was used to detect the HLA typing of 50 AA patients and 183 normal healthy individuals as controls in Chinese Han population of northwestern plateau.</p><p><b>RESULTS</b>The frequency of HLA-A* 0201 (45.0%), B* 1501 (11.0%), B* 5501 (9.0%) and DRB1* 0901 (19.0%) gene frequences in AA patients were significantly higher than those in controls (Odds Ratio: OR=1.657, 2.138, 2.314 and 1.932, x2=4.882, 3.876, 3.863 and 4.473 (P<0.05). In contrast, A* 0301 gene frequency (4.0%) in AA was significantly lower than that in controls, OR=0.349, x2=4.154 (P<0.05). The male HLA-A* 0201 gene frequency was lower than that in female (38.2% vs 59.4%), and the difference was statistically significant (P<0.05). Concludsion: The HLA-A* 0201, B* 1501, B* 5501 and DRB1* 0901 genes may be considered as the risk markers while A* 0301 gene as a protective marker of AA, the HLA-A* 0201 also shows the sex differences.</p>

Effects of the interleukin-21 expression in patients with immune thrombocytopenia and its regulation by high-dose dexamethasone / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of immunologic thrombocytopenia(ITP) pathogenesis with the abnormal expression of IL-21, and to explore the association of high-dose dexamethasone (HD-DEX) treatment with the IL-21 expression.</p><p><b>METHODS</b>26 newly diagnosed ITP patients and 24 healthy controls were enrolled in this study. The mononuclear cells and serum were obtain from density gradient centrifugation in the newly diagnosed ITP patients before HD-DXM treatment, and the samples of healthy controls were also used for assays. The protein and mRNA expression of IL-21 on peripheral blood mononuclear cells(MNC) were determined by flow cytometry and real-time reverse-transcription polymerase chain reaction. Plasma levels of IL-21, IFN-γ and IL-4 were determined by enzyme-linked immunoabsorbent assay (ELISA).</p><p><b>RESULTS</b>IL-21 expression on mononuclear cells was significantly higher in ITP patients (13.07%) than that in normal controls (8.2%), the ratio of IL-21/GAPDH mRNA expression on MNC was significantly higher in ITP patients (9.524±0.97) than that in normal controls (3.701±0.60, P<0.01). After HD-DXM therapy, the ratio of IL-21/GAPDH mRNA decreased significantly (5.87±1.21) as compared with the level before treatment. Significantly high levels of serum IL-21, IFN-γ and lower IL-4 were found in ITP patients, as compared with healthy controls. Serum IL-21 and IFN-γ levels in ITP patients decreased significantly after HD-DXM administration (P<0.01), while post-treatment levels of IL-4 were increased significantly, compared with the levels before treatment (P<0.01).</p><p><b>CONCLUSION</b>Therapeutic effect of DXM on ITP associates with down-regalation of IL-21 expression. The increased expression of IL-21 involves in the pathogenesis of ITP.</p>

Curative efficacy for nasal type extranodal NK/T-cell lymphoma by autologous peripheral blood stem cell transplantation after sequencing chemotherapy and radiotherapy / 中国实验血液学杂志

