OBJECTIVETo investigate the irregular antibody production and its relationship with Rh factor genotypes and the loci of thalassemia gene mutations for the β-thalassemic children with long-term transfusion, so as provide experimental basis for clinical safe and effective transfusions for thalassemic children.

METHODSThe peripheral blood from 246 children with β-thalassemia was collected in our hospital; the extraction of genomic DNA and Rh factor (C/c, E/e) genotypes were assayed by PCR-SSP method, the irregular antibodies were screened and identified by serological method, the genotypes for thalassemia and gene mutations were analysed by PCR-RD method.

RESULTSThe genotypes of Rh factors classified by PCR- SSP in the 246 cases of β-thalassemia children were as follws: Ce/Ce (143/246, 58.1%), CE/ce (59/246, 24%), cE/cE (14/24, 5.7%), Ce/ce (12/246, 4.9%); The positive rate of irregular antibody was 7.7% (19/246), including anti-E (7/19), anti-c (5/19), anti-C (2/19), anti-E and anti-c (2/19), anti-e (1/19), anti-D (2/19); Of the 19 cases with positive irregular antibody, the genotypings of Rh factor were: Ce/Ce (11/19), CE/ce (2/19), cE/cE (2/19), Ce/ce (2/19), cE/ce (2/19); the gene mutations location of thalassemia for 19 cases with positive irregular antibody: CD41-42M (13/19), CD71-72M (2/19), IVS-II-654M (3/19), -28M (1/19).

CONCLUSIONThe irregular antibody production for β-thalassemic children with long-term transfusion may have some relevance with Rh factor genotypes and thalassemia genetic mutations. This study possesses a certain significance for effective prevention of RBC alloimmune response of β-thalassemia children and improvement of efficacy and safety of clinical transfasion blood.

Blood Group Antigens ; Blood Transfusion ; Child ; Genotype ; Histocompatibility ; Humans ; Mutation ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System ; Rho(D) Immune Globulin ; beta-Thalassemia

OBJECTIVETo detect the expression levels of CD4(+) CD25(+) CDl27(low) Treg cells, TGF-β and Notch1 mRNA in peripheral blood of the patients with idiopathic thrombocytopenic purpura (IPT) before and after treatment, and to investigate their significance in the pathogenesis of ITP.

METHODSPeripheral blood was collected from 30 newly diagnosed patients with ITP and 20 normal controls, then the number of CD4(+) CD25(+) Treg and CD4(+) CD25(+) CDl27(low) Treg were detected by the flow cytometry. Plasma TGF-β level was determined by ELISA. Total RNA was extracted and the expression level of Notch1 mRNA was measured by real-time Q-PCR.

RESULTSThe expression levels of CD4(+) CD25(+) CDl27(low) Treg and CD4(+) CD25(+) Treg in newly diagnosed ITP group were significantly lower than those in normal controls. After treatment, the proportion of Tregs increased to (5.17% ± 0.74%) and (4.16% ± 0.68%), and was higher than that in newly diagnosed patients. The TGF-β level in peripheral blood of newly-diagnosed patients was obviously lower than that in normal controls, and was (961.53 ± 60.10) ng/L after treatment and was significantly higher than that in newly diagnosed patients; the expression level of Notch1 mRNA in peripheral blood of patients in newly-diagnosed group was obviously lower than that in control, and was (1.35 ± 0.10) after treatment that was higher than that in newly-diagnosed group. After treatment, the proportion of Treg cells, level of TGF-β and erpression level of Notch1 mRNA in effective group were higher than those in effective group, improved group and ineffective group, and there was significant difference (P <0.01). The expression level of TGF-β and Notch1 mRNA in ITP patients possitively correlated to CD4(+) CD25(+) CDl27(low) (P <0.01).

