The aim was to study the roles that the bone marrow mesenchymal stem cells (MSC) and cytokines play in cord blood CD34(+) cell expansion ex vivo and the influence of culture ex vivo on expression of the adhesive molecule of CD44. CD34(+) cells sorted from cord blood cells had been cultured in each well of 24 well culture plates containing culture medium supplemented with mesenchymal stem cells layer or/and cytokines for a week, and then all kinds of indexes of different groups were compared. The results showed that as for cord blood cell expansion, there was no significant difference between the groups with cytokines SDF-1alpha + SCF + TPO + FL and SCF + TPO + FL no matter if MSC layer existed or not. The groups with MSC layer and cytokines were superior to the corresponding groups without MSC layer. In addition, the expression of the adhesion molecule CD44 had no distinct change after culture. It is concluded that SDF-1alpha has no distinct influence on the effect of cytokines SCF + TPO + FL on cord blood cell expansion ex vivo. MSC enhance the effect of cytokines on cord blood cell expansion ex vivo. Such expansion ex vivo may not influence the expression of the adhesive molecule CD44 on cord blood cells.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; pharmacology ; Female ; Fetal Blood ; cytology ; drug effects ; immunology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; drug effects ; immunology ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Pregnancy

OBJECTIVETo explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT).

METHODSMature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia.

RESULTSMature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05).

CONCLUSIONImmunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; Bone Marrow Transplantation ; Cancer Vaccines ; immunology ; Cell Differentiation ; Dendritic Cells ; immunology ; Female ; Graft vs Leukemia Effect ; Immunotherapy ; Leukemia, Experimental ; immunology ; surgery ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Survival Rate ; Transplantation, Homologous

The aim of this study was to explore the effect of muramyl dipeptide (MDP) on proliferation of dendritic cells (DCs) from bone marrow of children with acute leukemia in vitro. The mononuclear cells were isolated from bone marrow of children with acute leukemia to induce dendritic cells. The experiment was divided into 4 groups. The control group: MNC + RPMI 1640 medium; test group 1: MNC + MDP; test group 2: MNC + rhGM-CSF + IL-4 + rhTNFα; test group 3: MNC + rhGM-CSF + IL-4 + rhTNFα + MDP. The growth of DCs was observed by inverted microscope every day; the number of DCs in different groups were counted, the immunophenotypes of DCs were detected by flow cytometry on day 8 of culture. The results indicated that a certain number of typical DCs could be detected in all experimental groups. The DC number in control and 3 test groups were (0.85 ± 0.23) x 10⁵/L, (2.31 ± 0.24) x 10⁵/L, (3.26 ± 0.37) x 10⁵/L and (4.16 ± 0.34) x 10⁵/L, respectively, among which DC number is in all 3 test groups were higher than that in control group (p < 0.01), the DC number in test group 1 was lower than that in test groups 2 and 3 (p < 0.01), while it in test group 3 was higher than that in test group 2 (p < 0.01). The percentages of HLA-DR in control, test group 1, 2 and 3 were 19.98 ± 3.74, 37.24 ± 4.32, 58.81 ± 2.08 and 77.48 ± 5.57 respectively; the percentages of CD1a and CD83 in control, test group 1, 2 and 3 were 11.46 ± 2.43, 28.71 ± 6.64, 46.92 ± 4.78 and 57.03 ± 3.07, as well as 13.05 ± 5.70, 36.32 ± 5.61, 54.95 ± 7.83 and 75.70 ± 6.67 respectively. The comparison of HLA-DR, CD1a and CD83 levels in control and test group 1, 2 showed that their results were consistent with results of DC numbers. It is concluded that MDP not only promotes the proliferation of DCs derived from bone marrow of children with acute leukemia in vitro, cooperates with rhGM-CSF, rhIL-4 and rhTNFalpha in promoting of the proliferation and maturation of DCs, while the promotive effect of MDP alone on the proliferation of DCs is not as good as its combination with cytokines.
Acetylmuramyl-Alanyl-Isoglutamine ; pharmacology ; Bone Marrow Cells ; cytology ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Child ; Dendritic Cells ; cytology ; drug effects ; Flow Cytometry ; Humans ; Leukemia ; immunology ; pathology ; Tumor Cells, Cultured

OBJECTIVETo study the immunomodulatory effects and mechanisms of mesenchymal stem cells (MSC) derived from the bone marrow in acute leukemia patients in vitro.

