As a functionally and phenotypically distinctive T cell subpopulation, CD4+CD25+ regulatory T cells are anergic and retain their ability to suppress antigen-driven response of CD4+CD25- cells in a contact-dependent manner or through a way of secreting immunosuppressive cytokines such as IL-10 and TGF-beta. Graft-versus-host disease (GVHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Recently, some researches on the relationship between donor CD4+CD25+ regulatory T cells and GVHD severity produced two contradictory conclusions: one is CD4+CD25+ regulatory T cells that can prevent GVHD efficiently; the other is that GVHD is associated with the increased numbers of peripheral blood CD4+CD25+ regulatory T cells. The answer to this question will provide a new idea for clinic therapy of GVHD. In this review some new research progresses in the related area, such as the CD4+CD25+ regulatory T cells, the phenotype, characteristics, immunoregulatory mechanisms of CD4+CD25+ regulatory T cells, as well as the relation of CD4+CD25+ with GVHD were presented.
CD4 Antigens ; analysis ; Graft vs Host Disease ; etiology ; immunology ; Humans ; Interleukin-2 Receptor alpha Subunit ; analysis ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism

5-hydroxtryptamine (5-HT, serotonin) has been recognized not only as a neurotransmitter and vasoactive agent, but also as a growth factor. 5-HT mainly binds to 5-HT(2) receptors or 5-HT(1) receptors on cell surface to stimulate cell proliferation through Ras or MAPK pathway in many cell types. It has been reported that 5-HT stimulates megakaryocytopoiesis via 5-HT receptors. The possible mechanism of 5-HT on the proliferation and differentiation of megakaryocytes (MK) has been discussed in this review article. In early stage of megakaryocytopoiesis, 5-HT may bind to 5-HT(2B) receptor on megakaryocytes, and promotes their proliferation and differentiation. In the late stage, 5-HT may involve in the platelet release procedure by inducing nitric oxide (NO) synthesis via 5-HT(2A) receptors. 5-HT can also antagonize the apoptotic effect induced by thrombospondin-1 (TSP-1) which is a platelet alpha granule protein and has synergic effect with platelet-derived growth factor (PDGF) to enhance megakaryocytes proliferation. Therefore, 5-HT is likely to be an important substance in the feedback regulation of thrombopoiesis. In this review the 5-HT and its receptors, 5-HT as cell growth factor, pathway of 5-HT stimulating cell proliferation and influance of 5-HT on MK-progenitor cells were summarized.
Humans ; Megakaryocytes ; physiology ; Receptors, Serotonin ; metabolism ; Receptors, Serotonin, 5-HT2 ; metabolism ; Serotonin ; metabolism ; pharmacology ; Thrombopoiesis ; physiology ; Thrombopoietin ; physiology

To investigate the leukemia relapse of AL patients after HLA haploidentical bone marrow transplantation (HLA HBMT), 2 relapsed leukemia patients received HLA HBMT were studied, peripheral blood simples and bone marrow smear were examined, morphologic change of bone marrow cells was observed, while the HLA genotype and chromosome karyotye were analyzed by PCR and routine G-banding methods, respectively. The results indicated that the two cases were diagnosed primarily as acute lymphocytic leukemia (common cell subtype) and acute megakaryocytic leukemia, in which chromosome abnormalities or activation of protooncogene in leukemic cells were observed. The complete hematopuietie reconstitution of donor origin was obtained in these 2 cases after HLA HBMT, but the leukemic cells in these 2 leukemia patients were confirmed to be donor origin after relapse, their blood groups and HLA genotype were found to be originated from donor. These 2 relapsed leukemia patients were diagnosed as acute lymphocytic leukemia (B cell subtype) and acute megakaryocytic leukemia. It is suggested that high-dose of immunosuppressive agents used in transplantation may contribute to leukemia relapse of donor origin in these patients. Abnormalities in hematopoietic microenvironment may be also involved in the leukemia development. Donor-cell leukemia after allogeneic hematopoietic stem cell transplantation can be an ideal model to investigate the related events in human leukemogenesis.
Cell Transformation, Neoplastic ; Child ; HLA Antigens ; immunology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Histocompatibility ; Humans ; Infant ; Leukemia, Megakaryoblastic, Acute ; blood ; therapy ; Male ; Neoplasms, Second Primary ; etiology ; pathology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; blood ; therapy ; Recurrence ; Transplantation, Homologous

To explore the efficacy and safety of high-dose of etoposide with granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells, 10 patients with hematologic malignancies including 6 patients with multiple myeloma and 4 with non Hodgkin' s lymphoma received an etoposide dose of 1.6 g/m2. The total dose of undiluted etoposide was given on day 1 as a continuous intravenous infusion via a central vein for 10 hours. G-CSF 5 microg/kg was used on day 3 and given daily subcutaneously until leukopheresis was completed. The results showed that leukopheresis was started at days 11 (range 9-13 days) following etoposide therapy, the mean number of CD34+ cells collected in all 10 patients was 9.4 x 10(6)/kg (range 4.2 - 17.3 x 10(6)/kg), by an average of 2.6 leukophereses (range 1-4) times. Mobilization procedure that produced yields of greater than 4.0 x 10(6)/kg were achieved in every patient. Toxicity showed oropharyngeal mucositis, faucitis and urethritis respectively in 3 patients. It is concluded that high-dose etoposide with G-CSF is an effective and safe mobilizing regimen for autologous peripheral blood stem progenitor cells in patients with hematologic malignancies.
Adolescent ; Adult ; Aged ; Antineoplastic Agents, Phytogenic ; administration & dosage ; Etoposide ; administration & dosage ; Female ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; Hematologic Neoplasms ; blood ; therapy ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; methods ; Transplantation, Homologous