CONCLUSIONSThe levels of CD4(+) CD25(+) CDl27(low) Treg, TGF-βand Notch1 mRNA in peripheral blood of the patients with ITP are significantly lower than those of normal control, suggesting that there is significant abnormal immunoregulation in ITP patients. In the ITP patients the levels of CD4(+) CD25(+) CDl27(low) Treg postively correlated with Notch1 mRNA expression, indicating that Notch signal may be revalent to Treg's immunosuppression function.

Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Purpura, Thrombocytopenic, Idiopathic ; RNA ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Receptor, Notch1 ; T-Lymphocytes, Regulatory ; Transforming Growth Factor beta

OBJECTIVETo investigate the inhibitory effect of the copolymer of magnetic nanoparticles of Fe(3)O(4) (MNPs-Fe(3)O(4)) and artesunate (ART) on myelodysplastic syndromes (MDS) cell line SKM-1 cells and the potential mechanisms.

METHODSThe protein expression levels of BCL-2, BAX, Caspase-3, and Survivin in SKM-1 cells treated with or without the co-polymer were measured by Western blot. The co-polymer-induced apoptosis rate of SKM-1 cells was measured by flow cytometry.

RESULTSThe apoptosis rate of SKM-1 cells in the copolymer groups was higher than that in both MNPs-Fe(3)O(4) and artesunate groups alone. The MNPs-Fe(3)O(4) may enhance ART-induced cell apoptosis. Western blot assay showed that the expression of survivin and BCL-2 protein were down-regulated in the ART group, and this down-regulation was even more significant in the group of copolymer of ART with MNPs-Fe(3)O(4). The levels of BAX were increased both in ART group and the copolymer of ART with MNPs-Fe(3)O(4) group, as compared with control group and MNPs-Fe(3)O(4) group. The levels of active-caspase-3 were obviously up-regulated when the ART was combined with the MNPs-Fe(3)O(4). The copolymer of ART with MNPs-Fe(3)O(4) could trigger changes in the expression levels of apoptosis-related genes in SKM-1 cells, among which up-regulation of BAX and down-regulation of survivin and BCL-2 are the 2 major alterations.

CONCLUSIONArtesunate can induce the apoptosis of SKM-1 cells, and MNPs-Fe(3)O(4) may enhance the cell apoptosis induced by ART.

Apoptosis ; Artemisinins ; Caspase 3 ; Cell Line, Tumor ; Down-Regulation ; Ferric Compounds ; Humans ; Inhibitor of Apoptosis Proteins ; Magnetite Nanoparticles ; Up-Regulation

OBJECTIVETo establish a nested case-control study cohort in myelodysplastic syndrome (MDS) patients and investigate the clinical characteristics, WHO subtype and risk factors associated with MDS evolution to leukemia of this cohort.

METHODSAll patients, ≥18 years of age, provided by 24 Shanghai hospitals with initial clinical findings consistent with a hematopoietic abnormality between June 2003 and April 2007, were the candidates for inclusion in this study. The blood and bone marrow samples of every patient should be provided at baseline. Diagnosis was made by incorporating morphologic, immunophenotypic, cytogenetic and molecular features according to WHO classification criteria. Cytogenetic analysis was performed using conventional G-banding karyotyping and fluorescence in situ hybridization (FISH) techniques. Cumulative risk of evolution was estimated by Kaplan-Meier method. Prognostic factors were evaluated by univariate Log-rank method and multivariate Cox proportional hazard models.

RESULTSA total of 435 patients were diagnosed as MDS. The median age of MDS onset was 58(18-90) years, with 248 male patients and 187 female patients (male: female 1.33: 1). The percentage of cases with refractory cytopenia with multilineage dysplasia (RCMD) was the highest (65.5%), while that of refraetory anemia (RA) (2.3%), refractory anenia with ring sideroblast (RARS) (1.1%) and 5q-syndrome (0.5%) was lower. Trisomy 8 (+8) was the most common chromosome abnormalities (71 cases, 12.7%). The mean follow-up time was 20.3 (4.2-57.1) months. Cases were patients with evolution by the end of follow-up, while controls were patients without evolution by that time. Case group included 41 patients and control group included 342 patients. Univariate analysis showed that the age, sex, WHO subtype, WBC count, absolute neutrophil count (ANC), IPSS cytogenetic subgroup, IPSS group and bone marrow blast percentage were significant risk factors for leukemia-free survival (LFS). Multivariate analysis of COX model showed that the age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast were independent risk factors for LFS.

