OBJECTIVETo explore the molecular mechanisms involved in the patient with congenital FV deficiency.

METHODSActivity of FV was determined by biochemical method. The PCR products of FV gene was analysed by directly sequencing or sequencing after cloned into T-vector. The mutative FV gene was analysed by restriction enzyme analysis in the proband and her family members.

RESULTSA homozygous missense mutation G5729T resulting in Gly1880Val was revealed in the proband and confirmed in the family screening. Structure-function studies of the factor V mutants (Gly1880Val) demonstrated the importance of Gly1880 for structural stability of the Factor V.

CONCLUSIONG5729T mutation of FV gene is related to the pathogenesis of congenital FV deficiency.

Adult ; DNA Mutational Analysis ; Factor V ; genetics ; metabolism ; Factor V Deficiency ; blood ; congenital ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Polymerase Chain Reaction

OBJECTIVETo study the phenotypes and genotypes of a protein C (PC) deficiency pedigree.

METHODSImmunoassay (ELISA) was used for PC antigen and activated PC (APC) detection, PCR for amplification of the fragment of protein C gene exon II to exon IX, single-strand conformation polymorphism (SSCP) for difference of denatured cDNA and DNA sequencing for gene mutation.

RESULTSFour members in the pedigree were found to be PC antigen levels between 34.3% - 67.8% and PC activity between 22% - 49% which are lower in comparison with normal references (80% - 120% and 70% - 130%, respectively). A G-to-A mutation in exon VII of the protein C gene at 6 219 position was identified in 9 members. This mutation resulted in the substitution of Arg for Gln at 169 amino acid.

CONCLUSIONThe proband is of heterozygosity. The G6219 A mutation in exon VII of the protein C gene leads to the substitution of Arg 169 Gln. This mutation is reported for the first time in China.

Adult ; DNA Mutational Analysis ; Female ; Humans ; Male ; Middle Aged ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; Protein C ; genetics ; metabolism ; Protein C Deficiency ; congenital ; genetics

OBJECTIVETo explore the molecular elements responsible for the enhanced apoptotic susceptibility in U937 cells mediated by silencing atactic telangiectasis mutation (ATM) gene.

METHODSTwo U937 cell mutants, U937-ASPI3K (ATM gene negative) and U937-pZEOSV2 (+) (ATM gene positive) were used as a cell model system. Apoptosis was examined by measuring free nucleosome concentrations in U937 cells. Western blotting was employed to measure nuclear protein abundances of cdc25A, cdc25B, cdc25C, total p34cdc2, p34cdc2 (Thr161) or p34cdc2 (Thr14, Tyr15). RT-PCR was used to estimate cdc25 transcript levels.

RESULTSU937-ASPI3K exhibited an enhanced apoptotic susceptibility to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-theronine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors could abolish the enhancement. Upon irradiation, p34cdc2 in U937-pZEOSV2 (+) was in an inactive state owing to phosphorylation of Thr 14 and Tyr15, which was associated with a dramatic decrease of nuclear cdc25A, cdc25B and cdc25C poteins. In contrast, p34cdc2 in U937-ASPI3K was in an active state owing to the low phosphorylation of Thr14 and Tyr15, which was associated with constant nuclear cdc25A, cdc25B and cdc25C protein abundance before and after irradiation. The responsive decrease of nuclear cdc25 proteins occurred at the post-transcription level.

CONCLUSIONSilencing ATM gene blocks the irradiation induced responsive decrease of nuclear cdc25 proteins, resulting in an abnormal activation p34cdc2 is the critical molecular mechanism for the enhanced apoptotic responses.

Apoptosis ; drug effects ; genetics ; radiation effects ; Ataxia Telangiectasia Mutated Proteins ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; Cycloheximide ; pharmacology ; DNA-Binding Proteins ; Enzyme Inhibitors ; pharmacology ; Gene Silencing ; drug effects ; radiation effects ; Humans ; Nuclear Proteins ; genetics ; metabolism ; Oxazoles ; pharmacology ; Phosphoprotein Phosphatases ; antagonists & inhibitors ; Protein Synthesis Inhibitors ; pharmacology ; Protein-Serine-Threonine Kinases ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Proteins ; U937 Cells ; cdc25 Phosphatases ; genetics ; metabolism

OBJECTIVETo explore the significance of nonmyeloablative allogeneic peripheral blood hematopoietic stem cell transplantation in the treatment of hematological diseases.

