BACKGROUND: During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. METHODS: CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. RESULTS: CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. CONCLUSION: These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.
Antigens, CD95* ; Apoptosis* ; Cytokines ; Erythrocytes ; Fetal Blood ; Granulocyte-Macrophage Progenitor Cells ; Granulocytes ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells* ; Humans ; Macrophages ; Monocytes ; Myeloid Progenitor Cells ; Stem Cells

OBJECTIVE: To investigate the effects of high dose vitamin C on proliferation and apoptosis of acute myeloid leukemia (AML) cell lines including HL-60, U937 and primary CD34 leukemia cells in AML. METHODS: CD34 cells were sorted by using immunomagnetic cell sorting system, then the primary CD34 leukemia cells, including HL-60 and U937 cell lines were cultured in vitro. Cells in each group were treated with different concentrations of vitamin C, the survival rate of cells was determined by MTT assay, the apoptosis rate of cells was evaluated by Annexin V/PI double staining, the expression of apoptotic proteins-including cleaved caspase 3, cleaved caspase-9 and cleaved PARP were detected by Western blot. RESULTS: The proliferation of HL-60 and U937 cells could be inhibited by high dose vitamin C, which showed a concentration-dependent manner (r=-0.9664; r=-0.9796). HL-60 and U937 cells were treated with different concentrations of vitamin C (8 and 20 mmol/L) for 24 hours, respectively, it was found that with the increasing of vitamin C concentration, cell apoptosis rate was significantly increased (r=0.9905; r=0.9971), and the expression of apoptosis related proteins including cleaved caspase 3, cleaved caspase-9 and cleaved PARP was aslo significantly increased with the increasing of concentration. In addition, it was found that with or without the mutation of TET2, high dose vitamin C could inhibit the proliferation (r=-0.9719; r=-0.9699) and promote the apoptosis (r=0.9998; r=0.9901) of primary CD34 leukemia cells in AML, which showed a dose-dependent manner, but it showed no effect on the proliferation (r=-0.2032) and apoptosis (r=0.1912) of normal CD34 cells. CONCLUSION High dose vitamin C can inhibit the proliferation and promote the apoptosis of acute myeloid leukemia cells, and selectively kill primary CD34 leukemia cells in AML.
Apoptosis ; Ascorbic Acid ; Cell Proliferation ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; U937 Cells

INTRODUCTION: The ABX Pentra DX 120 (Pentra DX 120, ABX Diagnostics, Montpellier, France) adopted new technologies to perform differential leukocyte and erythroblast counts. The double matrix can discriminate Large Immature Cell (LIC), Immature Granulocyte (IMG), Immature Monocyte (IMM), Immature Lymphocyte (IML), and Atypical lymphocyte (ALY) in addition to a routine 5-differential count. For erythroblast (ERB), a fluorescence method is employed. In this study, we evaluated the performance of the Pentra DX 120 in the performance of differential leukocyte and erythroblast counts. METHODS: Precision was evaluated using 3-level control materials. Comparison analysis was performed on 200 samples: 100 normal and 100 abnormal samples. We evaluated the 5 part differential count, LIC, IMG, IMM, IML, ALY, and ERB. These parameters were analyzed in comparison with the results from the reference method, manual differential count. RESULTS: The coefficients of variation (CVs) of precision were <5% for neutrophils and lymphocytes, <15% for eosinophils and basophils and <7% for erythroblasts. The correlation coefficients (r) were >0.9 except monocytes and basophils. IMG, ALY and erythroblasts were also well correlated with manual count (r=0.8315, 0.5602, 0.8144, respectively). The efficiency of flagging system was 84% for LIC, 80% for ALY, and 78.0% for increased ERB (>2/100WBCs). CONCLUSIONS: The Pentra DX 120 performed reliable differential leukocyte and IMG, ALY, and ERB results demonstrated comparable performance to manual count. And, the flagging system was efficient for detecting each abnormal cell population. We expect the Pentra DX 120 double matrix and erythroblast count can reduce microscopic review rate in routine laboratory and promote laboratory efficiency.
Basophils ; Eosinophils ; Erythroblasts ; Fluorescence ; Granulocytes ; Leukocytes ; Lymphocytes ; Monocytes ; Neutrophils

This study was aimed to investigate the inhibitory effect of flavonoids of puerarin (PR) in different concentrations on proliferation of 4 kinds of acute myeloid leukemia (AML) cell lines (Kasumi-1, HL-60, NB4 and U937), and to explore its possible mechanism. The MTT method was used to detected the inhibitory effect of PR on proliferation of AML cell lines. The flow cytometry was adopted to determine the change of cell cycle in vitro. The results showed that a certain concentration of PR could inhibit the proliferation of these 4 cell lines effectively in time-and dose-dependent manners, and the intensity of inhibition on 4 kinds of AML cell lines was from high to low as follows: NB4>Kasumi-1>U937>HL-60. Meanwhile, PR could also change cycle process, cell proportion in G1/G0 phase decreased, cells in S phase increased and Sub-diploid peak also appeared. It is concluded that PR can selectively inhibit the proliferation of 4 AML cell lines and block cell cycle process, especially for NB4 cells.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HL-60 Cells ; Humans ; Isoflavones ; pharmacology ; Leukemia, Myeloid, Acute ; classification ; pathology ; U937 Cells

Recently, we have experienced two cases of xanthogranuloma in adults presenting either a solitary papule or multiple papules. Laboratory findings including blood lipid substances such as cholesterol and triglyceride were within normal limits. In both cases, we could not find any other extracutaneous manifestations. Granulomatous infiltrates containing foam cells, foreign body giant cells and Touton giant cells as well as histiocytes and lymphocytes were revealed by histopathologic findings.
Adult* ; Cholesterol ; Foam Cells ; Giant Cells ; Giant Cells, Foreign-Body ; Histiocytes ; Humans ; Lymphocytes ; Triglycerides

