Biomimetics ; Blood Cells ; metabolism ; CD55 Antigens ; blood ; CD59 Antigens ; blood ; Cell Membrane ; Humans ; Male ; Submarine Medicine

Aged ; CD59 Antigens ; immunology ; Female ; Granulocytes ; immunology ; Humans ; Myelodysplastic Syndromes ; immunology ; pathology

Biomarkers, Tumor ; CD59 Antigens ; physiology ; Glycosylphosphatidylinositols ; physiology ; Humans ; Membrane Cofactor Protein ; physiology ; Neoplasms ; diagnosis ; therapy

OBJECTIVE: To analyze the effect of down-regulating the CD59 gene expression by RNAi lentivirus as vector on Jurkat cell line of acute T-lineage leukemia. METHODS: The expression of CD59 in Jurkat cell line of acute T-line leukemia was induced to decrease by RNAi lentivirus as vector. The transfection of RNA lentivirus and the localization of CD59 molecule were analyzed by laser confocal technique. The relative expression of CD59 gene in blank control, negative control and RNAi lentivirus transfected group was detected by real-time fluorescence quantitative PCR, and the enzyme-linked immunosorbent assay was used to detect the expression of TNF-β and IL-3 in supernatants of cultured cells in 3 groups. The expression levels of apoptosis-related molecules including Caspase-3, Survivin, BCL-2 and BCL-2-associated X protein (BAX) were measured by Western blot. RESULTS: The transfection efficiency for Jurkat cells was higher than 90%. CD59 was mainly located on the cell membrane. Compared with the blank control group and the negative control group, the expression level of CD59 mRNA and protein in the RNAi lentivirus transfected group significantly decreased (P<0.05). Compared with the blank control group and the negative control group, the expression of TNF-β and IL-3 in the RNAi lentivirus transfected group were significantly higher and lower (P<0.05) respectively. The expression levels of Survivin and BCL-2 in the RNAi lentivirus transfected group were significantly lower than those in the blank control group and the negative control group, while the expression levels of Caspase-3 and BAX in the RNAi lentivirus transfected group were significantly higher than those in the blank control group and the negative control group (P< 0.05). CONCLUSION The down-regulation of CD59 gene expression induced by RNAi lenti-virus can decrease the expression of proliferation and differentiation-promoting molecule such as IL-3 and increase the expression of TNF-related factor in Jurkat cell line of acute T-lineage leukemia, which also can increase the expression of apoptosis-related proteins such as Caspase-3 and BAX, and decrease the expression of anti-apoptosis-related proteins such as Survivin and BCL-2.
Apoptosis ; CD59 Antigens ; Cell Lineage ; Cell Proliferation ; Down-Regulation ; Humans ; Jurkat Cells ; Lentivirus ; Leukemia ; RNA Interference ; RNA, Small Interfering ; Transfection

OBJECTIVETo investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH.

METHODSCD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted.

RESULTSThe optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01).

CONCLUSIONSThe CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.

Adolescent ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; CD59 Antigens ; analysis ; Cell Separation ; Child ; Female ; Hematopoiesis ; Hemoglobinuria, Paroxysmal ; therapy ; Humans ; Male

To investigate the effects of sera on the growth of single CD34(+) cells from patients with paroxysmal nocturnal hematoglobinuria (PNH), sera from both PNH patients and normal individuals were added separately to the single cell culture system and semi-solid colony formation system. The growth of the normal CD34(+) and PNH CD34(+) CD59(+) and CD34(+) CD59(-) cells was evaluated. No growth difference was found for growth of the normal CD34(+) and PNH CD34(+) CD59(+) cells when PNH or normal sera were added to the culture media in either single cell culture or colony formation culture. While no difference was detected for single PNH CD34(+) CD59(-) cells growth when PNH or normal sera were added, more colonies were observed in semi-solid culture with PNH sera. A conclusion was reached that compared with those from normal controls, the sera from PNH patients had no significant influence on single hematopoietic stem/progenitor cells derived from normal subjects and from PNH patients, but the PNH sera might promote the colony formation of the CD34(+) CD59(+) cells in semi-solid culture
Antigens, CD34 ; analysis ; Blood Proteins ; pharmacology ; CD59 Antigens ; analysis ; Cell Division ; drug effects ; Cells, Cultured ; Hematopoietic Stem Cells ; drug effects ; pathology ; Hemoglobinuria, Paroxysmal ; immunology ; pathology ; Humans

