In order to investigate the effect of anti-TGF-beta1 monoclonal antibody on the expansion of cord blood CD34(+) cells, the purified cord blood CD34(+) cells were divided into three groups: blank control group: purified cord blood CD34(+) cells cultured on day 0; control group: cells cultured for 3 days in the culture system, containing SCF, IL-3, IL-6 and FLT3-L; test group: cells cultured for 3 days in the same culture system as control group, but with anti-TGF-beta1 monoclonal antibody. The mononuclear cell counting (MNC), the expression of CD34 and c-kit, and CFU-GEMM, BFU-E and CFU-GM counting were detected in all three groups. The result showed that the expansion of MNCs, CD34(+) cells and CD34(+)c-kit(+) cells in test group [(2.35 +/- 0.25) x 10(5), (1.16 +/- 0.29) x 10(5), (1.09 +/- 0.26) x 10(5)] was significantly higher than that in control group [(1.25 +/- 0.13) x 10(5), (0.55 +/- 0.19) x 10(5), (0.51 +/- 0.2) x 10(5)](p < 0.01). The expansion of more primitive CD34(+)c-kit(-) subpopulation in test group [(12.95 +/- 3.17) x 10(3)] was even significantly higher than in control group (1.71 +/- 0.83) x 10(3) (p < 0.01). Colony forming assay showed that the number of earlier progenitor colony CFU-GEMM, BFU-E in test group [(16.3 +/- 4.72) x 10(3), (60.5 +/- 20.96) x 10(3)] was higher than that in control group [(5.0 +/- 2.58) x 10(3), (16.25 +/- 7.93) x 10(3)] (p < 0.01). The number of relatively mature CFU-GM between test group and control group was not statistical significance [(6.33 +/- 2.85) x 10(3) vs (4.0 +/- 2.28) x 10(3)](p > 0.05), but both were higher than that in blank group (0.75 +/- 0.29) x 10(3). These results demonstrated that anti-TGF-beta1 monoclonal antibody promoted the expansion of MNC and CD34(+) cells, and even more marked expansion of the more primitive progenitor cells-CD34(+)c-kit(-) cells. Meanwhile, it enhanced the output of more immature colony CFU-GEMM and BFU-E, but had no evident influence on the mature myeloid colony CFU-GM. It is concluded that the anti-TGF-beta1 monoclonal antibody can synergize other cytokines to enhance the proliferation of cord blood CD34(+) progenitor cells effectively, and it is more important that can reserve more primitive progenitor cells.
Antibodies, Monoclonal ; immunology ; pharmacology ; Antigens, CD34 ; immunology ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; drug effects ; immunology ; Humans ; Transforming Growth Factor beta1 ; immunology

The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.
Antigens, CD34 ; Bone Marrow Cells ; cytology ; immunology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Division ; Cell Separation ; Cells, Cultured ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; immunology ; Humans ; Megakaryocyte Progenitor Cells ; cytology ; immunology

This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34(+) cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells (MNCs) were obtained from umbilical cord blood by Ficoll-Hyapaque density gradient separation. CD34(+) cells were enriched by magnetic cell sorting (MACS). The selected CD34(+) cells were seeded in serum-free medium stimulated with thrombopoietin (TPO, 50 ng/ml), interleukin 11 (IL-11, 50 ng/ml), and heparin (25 U/ml) for 14 days. The immunophenotyping (CD34(+), CD41a(+), CD61(+), CD34(+) CD41a(+) and CD34(+) CD61(+) cells) of amplificated products, matured megakaryocyte apoptosis, and expression of human leukocyte antigen (HLA) class I and class II molecules were measured by fluorescence-activated cell sorter (FACS). The number of colony-forming units-megakaryocyte (CFU-Mk) was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34(+) mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a(+) and CD61(+) cells were all at 14th days, while that of CD34(+) CD41(+) and CD34(+) CD61(+) cells were at 7th day [(3.41 +/- 2.80)% and (1.89 +/- 1.43)%, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day (20.66 +/- 32.79) and 10th day (435.62 +/- 482.65), respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were (19.48 +/- 9.64)%, (26.87 +/- 9.03)%, and (52.46 +/- 11.74)%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture (p > 0.05), while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days (p < 0.05, respectively). The expression of HLA class I and class II molecules on megakaryocytes decreased along with the prolongation of expansion time and sharply decreased in 0 to 10 days. It is concluded that the cytokines of TPO, IL-11, and heparin can promote the expansion of megakaryocyte progenitors from umbilical cord blood CD34(+) mononuclear cells effectively in vitro. The peaked expansion times of large CFU-Mk, the peaked expression ratios of CD34(+) CD41(+) and CD34(+) CD61(+) cells were all at 7th day. So the culture for 7 days appeared to be the optimal duration of expanding megakaryocyte progenitors.
Antigens, CD34 ; immunology ; Cell Differentiation ; Cell Division ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; immunology ; Humans ; Megakaryocyte Progenitor Cells ; cytology

