OBJECTIVETo understand the expression of complement regulatory proteins CD55 and CD59, which secreted by epithelial cells of normal and chronic sinusitis patients.

METHODSCell culture and flow cytometry were used to detect the expression of complement regulatory proteins CD55 and CD59.SPSS 18.0 software was used to analyze the data.

RESULTSCD55 was expressed both in normal nasal mucosa and mucosa of chronic sinusitis. The expression in normal group was 0.001 ± 0.001, significantly lower than that in CRS group which was 0.067 ± 0.028 (t = -10.535, P < 0.01). CD59 was also expressed in the two groups . In normal group, the expression of CD59 was 0.879 ± 0.005, significantly higher than that in CRS group which was 0.238 ± 0.034 (t = 83.416, P < 0.01).

CONCLUSIONSThe nasal mucosa in CRS patients showed low expression of CD55 and high expression of CD59. This mechanism may be involved in the occurrence of CRS.

CD55 Antigens ; metabolism ; CD59 Antigens ; metabolism ; Female ; Flow Cytometry ; Humans ; Rhinitis ; metabolism ; Sinusitis ; metabolism

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

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

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

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 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

Since flow cytometry was not feasible for sorting a huge amount of cells for clinical use, the method of double immunomagnetic positive sorting was used for selection of CD34(+)CD59(+) cells from bone marrow mononuclear cells in patients with paroxysmal nocturnal hemoglobinuria (PNH), which laid the groundwork for clinical ABMT/APBSCT of patients with PNH. Immunomagnetic positive selection was used for two times, the microbeads were removed from the CD34(+) cells selected firstly by means of overnight culture, then the sufficient CD34(+)CD59(+) cells were used for ex vivo expansion. The results showed that the survival, proliferation and colony-forming units of the selected CD34(+)CD59(+) cells by double immunomagnetic positive sorting had no significant difference as compared with that of CD34(+)CD59(+) cells selected by flow cytometry technique. It is suggested that the double immunomagnetic positive sorting promotes the use for separation and purification hematopoietic stem/progenitor cells and other cells with double or multiple markers cells for autologous hematopoietic stem cell transplantation in PNH patients.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; CD59 Antigens ; analysis ; Flow Cytometry ; Hematopoietic Stem Cell Transplantation ; Hemoglobinuria, Paroxysmal ; therapy ; Humans ; Immunomagnetic Separation ; Transplantation, Autologous