Bone Marrow Transplantation ; history ; methods ; HLA Antigens ; immunology ; Hematopoietic Stem Cell Transplantation ; history ; methods ; History, 20th Century ; History, 21st Century ; Hospitals, General ; Humans ; Peripheral Blood Stem Cell Transplantation ; history ; methods ; Singapore ; Transplantation Conditioning ; history ; methods

The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1 × 10(6) units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3(+)/CD8(+) cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73% ± 12.51%) was dramatically higher than that before treatment (29.20% ± 8.34%, P < 0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.
Adolescent ; Antineoplastic Agents ; therapeutic use ; Bone Marrow ; drug effects ; immunology ; pathology ; Child ; Child, Preschool ; Cytokine-Induced Killer Cells ; cytology ; immunology ; transplantation ; Dendritic Cells ; cytology ; immunology ; transplantation ; Female ; Humans ; Immunotherapy, Adoptive ; methods ; Injections, Subcutaneous ; Interleukin-2 ; therapeutic use ; Leukemia, Myeloid, Acute ; immunology ; pathology ; therapy ; Male ; Neoplasm, Residual ; Primary Cell Culture ; Recurrence ; Remission Induction ; Treatment Outcome

The purpose of the study was to investigate the influencing factors of mortality rate in the bone marrow transplantation in mice. The recipient mice receiving whole-body irradiation of gamma-ray were infused with the same strain of bone marrow cells or the mixture of the bone marrow cells and splenocytes respectively. Experiments were carried out in four batches, with different strains of mice used, respectively. The manifestations and the appearance of graft-versus-host disease (GVHD) were observed, as well as the mortality rate within 35 d of the transplantation in the recipient mice. The mortality rate of the first group of recipient mice was the lowest, the mortality rate of the second group of recipient mice was the highest and the obvious GVHD performance was observed before death. In the third group, the mortality rate declined and there was statistical significance compared to that of the second group. The mortality rate of the fourth group of mice was higher than that of the third group, but still lower than that of the second group of mice and there is a statistical significance. This evidence suggested that mouse genetic purity, splenocytes, the ratio of the bone marrow cells and splenocytes and the week-old of the mouse could be the important influencing factors of the mortality rate in mouse bone marrow transplantation.
Animals ; Bone Marrow Transplantation ; adverse effects ; methods ; mortality ; Cell Transplantation ; Female ; Graft vs Host Disease ; immunology ; mortality ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred ICR ; Mice, Transgenic ; Spleen ; cytology ; Survival Rate ; Whole-Body Irradiation

OBJECTIVETo investigate the effect of immature dendritic cells (inDC) genetically modified to express sTNFR I on acute graft-versus-host disease (aGVHD) and the graft-versus-leukemia (GVL) effect ofter allogeneic bone marrow transplantation (allo-BMT) in leukemic mice and its mechanism.

METHODSAn EL4 leukemia allo-BMT model was established with the BALB/c (H-2d) donor mice (DM)and C57BL/6 (H-2b) recipient mice (RM). The RM received DM bone marrow (BM) cells at a 1:1 ratio with spleen cells intravenously via tail vein at 4 h after TBI. Fifty DM were separated randomly into five groups: (1) Group A: total body irradiation (TBI) group, (2) Group B: lymphoma cell leukemia group, (3) Group C: allo-BMT group, (4) Group D: pXZ9-DC group, (5) Group E: sTNFR I-DC group. Acute GVHD scores, incidence of leukemic cell infiltration, histopathological analysis, survival rate, and survival rate of the recipients were estimated after allo-BMT. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (INF-gamma and IL-4 ) production. Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.