The purpose of this study was to explore the curative efficacy for nasal type extranodal NK/T-cell lymphoma by autologous peripheral blood stem cell transplantation (APBSCT) after sequencing chemotherapy and radiotherapy. A total 65 cases diagnosed as nasal type extranodal NK/T-cell lymphoma by pathology and immuno-histochemistry were treated with chemotherapy and radiotherapy in our hospital from January of 2000 to December of 2009. They were divided into observation group (34 cases) and transplantation group (31 cases). The 34 cases of observation group were ceased from treatment, the 31 cases in transplantation group received APBSCT after conditioning regimen with TBI combined VEMAC. Autologous peripheral blood stem cells were mobilized with chemotherapy combined rhG-CSF. The patients were followed up for 3-5 years. The results showed that some side-effects such as bone marrow suppression and injure of oral cavity mucosa were found in patients after sequencing chemotherapy and radiotherapy. All patients in transplantation group obtained hematopoietic reconstruction, and there were no any special side effect such as VOD. In transplantation group, the median time of ANC ≥ 0.5×10(9)/L was 14 (11-17) days, median time of WBC count ≥ 4.0×10(9)/L was 17 (16-20) days, median time of Plt count ≥ 50×10(8)/L were 25 (23-28) days. After chemotherapy and radiotherapy, effective rate of treatment was 91.2% in observation group, whereas was 90.3% in transplantation group, there were no obvious difference between two groups (P > 0.05). After following up about 1 year, effective rate of treatment was 76.5% in observation group, whereas was 96.8% in transplantation group, there were obvious difference between two groups (P < 0.05). After following up about 3 years and 5 years the disease-free survival (DFS) was 61.3%, 43.5% and 87.1%, 81.5% in observation group and transplantation group, there was significant difference between two groups (P < 0.05). It is concluded that treatment with APBSCT after sequencing chemotherapy and radiotherapy for nasal type extranodal NK/T-cell lymphoma may increase DFS efficiently.

Autophagy is involved in doxorubicin induced resistance of human myeloma cell line RPMI8226 / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of autophagy in doxorubicin (DOX)-induced resistance of human myeloma cell line RPMI8226.</p><p><b>METHODS</b>We established doxorubicin induced resistant subline of myeloma cell line RPMI8226/DOX by drug concentration step-elevation method. Resistant index of DOX was measured by MTT assay. Autophagy of myeloma cell lines RPMI8226/s and RPMI8226/DOX was detected by transmission electron microscopy, immunofluorescence (LC3-FITC) and western blot respectively. Apoptosis of RPMI8226/DOX cells induced by DOX combined with autophagic inhibitor hydroxychloroquine or 3-MA was identified by AnnexinV-FITC/PI double fluorescence dyeing.</p><p><b>RESULTS</b>Resistant index of RPMI8226/DOX was approximately 10.8 fold of that of RPMI8226/S. Electron microscopic studies revealed that most of RPMI8226/DOX cells displayed viable attributes and contained numerous autophagic vacuoles. Fluorescent images of RPMI8226/DOX cells showed a punctuate distribution in LC3 protein. Increased LC3-II protein in RPMI8226/DOX cells was determined by immunoblotting. There were no differences among 8 μmol/L HCQ (3.24±1.08)%, 10 mmol/L 3-MA (2.81±0.80)% or control \[(2.12±1.24)%\] (P>0.05) in terms of AnnexinV-FITC/PI double fluorescence dyeing; Compared with apoptosis of (9.75±2.15)%, (24.36±2.16)% and (40.51±3.14)% of RPMI8226/DOX cells under 2, 4 and 6 μmol/L DOX, apoptosis increased significantly after 24 h incubation under 2, 4 and 6 μmol/L DOX combined with 8 μmol/L HCQ as of \[(16.56±1.89)%, (36.44±2.91)% and (62.68±3.75)%, respectively\], or under 2, 4 and 6 μmol/L DOX combined with 10 mmol/L 3-MA as of \[(15.47±1.85)%, (39.28±3.06)% and (55.46±4.07)%, respectively\] (P<0.05).</p><p><b>CONCLUSION</b>Autophagy was involved in doxorubicin-induced resistance of myeloma cell line RPMI8226, DOX resistance in myeloma cells was reversed partly by autophagy inhibitor hydroxychloroquine or 3-MA, and autophagy may be one of mechanisms for drug resistance.</p>

Efficacy of adoptive immunotherapy after mixed hematopoietic stem cell transplantation on acute myeloid leukemia / 中国实验血液学杂志