METHODSA nonmyeloablative conditioning regimen consisted of CD(3) monoclonal antibody, cyclosporine A, cyclophosphamide and cytarabine was used for allogeneic stem cell transplantation in 33 patients with hematological diseases. Of them, 11 were acute leukemia (AL) in first complete remission (CR(1)), 4 AL-CR(2) approximately 3, 3 refractory AL, 4 severe aplastic anemia (SAA), 7 chronic myeloid leukemia (CML), 2 myelodysplastic syndrome, 1 each of chronic lymphocytic leukemia (CLL) and myelofibrosis.

RESULTSAll 33 patients passed the hematopoietic suppression stage smoothly and achieved engraftment of the donor cells. There were 24 cases of full donor chimerism (13 cases converted from mixed chimerism), 4 mixed chimerism (MC) and 5 developed graft rejection. Of the 33 cases, 7 (21.2%) developed acute GVHD and chronic GVHD, 25 (75.8%) still live and 8 (24.2%) died.

CONCLUSIONSNonmyeloablative allogeneic peripheral blood stem cells transplantation is a safe, less toxic and curative approach for patients with hematological disease.

Acute Disease ; Adolescent ; Adult ; Chronic Disease ; Female ; Follow-Up Studies ; Graft vs Host Disease ; etiology ; Hematologic Diseases ; mortality ; therapy ; Humans ; Leukocyte Count ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; adverse effects ; Platelet Count ; Survival Analysis ; Survival Rate ; Transplantation Chimera ; blood ; Transplantation Conditioning ; methods ; Transplantation, Homologous ; Treatment Outcome

OBJECTIVESTo investigate the engraftment, survival and graft-versus host disease (GVHD) after transplantation of unrelated cord blood for the treatment of childhood and adult hematological malignancies.

METHODSSeventeen patients (13 children and 4 adults) with hematological malignancies were enrolled in this study. Twelve patients were transplanted with one unit and 5 with 2 units of cord blood. There were HLA-matched in 6 and HLA-mismatched at 1 approximately 2 loci in 11 patients. Ten patients were transplanted at stable status, 7 at advanced stage of leukemia. Conditioning regimens were BU/CY for 13 and CY/TBI for 3 patients. Most patients received additional ATG at a dose of 15 approximately 20 mg x kg(-1) x d(-1) for 3 days. CsA, mycophenolate mofetil (MMF) and methylprednisolone were used for GVHD prophylaxis.

RESULTSFourteen patients survived more than 40 days after transplantation were evaluated for engraftment. At day 60 after UCBT, 86% and 71% of the patients showed neutrophil and platelet engraftment, respectively. The time for an absolute neutrophil count > or = 0.5 x 10(9)/L was (21.0 +/- 1.3) days and platelet > or = 20 x 10(9)/L was (39.0 +/- 10.3) days. Four patients developed grade II acute GVHD and 2 chronic GVHD. Of the 17 patients, 11 were still alive and 8 of them were in event-free status. For the 10 patients transplanted at stable status, 2 year overall survival is 90%, and event-free survival (EFS) 70%. However, for the 7 patients transplanted at advanced stage of leukemia, only 2 survived without relapse. Of the 4 adult patients, 2 had sustained engraftment and survived for 18 and 14 months, respectively.

CONCLUSIONSHLA-matched or 1 approximately 2 loci-mismatched UCBT is a feasible procedure to cure a significant proportion of children or adults with leukemia, especially if performed in a favourable phase of disease. Two units of CBT can be used for adult patients if the cell number of one unit is not enough.

Adolescent ; Adult ; Child ; Child, Preschool ; Cord Blood Stem Cell Transplantation ; adverse effects ; Female ; Graft vs Host Disease ; etiology ; Hematologic Neoplasms ; immunology ; mortality ; therapy ; Histocompatibility Testing ; Humans ; Male ; Survival Analysis ; Survival Rate ; Transplantation Conditioning

OBJECTIVETo study the expression of gelatinases, including matrix metalloproteinase-9 (MMP-9, gelatinase B) and matrix metalloproteinase-2 (MMP-2, gelatinase A), in CD(34)(+) cells and leukemic cell lines, and explore the significance of gelatinase in migrating and homing capacity of CD(34)(+) cells, as well as the role of gelatinase in leukemia pathogenesis.