CONCLUSIONA nested case-control study cohort of MDS patients is established. The clinical characteristics and WHO subtype of MDS patients in Chinese Shanghai are different from that in Western countries. The independent risk factors for MDS evolution are age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast percentage.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; Bone Marrow ; Case-Control Studies ; China ; Chromosome Aberrations ; Chromosome Deletion ; Chromosomes, Human, Pair 5 ; Chromosomes, Human, Pair 8 ; Cri-du-Chat Syndrome ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia ; Male ; Middle Aged ; Myelodysplastic Syndromes ; Proportional Hazards Models ; Risk Factors ; Trisomy ; Young Adult

OBJECTIVETo evaluate the treatment value of adoptive immunotherapy (dendritic cells and cytokine-induced killer cells, DC-CIK) combined with chemotherapy on patients with multiple myeloma (MM) and its effect on secreting function of T lymphocytes in MM patients.

METHODSA total of 36 patients with MM were randomly divided into two groups, among them 28 patients in chemotherapy group were treated by chemotherapy only, 28 patients in combined therapy group were treated by adoptive immunotherapy (DC-CIK) combined with chemotherapy, and the clinical outcomes and the levels of IL-2, IFN-γ, IL-4, IL-10 secreted by T lymphocytes between two groups were compared.

RESULTSAfter treatment, the quality of life, clinical index and survival in combined therapy group were better than those in chemotherapy group (P <0.05); the levels of IL-2 and IFN-γ in combined therapy group was higher than these in chemotherapy group (P <0.05), and the levels of IL-4 and IL-10 in combined therapy group were lower than those in chemotherapy group (P <0.05).

CONCLUSIONDC-CIK combined with chemotherapy can be an effective and promising treatment for patients with MM, and it maybe strengthen the anti-tumor action of bodies by regulating the balance between Th1 and Th2 reaction.

Cytokine-Induced Killer Cells ; Dendritic Cells ; Humans ; Immunotherapy ; Immunotherapy, Adoptive ; Interleukin-10 ; Interleukin-2 ; Interleukin-4 ; Multiple Myeloma ; Quality of Life ; T-Lymphocytes

OBJECTIVETo investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of multiple myeloma H929 cell line and its mechanisms.

METHODSH929 cells were treated with different concentrations of GI254023X, the proliferation-inhibitive curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V/7-AAD double staining. The cleavage of Notch1 protein (cleaved notch1) was determined by Western blot. The transcripts of Notch1 target gene Hes-1 were detected by real-time PCR.

RESULTSThe GI254023X inhibited the proliferation of H929 cells in the time- and dose- dependent manners. As compared with the control group, the apoptosis of cells increased along with enhancement of GI254023X concentration; The expression of cleaved Notch1 was down-regulated after the treatment with GI254023X. The levels of Hes-1 mRNA transcripts in H929 cells was reduced in GI254023X treated group.

CONCLUSIONGI254023X can remarkably inhibit the proliferation and induce the apoptosis of H929 cells. Its mechanism may be associated with inbihition of Notch1 activation.

ADAM Proteins ; ADAM10 Protein ; Amyloid Precursor Protein Secretases ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Dipeptides ; Down-Regulation ; Humans ; Hydroxamic Acids ; Membrane Proteins ; Multiple Myeloma ; Receptor, Notch1

OBJECTIVETo investigate the effects of FTY720 on apoptosis in multiple myeloma cell line U266 and to clarify the molecular mechanism of apoptosis induced by FTY720.