To determine whether addition of vitamin C (Vit C) to single-unit plasma could influence the efficacy of inactivating viruses and could maintain the activity of plasma proteins by methylene blue (MB)-light treatment. Vesicular stomatitis virus (VSV) Indiana strain was used as the indicating virus. Human plasma containing VSV was added with different concentrations of Vit C and final concentration 1 micromol/L MB and irradiated by fluorescence at an intensity of 40,000 lx, samples were collected at different times for detection. Cytopathic effect was used to test the effect of virus inactivation. A segment of the nucleic acid encoding capsid protein of VSV was amplified with RT-PCR. Some methods, such as the Clauss method, the one-stage method, microimmunoelectrophoresis, were used to investigate the changes of plasma components. The results showed that when the VSV plasma was added with 240 micromol/L Vit C and treated by MB-light irradiation for 60 min, the titer of VSV decreased by more than 8 lg TICD50/ml. Meanwhile, target segment amplification of VSV was also negative. The recovery rates of fibrinogen and coagulation factor VIII (FVIII: C) were 83.55% and 81.67% respectively, which had significant difference comparing with the routine MB-fluorescent light treatment. Most of plasma proteins were not affected significantly. No change in immunogenicity of these proteins was observed by using microimmunoelectrophoresis. It is concluded that virus inactivation is not influenced and plasma proteins are effectively protected by Vit C. Vit C can be used as a protector and is beneficial to improving the quality of plasma subjected to MB- photodynamic treatment.
Ascorbic Acid ; pharmacology ; Blood Proteins ; metabolism ; Humans ; Light ; Methylene Blue ; pharmacology ; Plasma ; virology ; Vesicular stomatitis Indiana virus ; drug effects ; Virus Inactivation ; drug effects

This study was aimed to construct the CD14 eukaryotic expression vector, establish the transgeneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M(5)). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone techniques. Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases and ligating with T4 ligase. A murine melanoma cell line B16 was transfected with the pcDNA3.1(+)/CD14 recombinant with Superfect transfection reagent. Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry (FCM). The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank database. The recombinant pcDNA3.1(+)/CD14 was identified with double-enzyme cleaving. The expression of the human CD14 on the transfectant (B16/CD14) was confirmed by FCM. In conclusion, the murine cell line B16/CD14 fransfected with human CD14 gene has been established which can be used for the study of human AML-M(5) antibody targeting therapy with mouse model.
Animals ; Antigens, Neoplasm ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Leukemia, Monocytic, Acute ; genetics ; Lipopolysaccharide Receptors ; genetics ; Melanoma, Experimental ; genetics ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Transfection ; Tumor Cells, Cultured

To construct a plasmid expressing MDR1 short hairpin RNA (shRNA) mediated by pEGFP-C1/U6 vector, two coding sequences of 19 nucleotides were selected from MDR. Two pairs of oligonucleotides were designed for these two fragments. After annealing the formed double-stranded DNAs were ligated with plasmid pEGFP-C1/U6 (pEGFP-C1 vector with U6 promoter). The plasmids producing MDR1 shRNA were constructed from the inverted motif containing 9 spacers and four Ts. The results showed that the constructed plasmids were named pEGFP-C1/U6/A and pEGFP-C1/U6/B, and the constructs were identified by restriction and sequence analysis, no any base mutation was observed. It is concluded that plasmids of pEGFP-C1/U6/A and pEGFP-C1/U6/B expressing MDR1 shRNA were successfully constructed, providing a highly effective method for reversing the multidrug resistance in clinic.
ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; DNA-Binding Proteins ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Plasmids ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; biosynthesis ; genetics ; RNA, Small Nuclear ; genetics

Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; isolation & purification ; Hematopoiesis ; genetics ; Humans ; Species Specificity ; Stem Cell Factor ; biosynthesis ; genetics ; isolation & purification

This study was aimed to investigate the efficiency of modified methylation-specific polymerase chain reaction i.e. nested methylation-specific polymerase chain reaction, used to detect the promoter methylation of p16 gene in six hematological malignant cell lines, and to explore the application in selection of hematological malignant cell lines with promoter hypermethylation, and make them to be an idel cell models for studying the relationship between gene methylation and expression. DNAs were denatured by NaOH and then were subjected to bisulfite modification and a nested-MSP was used to amplify the promoter region, nested MSP product of p16 gene promoter was analyzed and sequenced. The results showed that the hypermethylation of p16 gene was detected in CA46 and U266, however, Molt4, K562, HL-60 and Jurkat cell lines were unmethylated. In conclusion, p16 gene methylation in hematological malignant cell lines can be perfectly detected by nested-MSP method, which is simple, sensitive and specific for screening all kinds of hematological malignant cell lines with p16 gene methylated.
Base Sequence ; Cell Line, Tumor ; DNA Methylation ; Genes, p16 ; HL-60 Cells ; Hematologic Neoplasms ; genetics ; pathology ; Humans ; K562 Cells ; Lymphoma ; genetics ; pathology ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.
Dependovirus ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; biosynthesis ; genetics ; Platelet Membrane Glycoprotein IIb ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thrombasthenia ; metabolism ; therapy