The purpose of this study was to investigate the efficacy of treatment with haploidentical donor's lymphocyte infusion(hiDLI) combined with interleukin-2 (IL-2) after transplantation of autologous peripheral blood stem cells mixed with haploidentical allogeneic bone marrow (mix-HSCT) for acute myeloid leukemia (AML). 49 patients diagnosed as AML were enrolled in this study. After preconditioning with TBI plus VEMAC regimen, all patients received mix-HSCT. Autologous peripheral blood hematopoietic stem cells were mobilized with chemotherapy-combined G-CSF, and haploidentical allogeneic bone marrow cells were not mobilized with G-CSF. 33 patients in test group were treated with hiDLI plus IL-2 for 1-8 times after hematopoietic reconstruction, 16 patients in control group received mix-HSCT only. All the patients were followed-up for more than 3 years. The results showed that all the patients obtained hematopoietic reconstruction, and no graft-versus-host disease (GVHD) was found. In two groups, the median time of absolute neutrophil count (ANC) ≥ 0.5×10(9)/L was 14 (12 - 18) and 14 (11 - 16) days, and WBC count ≥ 4.0×10(9)/L was 17 (16 - 22) and 18(17 - 20) days, Plt count ≥ 50×10(8)/L were 25 (24 - 29) and 25 (23 - 26) days. 9 patients in test group formed mixed chimerism (46XX/46XY) and sustained about 3 - 12 months; disease-free survival (DFS) was 63.6%, 3 patients in control group formed mixed chimerism (46XX/46XY), persistent about 3-6 months; DFS was 50.0%. It is concluded that treatment with hiDLI plus IL-2 after mix-HSCT for AML patients may increase DFS efficiently.

Expression of autophagy related gene Beclin1 and MAPLC3 in bone marrow mononuclear cells isolated from acute leukemia patients and its significance / 中国实验血液学杂志

This study was purposed to detect the expression of autophagy-related gene Beclin1 and microtubule-associated protein 1 light chain 3 (MAPLC3) in bone marrow mononuclear cells (BMMNC) isolated from acute leukemia (AL) patients, and to explore its significance. Transmission electron microscopy and RT-PCR were used to detect the autophagy activity and the expression level of Beclin1 and MAPLC3 mRNA in BMMNC isolated from 27 AL patients with de novo, refractory or relapse AL and completely remission and 31 normal persons respectively. The results showed that autophagy activity and expression levels of Beclin1 and MAPLC3 mRNA in BMMNC from de novo AL patients were 80%, 0.68 ± 0.18, 0.24 ± 0.06, respectively; those in BMMNC from refractory or relapse AL patients were 100%, 0.79 ± 0.09, 0.30 ± 0.07, respectively; those in BMMNC from CR patients were 40%, 0.52 ± 0.15, 0.16 ± 0.04, respectively, while those in BMMNC from normal persons were 20%, 0.57 ± 0.13, 0.16 ± 0.05, respectively. The autophagic activity and expression levels of Beclin1 and MAPLC3 mRNA in de novo and refractory or relapse AL patients were higher than those in normal persons, with statistical significance (p < 0.05), while the comparison between CR patients and normal control showed no statistical difference (p > 0.05). It is concluded that autophagy activity and Beclin1 and MAPLC3 mRNA expression level of in de novo and refractory or relapse patients are higher than those in normal control, and the up-regulation of autophagy activity and expression of Beclin1 and MAPLC3 mRNA in refractory or relapse patients is especially significant. This may be related to the genesis, development and drug resistance of AL.