OBJECTIVESTo investigate the stroma-independent growth ability, multilineage differentiation and expansion of the single hematopoietic stem/progenitor cell from patients with paroxysmal nocturnal hematoglobinuria (PNH).

METHODThe CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) CD(59)(+) cells from normal volunteers were sorted as each single cell into a well of 96 well culture plates containing culture medium supplemented with SCF, IL-3, Epo, GM-CSF, G-CSF, IL-6, Tpo and Flt-3 ligand.

RESULTS(1) Single PNH CD(34)(+) CD(59)(-) cell had a higher capacities for plating efficiency, colony (>/= 50 cells) formation and cell expansion than that of the PNH CD(34)(+) CD(59)(+) cells (P < 0.05); (2) Both the single CD(34)(+) CD(59)(-) cells from PNH patients and the single CD(34)(+) CD(59)(+) cells from normal controls had similar capacities for cell plating efficiency and colony and large colony formation. The PNH CD(34)(+) CD(59)(-) cells had a lower average cell production and cell expansion capacity. (3) The single CD(34)(+) CD(59)(+) cells from both PNH patients and normal controls showed the same capacities for cell plating efficiency and colony formation. The PNH CD(34)(+) CD(59)(+) cells exhibited much lower capacity for large colony formation, average cell production and total cell expansion. (4) A diminished secondary colony formation ability was also observed in the PNH CD(34)(+) CD(59)(+) and CD(34)(-) CD(59)(-) clones.

CONCLUSIONThe single PNH CD(34)(+) CD(59)(-) cells had growth advantage over the single PNH CD(34)(+) CD(59)(+) cells to some extent, but they both had impaired growth abilities as compared with CD(34)(+) cells from normal volunteers.

Antigens, CD34 ; immunology ; CD59 Antigens ; immunology ; Cell Culture Techniques ; Cell Division ; physiology ; Colony-Forming Units Assay ; Hematopoietic Stem Cells ; immunology ; pathology ; Hemoglobinuria, Paroxysmal ; physiopathology ; Humans

OBJECTIVETo explore in vitro expansion of CD34+CD59+ cells from patients with PNH, and compare the capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control.

METHODSCD34+CD59+ cells from patients with PNH and CD34+ cells from normal control were selected from the bone marrow mononuclear cells by means of two-step sorting method with immunomagnetic microbead-flow cytometry, then underwent in vitro expansion for two weeks and semi-solid culture in vitro before and after expansion.

RESULTS(1) CD34+CD59+ cells from patients with PNH can be expanded effectively in vitro, and the biggest expansion of CD34+CD59+ cells was about 23.49 fold on the 7th day. (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, such as: the best combination of hematopoietic factors for in vitro expansion was SCF+ IL-3 + IL-6 + FL + Tpo + Epo, and the seventh day was the most suitable in course of 4-14 days for in vitro expansion, and after in vitro expansion, the cells remained CD59 positive and strong capability of performing colony-forming. (3) CD34+ cells from normal control had better proliferation, expansion and stronger potential to survive than CD34+CD59+ cells from patients with PNH.

CONCLUSIONS(1) In vitro expansion of CD34+CD59+ cells from patients with PNH can be performed. The present study showed the possibility of performing ABMT or APBSCT clinically for patients with PNH. (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, but the latter had better proliferation, expansion and stronger potential to survive than the former. CD34+CD59+ cells from patients with PNH were not completely normal cells.

Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Cell Differentiation ; Cell Division ; Cell Survival ; Cells, Cultured ; Hemoglobinuria, Paroxysmal ; immunology ; pathology ; Humans ; Immunophenotyping

OBJECTIVETo analyse the expression of glycosylphosphatidylinositol (GPI)-anchored protein and functional transition of lymphocyte subsets in paroxysmal nocturnal hemoglobinuria (PNH) patients.