METHODSU266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the apoptotic rates were tested by flow cytometry with Annexin-V-FITC/PI staining. Then U266 cells were treated with 20 µmol/L FTY720 for 0, 6, 16 and 24 hours, the apoptotic rates were tested. U266 cells were treated with DMSO and FTY720 separately and then were stained with DAPI for 5 min. Drop the cells to the slides and cover the slide with the glass. The cells were observed by fluorescence microscopy. U266 cells were treated with 5 µmol/L FTY720 or together with different doses of Z-VAD-fmk (12.5, 25, 50 µmol/L), a pancaspase inhibitor, for 24 hours, then the cell viability was tested by CCK-8. U266 cells were treated with 2.5, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expression of cleaved caspase-3 was tested by Western blot. U266 cells were treated with 0, 5, 10 and 20 µmol/L of FTY720 for 24 hours, the expressions of MCL-1, survivin, BCL-2, BID, BAX, BAK, P-ERK were tested by Western blot.

RESULTSThe apoptotic rate increased in U266 cells treated with FTY720 and showed the characteristic of time-dependent and dose-dependent manner. Karyopyknosis and nuclearfragmentation could be observed in U266 cells treated with FTY720 after being stained with DAPI under fluorescent microscope. The same effect was not observed in the cells treated with DMSO. Z-VAD-fmk could rescue the apoptosis in U266 cells treated with FTY720 in dose-dependent manner. The expression of MCL-1, survivin and BCL-2 decreased in U266 cells treated with FTY720. The cleavage of BID could be observed in U266 cells treated with FTY720. FTY720 had no effect on the expression of BAX, BAK and P-ERK.

CONCLUSIONFTY720 can induce the apoptosis in U266 cells, the apoptosis was Caspase-3-depended. The apoptosis induced by FTY720 is due to the decrease of MCL-1, survivin and BCL-2, which are the inhibitors of apoptosis. Meanwhile, the apoptosis was also due to the activation of BID, which is pro-apoptotic protein.

Amino Acid Chloromethyl Ketones ; Apoptosis ; Caspase 3 ; Cell Line, Tumor ; Cell Survival ; Fingolimod Hydrochloride ; Humans ; Inhibitor of Apoptosis Proteins ; Multiple Myeloma

OBJECTIVETo retrospectively analyze the safety and efficacy of busulfan (BU) combined with cyclophosphamide (CY) as the conditioning regimen of autologous hematopoietic stem cell transplantation (auto-HSCT) in patients with multiple myeloma (MM).

METHODSThe safety and efficacy of the BUCY regimen were evaluated through observing the adverse reactions, recovery of hematopoietic reconstitution, response and survival in 20 patients after auto-HSCT.

RESULTSIn 20 MM patients with median age 52.5 (38-66), the neutrophil and platelet counts recovered at 10(8-18) d and 10 (8-17) d after auto-HSCT respectively, the treatment related mortality during 100 days after auto-HSCT was 0, the partial remission (PR) rate decreased from 31.58% to 0 (P < 0.05) after auto-HSCT, only 1 patient was in progression of disease, all patients were alived.

CONCLUSIONFor patients with MM treated with Auto-HSCT, the BUCY regimen is ideal in safety and response, but the long-term effect still should be observed.

Busulfan ; Cyclophosphamide ; Hematopoietic Stem Cell Transplantation ; Humans ; Multiple Myeloma ; Retrospective Studies ; Transplantation Conditioning ; Transplantation, Autologous

OBJECTIVETo study the non-Hodgkin's lymphoma treated with enhanced chemotherapy regimen and increase of treatment courses, including number of treatment courses, short-term efficacy, long-term survival and safety.

METHODSAll the 254 cases of NHL in our hospital from January 2004 to February 2014 received a variety of intensive enhanced chemotherapy regimen, such as CHOPE, MAED, MMED and TAED. The median number of treatment course was 14, including 8 in the 1st year, 4 in the 2nd and 2 in the 3rd.