X-ray induces autophagy in human mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the autophagy in human bone marrow mesenchymal stem cells (hBMMSC) exposed to irradiation.</p><p><b>METHODS</b>The apoptosis and necrosis rate were assessed by Annexin V and propidium (PI) staining in hBMMSC at 4h after irradiated with X-ray at 0, 2, 4, 8 and 10 Gy. The autophagy was observed by transmission electron microscopy. The mRNA expression of Beclin1 and microtubule-associated protein 1 light chain 3 (MAPLC3 or LC3) was analyzed by RT-PCR in hBMMSC at 4h after X-ray irradiation at 0, 8 and 10 Gy.</p><p><b>RESULTS</b>The apoptosis rate of hBMMSC was markedly decreased while the necrosis and death rate were slowly increased with the increase of irradiation dose when under 8 Gy. The apoptosis rate was significantly increased and reached a peak while the necrosis and whole death rate were obviously increased when irradiated with 10 Gy X-rays. In addition, the change of apoptosis rate was more significant than that of necrosis rate. By electron microscopy, a mass of autophagic vacuoles (autophagosome and autolysosome) were observed in irradiation and positive control groups, but were only occasionally seen in normal control group. The proportion of hBMMSC with autophagic vacuoles in 8 Gy irradiation group was higher than that in 10 Gy one. The mRNA expression of Beclin1 and LC3 in irradiation groups and positive control group was significantly higher than in normal control group, and so did in 8 Gy irradiation group than that in 10 Gy group.</p><p><b>CONCLUSION</b>Irradiation may induce the autophagy in hBMMSC, and autophagy could protect hBMMSC from irradiation injury in a certain dose range.</p>

PI3-kinase mediates thrombin-induced platelet aggregation through mDia1 pathway / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of mDia1 (mammalian diaphanous 1)in platelet and the role of mDia1 or phosphatidylinositol 3-kinase (PI3K) in the process of thrombin-induced platelet aggregation.</p><p><b>METHODS</b>The extent of platelet aggregation was measured by a platelet aggregation system and the expression of mDia1 and its relation with F-actin in quiescent, spreading or aggregated platelets by Western blot.</p><p><b>RESULTS</b>There was no significant difference in mDia1 expression level between quiescent and activated platelets. mDia1 moved from a Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction after thrombin induced platelets aggregation. Anti-mDia1 antibody could inhibit this aggregation. PI3K inhibitor Wortmannin or Ly294002 inhibited the thrombin induced platelet aggregation and the above mentioned mDia1 translocation.</p><p><b>CONCLUSION</b>PI3-kinase mediates the thrombin-induced platelet aggregation through mDia1 pathway.</p>

Bortezomib-induced BiP expression and apoptosis in multiple myeloma cells / 中国实验血液学杂志

This study was aimed to explore the effect of bortezomib on the apoptosis and expression of the molecular chaperone BiP in human multiple myeloma cell line NCI-H929 (H929). After treatment of H929 cells with different concentrations of bortezomib for 24 hours, cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression levels of BiP mRNA and protein were detected by RT-PCR and Western blotting analysis. The results showed that bortezomib of different concentrations (20, 40 and 80 nmol/L) induced apoptosis of H929 cells in dose-dependent manner, with apoptotic rates (15.73 +/- 0.67)%, (27.83 +/- 1.26)% and (44.17 +/- 2.25)% respectively, which were significantly higher than that in control (1.21 +/- 0.07%) (p < 0.05). Bortezomib-induced up-regulation of BiP mRNA levels was almost on a parallel with BiP protein when compared with control. Under the similar apoptosis-stimulating conditions with apoptotic rates varying from 40% to 50%, expression levels of BiP mRNA and BiP protein induced by the classical endoplasmic reticulum stressor Brefeldin A (500 ng/ml, 24 h) were almost consistent with those by bortezomib (80 nmol/L, 24 h). It is concluded that bortezomib-induced apoptosis in H929 cells correlates closely with endoplasmic reticulum stress.

PI3-kinase mediates activity of RhoA and interaction of RhoA with mDia1 in thrombin-induced platelet aggregation / 中国实验血液学杂志

The aim of this study was to investigate the role of RhoA/mDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with mDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with mDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with mDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with mDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-mDia1 pathway.

CMV pp65 gene modified dendritic cells activate autologous T cells / 中国实验血液学杂志

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.

Tyrosine kinase inhibitor reverses adriamycin resistance mediated by cell adhesion in RPMI8226 cells / 中国实验血液学杂志

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.