To detect the cellular immunity state of New Zealand white rabbit immunized by pig type II collagen. The New Zealand white rabbit was immunized by type II collagen for sixty days. The plasma was collected at a regular interval and the anti-type II collagen antibodies were examined. At the sixtieth day, the peripheral circular lymphocytes and the lymphocytes separated from spleen cells of rabbit and lymph nodes were collected and were stimulated by type II collagen in vivo again. The regulation of reactive cellular proliferation caused by the stimulation was detected. The experiment samples were divided into two groups. The first group was the positive control group by adding different concentrations of PHA and the non-specific immunity was assayed. The different concentrations of type II collagen were added to the second group and the specific immunity was assayed. The lymphocytes of normal rabbits showed proliferation by PHA stimulation but no proliferation by the first stimulation of type II collagen. Obvious proliferation due to the stimulation of both PHA and type II collagen in the immunized rabbit were observed. It shows that certain concentration of heterogeneous collagen may cause an increase of anti-type II collagen antibody in immunized rabbit and may cause a proliferation of lymphocytes in rabbit spleen and peripheral blood. The heterogeneous type II collagen causes cellular immunity in vivo.
Animals ; Cell Division ; drug effects ; Collagen Type II ; administration & dosage ; immunology ; Cytotoxicity, Immunologic ; Female ; Histocompatibility Antigens ; Lymphocytes ; cytology ; immunology ; Male ; Rabbits ; Spleen ; cytology ; Swine ; Transplantation, Heterologous

OBJECTIVETo expand cord blood megakaryocyte progenitor cells in vitro.

METHODSCord blood CD34+ cells were selected by magnetic cell sorting (MACS), and thrombopoietin (TPO), interleukin-11 (IL-11), and heparin were used in the expansion system of megakaryocyte progenitor. The expansion efficiency was measured by fluorescence-activated cell sorting (FACS) using the megakaryocytic specific monoclonal antibodies (CD34+, CD41a+, CD61+, CD34+CD41a+, CD41a+CD61+) and colony-forming units-megakaryocyte (CFU-MK) analysis. The expanded megakaryocyte progenitor were determined by histochemistry staining using CD41a and the observation of the ultrastructure of megakaryocyte (MK) by electron microscopy. The megakaryocyte function were examined by the platelet activation in vitro and nonobese diabetic/severe combined immunodifficiency (NOD/SCID) mice transplantation in vivo.

RESULTSCD34+CD41a+ cells was expanded (4.0 +/- 1.7) folds on day 7 in TPO (50 ng/ml) group and (10.5 +/- 4.8) fold in TPO combined with IL-11 group; after heparin was joined in on day 0, a more significantly elevated expansion was found in the heparin, TPO, and IL-11 group [(29.9 +/- 6.4) folds than the above two groups; P < 0.05]. Meanwhile, the large CFU-MK colony (> 50 cells/colony) was (106.8 +/- 26.9) folds on day 7 (P < 0.05). The megakaryocyte expanding with TPO, IL-11 and heparin for 7 days in vitro transplanted the NOD/SCID mice fasten the recovery of platelet and white blood cell account and improved the survival. Megakaryocyte under culture displayed certain development of territories membrane. Platelet activation test comfirmed that the expanding megakaryocyte progenitor had the normal function.

CONCLUSIONTPO, IL-11, and heparin combination system for ex vivo expansion is an effective expansion system of megakaryocyte progenitor.

Animals ; Antigens, CD34 ; Cell Differentiation ; drug effects ; immunology ; Cell Division ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; drug effects ; immunology ; Heparin ; pharmacology ; Humans ; Interleukin-11 ; pharmacology ; Male ; Megakaryocytes ; cytology ; immunology ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Thrombopoietin ; pharmacology

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1beta, fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.
Antigens, CD34/analysis ; Cell Differentiation/drug effects ; Cell Division/drug effects ; *Cell Separation ; Cells, Cultured ; Cytokines/pharmacology ; *Endothelial Cells/immunology ; Fetal Blood/*cytology ; Fetus ; Flow Cytometry ; Humans ; Research Support, Non-U.S. Gov't ; *Stem Cells/*immunology ; Thrombopoietin/pharmacology

Animals ; Breast Neoplasms ; pathology ; Cell Division ; physiology ; Cells, Cultured ; Humans ; Macrophages ; cytology ; Signal Transduction ; immunology ; physiology ; Space Flight ; Tumor Cells, Cultured ; Weightlessness

BACKGROUNDAirway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation.

METHODSHASMCs in culture were passively sensitized with 10% serum from asthmatic patients, with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-alpha in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction.