RESULTS(1) In 254 assessable patients, 182 patients (71.7%) achieved complete responses (CR), 30 patients (11.8%) achieved partial responses (PR), 22 patients (8.7%) achieved stable disease (SD), 20 patients (7.9%) achieved progressive disease (PD), 212 patients (83.5%) achieved response rate (RR). The median time of following-up was 56.5 months, the overall survivals (OS) of 1, 3 and 5 years were 90.1%, 74.5% and 61.1% respectively, the median survival time was 69 months, and the disease-free survivals (DFS) were 81.8%, 65.4% and 54.7% respectively, the median DFS was 65 months. (2) In therapeutic effects at early phase, the 3-year OS of patients who achieved CR, PR, SD and PD were 92.2%, 56.0%, 20.2% and 0% respectively; The 5-year OS of patients who achieved CR through ≤4 cycle treatments and the 5-year OS of patients who achieved CR through >4 cycles treatments were 83.1% and 6.8%, their DFS were 72.4% and 0% respectively. (3) The relapse rates of patients who received < 6, 6-8, 9-10, 11-13, 14, 15 and 20 cycle treatments were 82.5%, 78.9%, 71.9%, 65.8%, 41.8%, 30.4% and 16.7%. The response rate (RR) of patients who received 6-8 traditional chemotherapy cycle as CHOP or CHOP-like regimen were 50%-60% and relapse rate > 70%.

CONCLUSIONCompared with traditional chemotherapy regimens, the dose-escalated, intensive and modified chemotherapy regimen can significatly improve the therapeutic efficiency for patients with NHL, including CR, long-term survival rate, and a good tolerance for patients. The chemotherapy intensity has been confirmed to be an important factor that associated with therapeutic efficiency. On the conditions tolerated by patients, the number of treatment cycles for NHL patients can be increased at lest 14, with 8 in the first year, 4 in the second year and 2 in the third year. The increase of chemotherapy cycle can obviously reduce the relapse rate and improve the long-term prognosis of patients. It is worth to further explore.

Antineoplastic Combined Chemotherapy Protocols ; Cyclophosphamide ; Disease-Free Survival ; Doxorubicin ; Etoposide ; Humans ; Lymphoma, Non-Hodgkin ; Prednisone ; Prognosis ; Recurrence ; Remission Induction ; Vincristine

OBJECTIVETo explore the value of BCL-2 protein for evaluating the prognosis of patients with diffuse large B cell lymphama (DLBCL).

METHODSThe clinical data of 128 patients with DLBCL including clinical features, BCL-2 protein expression, therapeutic outcome and so on were analyzed retrospectively in departenent of hematology, Chinese PLA general hospital from January 2008 to December 2010, and the prognosis of DLBCL patients with different expression levels of BCL-2 protein was compared, including overall survival (OS) and progression-free survival (PFS) rates.

RESULTSThe BCL-2 expression postive was found in 83 cases (64.8%), while BCL-2 expression negative was observed in 45 cases (35.2%). The OS rates in BCL-2 expression positive and negative groups were 76.6% vs 76.8% in 3 years (P >0.05), and the PFS rates in BCL-2 expression positive and negative groups were 57.1% vs 70.5% (P >0.05) in 3 years, suggesting that BCL-2 expression level had no significant impact on OS and PFS rates in all DLBCL patients. However, among the older patients aged ≥ 60 years, there was singnificant different of 3 year OS rates in BCL-2 expression positive and negative groups (66.7% vs 76.4%, P >0.05), while 3-year PFS rate in BCL-2 expression positive group was obviosusly lower than that in BCL-2 expression negative group (35.8% vs 83.3%, P < 0.05).

CONCLUSIONThe positive expression of BCL-2 protein is a poor prognostic factor for older patients aged ≥ 60 years, thus this indicator possesses the prognostic value for these patients with DLBCL.

B-Lymphocytes ; Disease-Free Survival ; Humans ; Lymphoma, Large B-Cell, Diffuse ; Prognosis ; Proto-Oncogene Proteins c-bcl-2 ; Retrospective Studies ; Survival Rate