Osteoclasts, derived from multipotent myeloid progenitor cells, play homeostatic roles in skeletal modeling and remodeling, but may also destroy bone in pathological conditions such as osteoporosis and rheumatoid arthritis. Osteoclast development depends critically on a differentiation factor, the receptor activator of NF-kappaB ligand (RANKL). In this study, we found that the hexane soluble fraction of the common fig Ficus carica (HF6-FC) is a potent inhibitor of osteoclastogenesis in RANKL-stimulated RAW264.7 cells and in bone marrow-derived macrophages (BMMs). HF6-FC exerts its inhibitory effects by suppression of p38 and NF-kappaB but activation of ERK. In addition, HF6-FC significantly decreased the expression of NFATc1 and c-Fos, the master regulator of osteoclast differentiation. The data indicate that components of HF6-FC may have therapeutic effects on bone-destructive processes such as osteoporosis, rheumatoid arthritis, and periodontal bone resorption.
Arthritis, Rheumatoid ; Bone Resorption ; Carica ; Ficus ; Macrophages ; Myeloid Progenitor Cells ; NF-kappa B ; Osteoclasts ; Osteoporosis ; Receptor Activator of Nuclear Factor-kappa B

The effects of the Smad3- knockout on the hematopoiesis of mouse were investigated in this work. Five pairs of wild type and Smad3- null mice were studied. White blood cell(WBC), red blood cell(RBC) and platelet (PLT) counting of peripheral blood cells were performed with blood obtained from tails. And white blood cells were classified by their morphology. Bone marrow nucleated cells (BMNCs) were counted and classified. The CFU-GM, BFU-E, CFU-GEMM yields were measured in each pair of mice. CFU-S yield of each mouse was measured by injecting bone marrow cells into lethally irradiated 8-10 weeks old wild type female mice. And the pathomorphism of their bone marrows, spleens and livers were observed. As a result, WBC and PLT of Smad3- null mice were significantly higher than those in wild type mice. Smad3- null mice had much more proportion of granulocytes in classification. There wasn't any difference in RBC counting and BFU-E measurement. The yield of CFU-GM increased, while the yields of CFU-GEMM and CFU-S markedly reduced. Bone marrows are actively proliferative, with granulocytosis. The granulocyte/erythrocyte ratio increased. There were no obviously alterative in spleen and liver. Thus Smad3- knockout results in a decreased number of stem and progenitor cells. Moreover hematopoietic differentiation is abnormal with a tendency to forming more granulocytes and platelets. The effect of Smad3 on hematopoiesis is correlative to that of TGF-beta.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Erythrocytes ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; metabolism ; Granulocytes ; cytology ; metabolism ; Hematopoiesis ; genetics ; Mice ; Mice, Knockout ; Myeloid Progenitor Cells ; cytology ; metabolism ; Smad3 Protein ; genetics

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.
Apoptosis ; physiology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; T-Lymphocytes ; cytology ; Tumor Cells, Cultured ; U937 Cells

This study was aimed to explore the expression of survivin in leukemia cells and to investigate the effect of GM-CSF on survivin expression. The survivin expressions in 37 previously untreated leukemia patients and 10 normal persons as well as in three kinds of leukemia cell lines (K562, HL-60, U937) were analyzed by RT-PCR. The HL-60 cells were treated by the proper concentration of GM-CSF, and the changes of survivin mRNA and protein expression levels were detected by using RT-PCR and Western blot respectively. The results indicated that the positive rate of survivin gene in 37 leukemia patients was 67.6% (25/37) and significantly higher than that in normal control (20.0%). The expression level was higher in ALL cells than that in AML cells (73.3% vs 63.6%). Moreover, three kinds of leukemia cell lines all expressed survivin. After treating with the proper concentration of GM-CSF for 2 days, the expression of survivin obviously increased and the expression level of survivin mRNA was up-regulated by 26%, the expression level of survivin protein was up-regulated by 49%. It is concluded that the survivin gene extensively express in leukemia and its cell lines, and its expression can be obviously increased by GM-CSF.
Adolescent ; Adult ; Aged ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; K562 Cells ; Leukemia, Myeloid, Acute ; metabolism ; Male ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; U937 Cells

The triggering receptor expressed on myeloid cells (TREM) family, which is abundantly expressed in myeloid lineage cells, plays a pivotal role in innate and adaptive immune response. In this study, we aimed to identify a novel receptor expressed on hematopoietic stem cells (HSCs) by using in silico bioinformatics and to characterize the identified receptor. We thus found the TREM-like transcript (TLT)-6, a new member of TREM family. TLT-6 has a single immunoglobulin domain in the extracellular region and a long cytoplasmic region containing 2 immunoreceptor tyrosine-based inhibitory motif-like domains. TLT-6 transcript was expressed in HSCs, monocytes and macrophages. TLT-6 protein was up-regulated on the surface of bone marrow-derived and peritoneal macrophages by lipopolysaccharide stimulation. TLT-6 exerted anti-proliferative effects in macrophages. Our results demonstrate that TLT-6 may regulate the activation and proliferation of macrophages.
Adaptive Immunity ; Computational Biology ; Computer Simulation ; Cytoplasm ; Hematopoietic Stem Cells ; Humans ; Immunoglobulins ; Macrophages* ; Macrophages, Peritoneal ; Monocytes ; Myeloid Cells*