METHODSCD(34)(+) cells were isolated from umbilical cord blood and normal bone marrow by Mini MACS system. By zymogram analysis, MMP-2 and MMP-9 were detected in the serum free condition medium of CD(34)(+) cells and cell lines.

RESULTSOne brilliant band with molecular weight of 92 x 10(3) was detected in condition medium of cord blood CD(34)(+) cells. No band was detected in condition medium of bone marrow CD(34)(+) cells. Brilliant bands with molecular weight of 92 x 10(3) and 72 x 10(3) were detected in the condition medium of U937, KG-1a and HL-60 cell lines, but not in that of HEL, Namalva, CEM, K562 and LCL-H cell lines. In the condition media of J6-1 and J6-2 cells only the 92 x 10(3) band was detected.

CONCLUSIONSCord blood CD(34)(+) cells produced MMP-9, but bone marrow CD(34)(+) cells did not, partly explains the fact that cord blood CD(34)(+) cells possessed higher migrating capacity in comparison with bone marrow CD(34)(+) cells. The expression of MMP-9 and MMP-2 in leukemic cell lines varied.

Antigens, CD34 ; analysis ; Culture Media, Conditioned ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Fetal Blood ; cytology ; enzymology ; immunology ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; enzymology ; pathology ; Matrix Metalloproteinase 2 ; biosynthesis ; Matrix Metalloproteinase 9 ; biosynthesis ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVETo explore the ex vivo expansion characteristics of selected CD(34)(+) cells and mononuclear cells (MNC).

METHODSCD(34)(+) cells were isolated from umbilical cord blood MNC by MiniMACS system, expanded under the same conditions as that for MNC. The effects of re-isolation and the MNC supernatant (MNC-SN) on the selected CD(34)(+) cells were investigated. And the CD(34)(-) cells of MNC were cultured ex vivo.

RESULTSIn the culture of selected CD(34)(+) cells, both the colony density and the proportion of the CD(34)(+) cells declined continuously with the culturing, although they presented a high proliferation potential. However, in the culture of the MNC, from day 0 to day 7, the colony density and the proportion of CD(34)(+) cells were increased from 412 +/- 167/10(5) cells and (1.12 +/- 0.42)% to 1 162 +/- 566/10(5) cells and (4.17 +/- 1.44)%, respectively. It was found that both the total cells and the CD(34)(+) cells restored expansion potential by re-isolating. CD(34)(-) cells of MNC had the ability to form colony and could transform to CD(34)(+) cells. MNC-SN can promote colony forming ability and lead to CD(34)(+) cells differentiation at the same time.

CONCLUSIONSIn ex vivo culture, selected CD(34)(+) cells presented a high proliferation and differentiation potentials, and the CD(34)(-) cells produced during the cultivation had inhibition effect on CD(34)(+) cells expansion. CD(34)(-) cell population from cord blood MNC contained hematopoietic stem/progenitor cells and the cytokines secreted by CD(34)(-) cells could induce CD(34)(+) cells to more mature colony-forming cells.

Antigens, CD34 ; analysis ; Cell Count ; Cell Differentiation ; immunology ; Cell Division ; immunology ; Cells, Cultured ; Colony-Forming Units Assay ; Fetal Blood ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Time Factors

OBJECTIVETo study the in vitro antitumor activity of bcl-2 fully phosporothioated antisense oligodeoxynucleotide (bcl-2 ASODN) to malignant lymphoblastic cells.

METHODSProliferation and apoptosis of Raji cells incubated with bcl-2 ASODN were evaluated by MTT assay, flow cytometry (FCM) and electron microscopy, and the level of bcl-2 protein and mRNA expression were assessed by FCM and RT-PCR, respectively.

RESULTSMTT assay demonstrated that bcl-2 ASODN could partially inhibit the growth of Raji cells. After incubated with ASODN for 48 hours, Raji cells exhibited characteristic morphologic changes of apoptosis, including cytoplasm membrane blebbing, chromatin condensation crescents formation and nuclear fragmentation. The apoptosis rate of Raji cells treated with 20 micromol/L bcl-2 ASON for 72 hrs was 43.86% which is significantly higher than that of control (10.05%). The bcl-2 ASODN induced apoptosis of Raji cells was accompanied by declined expression of bcl-2 mRNA, which decreased to 0.88% at 72 hrs and was significantly lower than that of control (79.54%).

