Proliferation and anti-tumor activity of lymphocytes and their effect on normal phenotype of autologous bone marrow hematopoietic cells in patients with paroxysmal nocturnal hemoglobinuria / 中国实验血液学杂志

There is a hypothesis that paroxysmal nocturnal hemoglobinuria (PNH) hematopoitic stem cells are resistant to the cytotoxic effect of T cells because they lack glycosylphosphatidylinositol (GPI)-linked proteins. The aim of this study is to investigate proliferation and anti-tumor activity of lymphocytes in patients with PNH, and also to assay the effect on normal cell (CD59(+)) phenotype in bone marrow of PNH patient by autologous lymphocytes in vitro. MTT assay for detection of lymphocytes proliferation and its anti-tumor effect was driven to delineate T cell reactive function. The CD34(-) and CD34(+) bone marrow cells (selected by means of immunomagnetic method) as well as unsorted marrow cells in PNH patients were cultured together with autologous CD59(+) or CD59(-) lymphocytes, their cultured supernatant, extrinsic IFN-gamma and IL-2 in a liquid culture system. CD59(+) cells were counted by flow cytometry after 10 day culture in vitro. The results showed that there were no differences in the proliferation ability of lymphocytes between each group: controls 1.42 +/- 0.46, unsorted PNH lymphocytes 1.40 +/- 0.35, CD59(-) 1.30 +/- 0.40, and CD59(+) PNH lymphocytes 1.40 +/- 0.42. Anti-tumor effect of lymphocytes declined in PNH patients when compared with control [(50.00 +/- 28.67)% vs. (76.13 +/- 10.15)%]. The proportion of CD59(+) cells diminished significantly after culture with autologous lymphocytes, their supernatant, extrinsic IFN-gamma or IL-2 (P < 0.01) in groups of unsorted, CD34(+) and CD34(-) bone marrow cells. No significant difference was found between groups of CD59(-) and CD59(+) lymphocytes, or CD34(-) and CD34(+) marrow cells. It is concluded that turbulences of immune regulations may be involved in pathogenesis of PNH.

Application of double immunomagnetic positive sorting to ex vivo expansion of marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria / 中国实验血液学杂志

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.

Study of biological characteristics of the CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients' bone marrow / 中华血液学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>CD(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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSIONS</b>(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.</p>

A colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria / 中国实验血液学杂志

The purpose of the study is to establish a colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). RBCs from normal persons and patients with PNH and non-PNH anemia were treated with HEC toxin secreted by Aeromonas hydrophila J-1 strain and the absorbance at 630 nm was measured to quantitate the extent of hemolysis. The results demonstrated that the RBCs from PNH patients showed resistance to the toxin hemolysis, which was in accord with the percentages of CD59(-) cells, while the RBCs from normal persons and non-PNH anemic patients were nearly totally lysed. It is concluded that the method can be considered as a simple, specific and reliable method for the diagnosis of PNH.

Inhibition of the activation of transcriptional factor NFkappaB during sodium selenite-induced apoptosis in NB4 cells / 中国实验血液学杂志

In order to evaluate the effect of sodium selenite on the activation of NFkappaB during selenite-induced apoptosis in NB4 cells, Western blot was used to measure the level of P65 in nuclear extraction of NB4 cells treated with sodium selenite to reflect the activation of NFkappaB; the apoptosis of NB4 cells was determined by morphological observation, DNA ladder electrophoresis and flow cytometry; and MTT test was used to measure the growth inhibition of cells. Results showed that sodium selenite (>/=5 micro mol/L) suppressed the cell growth, induced apoptosis and inhibited the activation of NF kappaB in a concentration- and time-dependency pattern. It was concluded that inhibition of NF kappaB might be one of the mechanisms in selenite-induced apoptosis in NB4 cells.