METHODSFlow cytometer and multi-color McAbs were used to detect CD(59) expression on lymphocytes and their subsets. Stimulation test was used to examine the ability of affected T cells in converting from CD(45RA) phenotype to CD(45RO) phenotype.

RESULTSIn 14 PNH samples, CD(59) expression on affected lymphocytes was at a lower percentage than that on granulocytes and erythrocytes. B lymphocytes were widely affected than NK or T cells. CD(59)(-) deficient B lymphocytes existed in all PNH patients. More CD(8)(+) T cells were affected than CD(4)(+) T cells. Expression of HLA-DR and CD(45RO) on affected T cells were significantly lower than that on normal T cells (P < 0.01), whereas the expression of CD(45RA) was much higher on the former (P < 0.01). After stimulation with phytohemagglutinin (PHA), the expression of CD(45RA)(+) on affected T cells decreased sharply (the median value was from 82.7% to 26.6%), while the expression of CD(45RO)(+) increased significantly (the median value was from 19.6% to 64.3%).

CONCLUSIONSThe deficient CD(59) phenotype does involve in the different types of lymphocytes in PNH patients. Therefore affected B cells could be a good marker for diagnosis of PNH. The affected T cells were mainly comprised naive cells (CD(45RA)(+)HLA-DR(-)), which could acquire the memory phenotype (CD(45RO)(+)HLA-DR(+)) after stimulated by antigen.

Adolescent ; Adult ; CD59 Antigens ; analysis ; Female ; Hemoglobinuria, Paroxysmal ; blood ; immunology ; Humans ; Leukocyte Common Antigens ; analysis ; Lymphocyte Subsets ; cytology ; immunology ; Lymphocytes ; cytology ; immunology ; Male ; Middle Aged

OBJECTIVETo explore the characteristics of CD(34)(+) CD(59)(+) cells from paroxysmal nocturnal hemoglobinuria(PNH) patients' bone marrow and the possible reasons of hematopoietic clonal dominance of PNH clones.

METHODSCD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) cells from normal control were selected from the bone marrow mononuclear cells by means of immunomagnetic microbead-flow cytometry two step sorting method undergone ex vivo expansion in liquid culture for two weeks and performed semisolid cultures before and after expansion.

RESULTS(1) Cultivation for seven days was the optimum for ex vivo expansion of PNH CD(34)(+) CD(59)(+) cells and normal CD(34)(+) cells, both cell populations remained CD(59) positive after expansion. (2) Normal CD(34)(+) cells had higher capacities of proliferation and expansion, and stronger potential to survival than that of both PNH CD(34)(+) CD(59)(+) and PHN CD(34)(+) CD(59)(-) cells. (3) In terms of semisolid culture, there was no significant difference in the yields of CFU formation between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells. (4) In liquid culture with combinations of hematopoietic factors SCF + IL-3 + IL-6 + FL + Tpo or SCF + IL-3 + IL-6 + FL + Tpo + Epo, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells; but with combination of SCF + IL-3 + IL-6 + FL + Tpo + Epo + GM-CSF, CD(34)(+) CD(59)(-) cells had better proliferation and expansion capacities and stronger potential to survival than that of CD(34)(+) CD(59)(+) cells.

CONCLUSIONS(1) Normal CD(34)(+) cells had better proliferation, expansion capacities and stronger potential to survival than that of PNH CD(34)(+) CD(59)(+)cells. (2) In semisolid and liquid culture with hematopoietic factor combinations, there was no significant difference in the capabilities of survival, proliferation and expansion between CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells. It was suggested that CD(34)(+) CD(59)(-) cells had no clonal hemotopoiesis dominance. GM-CSF might be one of the reasons for PHN clones to possess clonal hematopoiesis dominance.

Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Cell Division ; Cell Survival ; Cells, Cultured ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; pathology ; physiopathology ; Humans