CONCLUSIONbcl-2 ASODN induced Raji cells apoptosis by downregulating bcl-2 protein.

Apoptosis ; drug effects ; Cell Division ; drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured ; drug effects ; metabolism

OBJECTIVETo investigate the clinical value of glycosylated G-CSF combined with middle-high dose cyclophosphamide (Cy) or conventional chemotherapy with increased dose of Cy for mobilizing peripheral blood progenitor cells in patients with tumor.

METHODSThirty patients from four hospitals in Beijing region were enrolled in this clinical study. Diagnoses of the patients were non-Hodgkin' lymphoma (n = 21), Hodgkin disease (n = 1), breast cancer (n = 7) and ovary cancer (n = 1). Autologous peripheral blood progenitor cells (APBPC) were mobilized by middle-high dose Cy or conventional chemotherapy with increased dose of Cy combined with G-CSF. G-CSF was given subcutaneously from the nadir of the white blood cell (WBC) count to the end of PBPC collection. The dosage of G-CSF was 250 microg/d in 29 patients and 500 microg/d in 1 patient. When WBC count was > 5 x 10(9)/L, APBPC were harvested with CS 3000 plus/COBE Spectra.

RESULTSThe average dosage of Cy was 3.95 g (2.3 g/m(2)). The doses of G-CSF were 3.1 approximately 6.4 microg x kg(-1) x d(-1). Thirteen patients (43%) were collected twice, 14 patients (47%) three times and 3 patients (10%) four times. All of the patients could tolerate the treatment regimens. Seven patients had bone pain after G-CSF injection and one was severe, one patient had headache and one had nausea and vomiting.

CONCLUSION250 microg glycosylated G-CSF combined with middle-high Cy or conventional chemotherapy with increased dose of Cy combined G-CSF is an optimal method for APBPC mobilization in tumor patients.

Adolescent ; Adult ; Antigens, CD34 ; analysis ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Colony-Forming Units Assay ; Cyclophosphamide ; administration & dosage ; Dose-Response Relationship, Drug ; Female ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; Hematopoietic Stem Cell Mobilization ; Humans ; Leukocyte Count ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Male ; Middle Aged ; Neoplasms ; blood ; drug therapy ; pathology ; Platelet Count ; Treatment Outcome

OBJECTIVETo study the effect of ex vivo expansion on the adhesion activities of umbilical cord blood hematopoietic stem and progenitor cells (HSPC).

METHODSFresh UCB CD(34)(+) cells were cultured in a serum and stroma-free culture system. At day 7, day 10 and day 14, CD(34)(+) cells were re-selected from the expanded products. The expression of adhesion molecules (CAMs) such as VLA-4, VLA-5, LFA-1, ICAM-1, HCAM, L-selectin and PECAM-1, and the adhesion activity of the expanded CD(34)(+) cells were evaluated and compared with those of precultured fresh CD(34)(+) cells.

RESULTS(1) The CD(34)(+) cells expressing homing-related CAMs were increased (from 15-fold increase for CD(34)(+) CD(54)(+) subset to 72-fold increase for CD(34)(+) CD(49e)(+) subset at day 14). (2) The expressions of CD(49d), CD(44), CD(11a) and CD(49e) on the expanded CD(34)(+) cells were increased or sustained the same levels as those on fresh UCB CD(34)(+) cells, while the expression of CD(62L), CD(54) and CD(31) on expanded CD(34)(+) cells declined with the cultivating. (3) Spontaneous adhesion and SDF-1-induced adhesion tended to be increased in the course of the first 10 day's culture.

CONCLUSIONSThe culture system used in this study could substantially support the expansion of HSPCs expressing the above CAMs, and the expanded HSPCs would sustain their intrinsic adhesion potentials.

Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Cell Adhesion ; Cell Adhesion Molecules ; biosynthesis ; Cell Division ; Fetal Blood ; cytology ; immunology ; metabolism ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; metabolism ; Humans ; Receptors, Lymphocyte Homing ; biosynthesis