METHODSBone marrow mononuclear cells from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) were obtained and cultured in low serum medium. The immunophenotypes were assessed by FACS and immunol histochemistry. The levels of cytokines were evaluated by enzyme linked immunosorbant assay (ELISA). T-cell suppression ability was evaluated by Transwell chamber assay. Moreover, the immunoregulatory ability of AML- and ALL-derived MSC was detected by mixed lymphocyte culture assay.

RESULTSALL-derived MSC showed a typical fibroblast-like morphology. They were positive for CD29, CD44 and CD105, the positive rate were 98.81%, 99.25% and 90.52%, respectively, while negative for CD31, CD45 and CD34. Moreover, ALL- and AML-derived MSC didn't express HLA-DR and co-stirnulatory molecules (CD40, CD80 and CD86). ALL and AML derived MSC could secret several cytokines, such as TGF-β1 (567.58 ± 52.64 and 357.15 ± 33.52), HGF (647.27 ± 102.54 and 219.67 ± 62.37), IL-6 (59.67 ± 15.69 and 54.35 ± 12.31) and IL-11 (102.58 ± 23.54 and 78.21 ± 9.67), the level of secretion of TGF-β1 and HGF were higher in ALL bone marrow derived MSC than that of in AML bone marrow derived MSC. ALL and AML derived MSC significantly suppressed T lymphocyte proliferation in a dose-dependent manner, the counts per minute (CPM) were (3.58 ± 0.54) × 10(4), (2.87 ± 0.33) × 10(4), (1.78 ± 0.51) × 10(4) and (1.15 ± 0.15) × 10(4) for AML derived MSC, and CPM were (1.96 ± 0.31) × 10(4), (1.57 ± 0.28) × 10(4), (0.91 ± 0.41) × 10(4) and (0.22 ± 0.11) × 10(4) for ALL derived MSC when MSC were 0.5 × 10(4), 1 × 10(4), 2 × 10(4) and 5 × 10(4). In addition, the CPM was (4.01 ± 0.72) × 10(4) in control group. The immunosuppressive ability was different between MSCs derived from AML and ALL. The immunosuppressive effect of ALL derived MSC could be reversed by anti-TGF-β1 and anti-HGF antibody.

CONCLUSIONALL-derived MSC show immunoregulatory effect in vitro and this effect is achieved through cytokines. But MSCs derived from AML display abnormal changes in T-cell suppression ability.

Bone Marrow ; immunology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cytokines ; pharmacology ; Humans ; Immunophenotyping ; Interleukin-11 ; metabolism ; Interleukin-6 ; metabolism ; Leukemia, Myeloid, Acute ; immunology ; Mesenchymal Stromal Cells ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo study the effect of Lycium bararum polysaccharides (LBPs) stimulation on the maturation of murine bone marrow derived dendritic cells (BMDCs).

METHODSMurine bone marrow cells were cultured in GM-CSF and IL-4 for 5 days, then were purified with a MACS column. Respectively, BMDCs were stimulated with LBPs, LPS and RPMI1640 for 2 days. Cell phenotypes and antigens uptake by BMDCs were analyzed by flow cytometry. Cytokines released by BMDCs were detected. The antigen presenting by BMDCs was evaluated by mixed lymphocyte responses.

RESULTCompared with to the BMDCs that only subjected to RPMI 1640, the expression of I-A/I-E, CD11c and secretion of IL-12 by BMDCs stimulated with LBPs were increased, the phagocytosis of FITC-dextran by BMDCs stimulated with LBPs was impaired but the activation of proliferation of allogenic lymphocytes by BMDCs was strengthened.