RESULTS(1) The mice in group A and group B all died of the BM failure and lymphoma cell leukemia, respectively. The mice in group C developed typical clinical signs of a GVHD after BMT with an average survival time(AST) of (11.50 +/- 3.50) d. The signs of aGVHD were less evident in the group D and E, and their AST (21.70 +/- 5.80 and 25.80 +/- 5.20 days, respectively) were all longer than that in group C (P < 0.05). AST of group E was the longest (P < 0.05). The mice in group B all died of leukemia within 18 days after engraftment of EL4 cells. There was was no significant difference in groups C, D and E in the incidence of leukemia (P > 0.05). (2) Serum IFN-gamma level reached peak value. At + 12 d, then decreased gradually in group C, D, and E, and then reached the nadir at +18 d post-BMT, with the lowest in group E (P < 0.05), and the level was significantly lower in group D than in group C (P < 0.05). After BMT, serum IL-4 level slightly decreased in group C, but gradually elevated in group D and E and reached their peak at +12 d, and even more significantly increased in group E (P < 0.05). There was no statistical significance in the pair wise comparison among three group (P < 0.05). (3) The average proportion of H-2d positive cells in RM was 95%-100% on day 30 post-BMT, with complete donor-type implantation.

CONCLUSIONImmature DC can induce immuno tolerance. Immature DC genetically modified to express sTNFR I has been shown to prevent acute GVHD in lethally irradiated mice reconstituted with allogeneic bone marrow grafts while maintaining the GVL response.

Animals ; Bone Marrow Transplantation ; adverse effects ; methods ; Dendritic Cells ; immunology ; Female ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; Immune Tolerance ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Transplantation, Homologous

Immune reconstitution is crucially relevant for patients receiving hematopoietic stem cell transplantation (HSCT). This study was purposed to investigate the ability of α-GalCer (α-galactosylceramide), a well-known activator of natural killer T cells (NK-T), to enhance immune and hematological reconstitution. Lethally irradiated BALB/c mice were transplanted with allogeneic C57BL/6 bone marrow cells and splenocytes. α-GalCer was administered immediately after HSCT. After transplantation, the weight, activity, hairs, diarrhea and survival time of mice were observed daily; the blood routine test was performed once weekly; the donor chimeras, amount of mononuclear cells in spleen (MNC) and relative levels of CD3(+), CD4(+), CD8(+), B220(+), CD11c(+), CD40(+), CD86(+) and CD80(+) cells were detected by FACS on day 2, 7, 14, 27, 70 after transplantation. The results indicated that the MNC counts and relative levels of CD3(+) and CD4(+) in group treated with α-GalCer on day 2 after transplantation were higher than those in control group; at the same time, the detected donor chimeras were complete recipient type chimeras, then gradually transformed into donor type, on day 7 - 14 donor chimeras in α-GalCer group were enhanced significantly as compared with control group, on day 27 the chimeras in two groups were complete donor type chimeras thereafter to day 70, the MNC count and relative levels of CD3(+), CD4(+), CD8(+), B220(+), CD40(+), CD86(+) cells in α-GalCer group were obviously higher than those in control group, at the same time, the hematopoietic reconstitution in α-GalCer group was accelerated as compared with control group. It is concluded that the α-GalCer administration after allogeneic bone marrow transplantations accelerates immune and hematological reconstitution.
Animals ; Bone Marrow Transplantation ; immunology ; methods ; Chimera ; Female ; Galactosylceramides ; pharmacology ; Leukocyte Count ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Natural Killer T-Cells ; drug effects ; immunology ; Postoperative Period