RESULTSThe percentage of S phase, absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30 +/- 2.68)%, 0.430 +/- 0.060 and (63.4 +/- 7.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [(10.01 +/- 1.38)%, 0.328 +/- 0.034 and (37.2 +/- 4.8)%, respectively] (P < 0.05). After HASMCs were passively sensitized with asthmatic serum, they were treated with PMA, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (20.33 +/- 3.39)%, 0.542 +/- 0.065 and (76.0 +/- 8.7)% respectively, which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA(P < 0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (11.21 +/- 1.56)%, 0.331 +/- 0.047 and (38.8 +/- 6.0)% respectively, which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 (P < 0.05). The relative ratio of value A of PKC-alpha mRNA and the positive percentage of PKC-alpha protein expression in passively sensitized HASMCs were 1.23 +/- 0.10 and (61.1 +/- 9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05 +/- 0.09 and (34.9 +/- 6.7)%, respectively] (P < 0.05).

CONCLUSIONSThe proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.

Asthma ; immunology ; pathology ; Cell Division ; physiology ; Cells, Cultured ; Humans ; Immunization, Passive ; Myocytes, Smooth Muscle ; pathology ; physiology ; Protein Kinase C ; physiology ; Protein Kinase C-alpha ; Signal Transduction ; physiology

OBJECTIVETo investigate the regulatory effect of IFN-gamma on recognition of target cells by human natural killer (NK) cells.

METHODSThe cytotoxic activity of human NK cell lines (NK92, NKL) was detected by MTT method. Expression of NK cell receptors (NKG2D, NKG2A/B, KIR2DL1 and KIR2DS1) and MICA on target cells (the ligand of NKG2D) was measured by RT-PCR.

RESULTSBoth NK92 and NKL cells exerted higher cytotoxicity to tumor cells with MICA expression, while tumors without MICA expression could resist NK cell lysis. IFN-gamma (> 1000 U/ml) inhibited NK lysis of tumor cells with MICA expression through down-regulating the expression of NKG2D, but up-regulating the expression of NKG2A/B and KIR2DL1.

CONCLUSIONIFN-gamma has a negative effect on activation and cytotoxicity of human NK cells by altering the balance between the expression of activating and inhibitory receptors on NK cells in favor of inhibition. This may serve to limit NK cell over-activation in vivo.

Cell Division ; drug effects ; Cytotoxicity, Immunologic ; drug effects ; Histocompatibility Antigens Class I ; analysis ; physiology ; Humans ; Interferon-gamma ; pharmacology ; Killer Cells, Natural ; immunology ; NK Cell Lectin-Like Receptor Subfamily C ; NK Cell Lectin-Like Receptor Subfamily K ; Receptors, Immunologic ; metabolism ; Receptors, KIR2DL1 ; Receptors, Natural Killer Cell ; Recombinant Proteins ; Tumor Cells, Cultured

OBJECTIVESTo explore the proliferative capacity of bone marrow hematopoietic stem cells and the function of T helper (Th) lymphocytes of patients with immuno-related pancytopenia (IRP).

METHODSTwenty-five untreated IRP patients, 15 IRP patients in complete remission (CR) and 10 normal controls were studied for in vitro yields of CFU-GM, CFU-E and BFU-E from bone marrow mononuclear cells (BMMNC). The mRNA expressions of IL-4, IL-10, IFN-gamma and IL-2 genes in unstimulated BMMNC from 25 untreated IRP patients,15 IRP patients in CR, 19 patients with other hematological diseases presenting pancytopenia and 10 normal controls were detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThere was no significant difference of the yields of CFU-E, CFU-GM or BFU-E among the untreated, and in CR IRP patients and normal controls (P > 0.05). The mRNA expressions of IL-4 and IL-10 of Th2 cells were significantly higher in untreated IRP patients than in the other groups. The mRNA expressions of IFN-gamma and IL-2 of the Th1 cells in all IRP patients were not higher than those in the other groups.

CONCLUSIONSThe cytopenia of IRP patients was not caused by the qualitative abnormality of the hematopoietic stem cells but by the destruction or suppression of hematopoietic stem cells from certain extrinsic insults. The imbalance of Th lymphocytes subtypes and overfunction of Th2 lymphocytes played important roles in the pathogenetic mechanism of IRP leading to increased and overfunctional B lymphocytes, which produced autoantibodies destructing or suppressing hematopoiesis in IRP.

Adolescent ; Adult ; Bone Marrow Cells ; cytology ; Cell Division ; Cells, Cultured ; Child ; Cytokines ; genetics ; Female ; Hematopoietic Stem Cells ; cytology ; Humans ; Male ; Middle Aged ; Pancytopenia ; immunology ; RNA, Messenger ; analysis ; T-Lymphocytes, Helper-Inducer ; physiology