CONCLUSIONLBPs promote not only the maturation of cultured murine BMDCs in vitro, but also the immune response initiation induced by BMDCs.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; CD11c Antigen ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Lipopolysaccharides ; pharmacology ; Lymphocyte Culture Test, Mixed ; Lymphocytes ; cytology ; drug effects ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Phagocytosis ; drug effects ; immunology

OBJECTIVETo investigate the synergic effects of rapamycin and donor bone marrow-derived immature dendritic cells (DCs) in inducing skin allograft tolerance in mice.

METHODSThe recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control group (without perioperative treatments), rapamycin group (receiving rapamycin at 1 mg.kg(-1).d(-1) by gavage for 7 consecutive 7 days after skin transplantation), immature DC group (receiving an injection of donor bone marrow-derived immature DCs of 2 x 10(6) via tail vein before skin transplantation), combined group (receiving an injection of the DCs of 2 x 10(6) before transplantation and rapamycin at 1 mg.kg(-1).d(-1) for 7 consecutive days after transplantation). The survival time of the skin allograft was observed in each group.

RESULTSThe survival time of the skin allograft in the control, rapamycin, immature DC and immature DC +rapamycin groups were 6.9-/+1.9, 12.3-/+3.0, 17.0-/+3.4 and 20.8-/+3.6 days, respectively, showing significant differences among the groups (P<0.05), and SNK test also indicated significant differences between every two groups.

CONCLUSIONSRapamycin and donor bone marrow-derived immature DCs have synergic effects in inducing skin allograft tolerance in mice.

Animals ; Bone Marrow Cells ; cytology ; immunology ; Dendritic Cells ; immunology ; Graft Survival ; drug effects ; immunology ; Immunosuppressive Agents ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Sirolimus ; pharmacology ; Skin Transplantation ; immunology ; methods ; Transplantation, Homologous

OBJECTIVETo investigate the influence of maturative agents, including lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) and interferon gamma(IFN-gamma) on the maturation of immature dendritic cells originated from murine bone marrow induced by low dose of granulocyte macrophage colony stimulating factor (rm GM-CSF).

METHODSDendritic cells from murine bone marrow progenitors were cultured in low and high doses of GM-CSF for 6 days, and then the suspending cells were harvested for the experiment. After 3 days of co-culture of the obtained DC with low dose rmGM-CSF (GM(low)DC) with LPS, TNF-alpha and IFN-gamma, the stimulatory capacity of inducing proliferation of non-sensitized splenocytes of GM(low)DC in mixed lymphocyte reaction (MLR) was observed and compared with that of GM(high)DC.

RESULTSGM(low)DC could not activate the non-sensitized splenocytes or induce it into proliferation after 3 days of co-incubation with LPS, TNF-alpha, IFN-gamma, with the stimulation index (SI) lower than 2. Whereas GM(high)DC could strongly activate naive splenocytes (SI = 4.71).

CONCLUSIONGM(low)DC was resistant to maturation and insensitive to the stimulation by LPS, TNF-alpha or IFN-gamma.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Interferon-alpha ; pharmacology ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology ; Tumor Necrosis Factor-alpha ; pharmacology

We investigated the immunostimulatory effects of a novel beta-glucan purified from Paenibacillus (P.) polymyxa JB115 on bone marrow-derived dendritic cells (DCs), a type of potent antigen-presenting cells. beta-glucan isolated from P. polymyxa JB115 enhanced the viability and induced the maturation of DCs. beta-glucan markedly increased the cytokine production of DCs and surface expression of DC markers. In addition, DCs treated with beta-glucan showed a higher capacity to stimulate allogeneic spleen cell proliferation compared to those treated with medium alone. These results demonstrate the effect of beta-glucan on DC maturation and may increase the use of beta-glucan.
Animals ; Bone Marrow Cells/cytology/*drug effects/*immunology ; Cell Survival/drug effects/*immunology ; Dendritic Cells/cytology/*drug effects/*immunology ; Flow Cytometry ; Immunophenotyping/methods ; Interleukin-12/analysis/immunology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/analysis/immunology ; Paenibacillus/*chemistry ; Tumor Necrosis Factor-alpha/analysis/immunology ; beta-Glucans/isolation & purification/*pharmacology