The comparison between the mechanisms of sodium selenite induced apoptosis and arsenic trioxide induced apoptosis in human acute promyelocytic leukemia cell line NB4 cells / 中国实验血液学杂志

In order to explore the differences between the mechanisms of selenite-induced apoptosis and arsenic induced apoptosis in NB4 cells, growth inhibition was determined by MTT test, apoptosis determined by DNA electrophoresis and analysis of intracellular DNA contents, reactive oxygen species and reduced glutathione in the cell were measured by Lucigenin dependent chemoluminescent (CL) test and spectrophotometry, and mitochondrial transmembrane potential was measured by flow cytometry. The results showed that: 5 micro mol/L sodium selenite similar to 1 micro mol/L arsenic trioxide could induce the apoptosis of NB4 cells after treatment for 24 hours. Both could elevate the level of reactive oxygen species and intensify mitochondrial transmembrane potential collapse, accompanied with decrease of reduced glutathione centent. The effect of selenium selenite on these aspects was more significant than those of arsenic trioxide. Elevation of intracellular glutathione in N-acytlcysteine pretreated NB4 cells could enhance the selenite induced apoptosis and oxidative stress, but ameliorate the arsenic trioxide induced apoptosis and oxidative stress. It was concluded that sodium selenite and arsenic trioxide can induce the apoptosis of NB4 cells, but there are significant differences between the mechanisms of selenite-induced and arsenic-induced apoptosis in NB4 cells, particularly in the influence of intracellular glutathione content on the drug action.

Comparison of capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control / 中国医学科学院学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>CD34+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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSIONS</b>(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.</p>

The effects of sera from patients with paroxysmal nocturnal hematoglobinuria on the growth of CD34(+) cells / 中国实验血液学杂志

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

Sodium selenite-induced oxidative stress and apoptosis in human acute promyelocytic leukemia NB4 cells / 药学学报

<p><b>AIM</b>To explore sodium selenite-induced oxidative stress and apoptosis in human promyelocytic leukemia NB4 cells.</p><p><b>METHODS</b>The growth inhibition of NB4 cells was measured by MTT test. Apoptosis was determined morphologically by Giemsa stain and by DNA ladder formation in electrophoresis. Quantitation of apoptosis was determined by percentage of PI stained cells containing subdiploid amount of DNA measured by flow cytometry. Generation of reactive oxidative species (ROS) in NB4 cells was determined by lucigenin dependent chemoluminescent (CL) test. Spectrophotometer was used to measure the level of reduced glutathione, superoxide dismutase (SOD) and glutathione peroxidase in the cell.</p><p><b>RESULTS</b>Sodium selenite was shown to inhibit the growth of NB4 cells. Sodium selenite induced apoptosis with dose and time dependency: the ratio of subdiploid cells in control group was 1.3% +/- 0.7%. The 5 mumol.L-1 group was 10.4% +/- 1.4%, 10 mumol.L-1 group was 16% +/- 1%, and the 20 mumol.L-1 group was 27.3% +/- 0.8%. Sodium selenite (> or = 5 mumol.L-1) enhanced the ROS level markedly in NB4 cells (in 20 mumol.L-1 group ROS level was increased by 17 times, compared with control group), accompanied with decrease of reduced intracellular glutathione. These effects were time and dose dependent. N-acytlcysteine as an antioxidant was found to inhibit sodium selenite-induced oxidative stress and apoptosis in NB4 cells.</p><p><b>CONCLUSION</b>Sodium selenite can induce apoptosis of NB4 cells and would possibly be used as an agent for the treatment of malignancy. The main mechanism of action might be related to oxidative stress induced by sodium selenite, thereby, leading to apoptosis as shown in NB4 cells.</p>

GPI-Pr Deficiency and Apoptosis of PNH Granulocytes / 中国实验血液学杂志

To study the relationship of Glycosyl phosphatidylinositol anchored proteins (GIP-Pr) and apoptosis of paroxysmal nocturnal hemoglobinuria (PNH) cells, we isolated peripheral granulocytes from 10 patients with PNH and 10 normal controls and measured apoptosis induced by serum starvation. The FCM analysis of phosphotidylserine (ps) externalization in granulocytes was determined using Annexin-V-FLUOS labeling. After the cells were induced for apoptosis in serum-free medium for 20 hours, the percentage of externalization was 78.6% in normal control cells but 39.5% in PNH cells. The results of FCM analysis of PI stained granulocytes showed that the PI positive rate was 51.5% in control cells and 30.2% in PNH cells. The gel electrophoresis analysis of DNA fragmentation all indicate that PNH granulocytes were relatively resistant to apoptosis as compared with normal controls. This resistance to apoptosis might not be related to the percentage of CD59 deficient granulocytes.