BACKGROUNDSmoking causes frequent asthma attacks, leading to a rapid decline in lung function in patients with asthma, and it can also reduce the therapeutic effect of glucocorticoids in patients with asthma. Therefore, the present study aimed to investigate the effect of cigarette smoke on the expression of myeloid differentiation factor 88 (MyD88) in marrow dendritic cells (DCs) in asthmatic rats, and to explore the molecular mechanism of cigarette smoke exposure on asthma by DCs.

METHODSForty Wistar rats were randomly divided into the following groups: control, smoke exposure, asthma, and asthma combined with smoke exposure. The animal model was established, and then rat bone marrow-derived DCs were collected. Additionally, rat spleen lymphocytes and bone marrow-derived DCs were cultured together for mixed lymphocyte responses. Interferon (IFN)-gamma and interleukin (IL)-4, IL-10, and IL-12 expressions were determined by enzyme-linked immunosorbent assay (ELISA). MyD88 expression was determined by Western blotting. The proliferation of lymphocytes was examined with methyl thiazolyl tetrazolium (MTT) colorimetric assay.

RESULTSMyD88 expression was decreased in the asthma combined with smoke exposure group compared to the asthma group (P < 0.01), and IL-10 and IL-12 expressions were decreased in the asthma combined with smoke exposure group compared to control group (P < 0.01). In addition, DCs stimulating activity on allogeneic lymphocytes were significantly decreased in the smoke exposure combined with asthma group compared to the control and asthma groups (P < 0.01). After allogeneic mixed lymphocyte responses, IL-4 expression was increased and IFN-gamma was decreased in the asthma group and the asthma combined with smoke exposure group compared to control group (P < 0.01). IL-4 expression was increased and IFN-gamma was decreased in the asthma combined with smoke exposure group compared to the asthma group (P < 0.01). The study also showed that MyD88 expression was positively correlated with IL-12 and IFN-gamma expressions and the activity of lymphocytes (P < 0.01), and negatively correlated with IL-4 expression (P < 0.01).

CONCLUSIONSSmoking aggravates asthma by weankening immunological mechanism. MyD88-dependent pathways may play a role in the immunological balance and activation of lymphocytes.

Animals ; Asthma ; immunology ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Dendritic Cells ; drug effects ; metabolism ; Lymphocyte Activation ; drug effects ; Male ; Myeloid Differentiation Factor 88 ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Smoking ; adverse effects

To study the effect of mesenchymal stem cell (MSC) on immune function, MSCs were isolated and cultured from human bone marrow cells. The purity of MSCs were identified with the spindle-fibroblastic morphology characterization by microphotograph and the phenotypes were tested by flow cytometry. MSCs were plated in 96-well plates (2,000/well and 1,000/well), and cocultured for 3 days with T cells isolated from cord blood. Cord blood T cells non-cocultured with MSC acted as control group. After cord blood T cells stimulated by PHA for 60 hours, [(3)H]-thymidine was added to each well and T cell proliferation was assessed by [(3)H] thymidine incorporation. The results showed that cord blood T cell proliferation was suppressed when 2,000 MSCs were plated each well and cord blood T cell proliferation was activated when 1,000 MSCs were plated. Our results suggested that the immunomodulatory function of MSC seemed dependent on cell dose. High concentration of MSC most often resulted in inhibition, while low concentration resulted in stimulation.
Antigens, CD ; analysis ; Bone Marrow Cells ; cytology ; Cell Division ; immunology ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Mesoderm ; cytology ; Phytohemagglutinins ; pharmacology ; Stem Cells ; cytology ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Thymidine ; metabolism ; Tritium