The aim of study was to investigate the modulation effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on adhesion molecule expression of memory T lymphocyte in the bone marrow grafts. rhG-CSF was administered in 41 donors by subcutaneous injection for 5 consecutive days. Bone marrow grafts were collected on day 4. The percentages of CD4+ and CD8+, and the expressions of CD49d, CD54, CD62L and CD11a on donor T cells of steady state-bone marrow grafts (SS-BM, n=11) and rhG-CSF primed bone marrow (G-BM, n=30) were analyzed by using multi-color flow cytometry. The results indicated that the percentages of CD4+ and CD8+ T cells were significantly lower in G-BM than those in SS-BM (p<0.05). There were no significant differences in the percentages of memory CD4+ and CD8+ T cells between SS-BM and G-BM (p>0.05). The expressions of CD49d on CD4+ and CD8+T cells were significantly lower in G-BM than that in SS-BM (p<0.05). Compared with SS-BM, the expressions of CD54 on CD4+, memory CD4+ T cells and CD8+ T cells were significantly lower in G-BM (p<0.05). The expressions of CD62L on CD4+ and CD8+ T cells and memory T cells were all significantly lower in G-BM (p values were all less than 0.001). The expressions of CD11a on CD4+, memory CD4+ T cells were significantly lower in G-BM than that in SS-BM (p<0.05). There were no significant differences in the expression of CD11a on CD8+, memory CD8+ T cells between SS-BM and G-BM (p>0.05). It is concluded that the expression of cell adhesion molecules on the CD4+ and CD8+ T cells in G-BM is down-regulated after rhG-CSF treatment of healthy donors.
Adolescent ; Adult ; Aged ; Bone Marrow ; Bone Marrow Cells ; immunology ; metabolism ; Bone Marrow Transplantation ; immunology ; methods ; Cell Adhesion Molecules ; metabolism ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Living Donors ; Male ; Middle Aged ; Recombinant Proteins ; T-Lymphocytes ; immunology ; metabolism ; Young Adult

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 evaluate the role of human leukocyte antigen G (HLA-G) in the better effect of allogenetic bone marrow transplantation than that of peripheral blood stem cell transplantation.

METHODSFlow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) on donor peripheral blood (PBC) or bone marrow (BM) mononuclear cells. The levels of soluble HLA-G (sHLA-G) in the plasma and bone marrow fluid were determined using enzyme-linked immunosorbent assay (ELISA) before and after granulocyte colony-stimulating factor (G-CSF) mobilization.

RESULTSThe mean levels of mHLA-G after G-CSF mobilization in the PBC and BM were significantly higher than that before G-CSF mobilization (P=0.001 and 0.000), but the plasma levels of sHLA-G showed no significant changes after the mobilization (P=0.279). The mean levels of sHLA-G in the BM fluid significantly increased (P=0.002) to a level higher than that in the PBC after G-CSF mobilization (P=0.004).

CONCLUSIONHLA-G plays an important role in immune tolerance after hematopoietic stem cell transplantation with G-CSF mobilization.

Adult ; Bone Marrow Transplantation ; immunology ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; HLA Antigens ; immunology ; metabolism ; HLA-G Antigens ; Hematopoietic Stem Cell Mobilization ; methods ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Antigens Class I ; immunology ; metabolism ; Humans ; Immune Tolerance ; Male ; Middle Aged

OBJECTIVETo study the effect of delayed sequential bone marrow transplantation on acute graft-versus-host disease (aGVHD) in major H-2 incompatible mouse transplantation.

METHODSC57BL/6 (H-2b) mice were used as donors and BALB/c (H-2d) mice as recipients. BALB/c mice were given 8.0 Gys total body irradiation (TBI) on day 0 and infused with a blend of bone marrow cells and spleen cells in different time. Transplantation was carried out as follows: group I TBI on day 0 and transplantation at 4 h after TBI; groups of II TBI on day 0 and transplantation at 4 h, d1, d2, d3 after TBI; groups III TBI on day 0 and transplantation at day 4 after TBI; groups IV TBI on day 0 and transplantation at day 4 through day 7 after TBI. Recipient's spleen H-2b cells were detected by flow cytometry and the level of serum cytokines (IL-2, IL4, IL-6, IL-10 and IFN-gamma) by ELISA. The survival, aGVHD and hematopoietic recovery were observed.

RESULTSaGVHD occurred in group I and the mice all died within 3 weeks after transplantation. The 60 day survival rates of groups of II and III were 30% and 50% respectively. The degree of aGVHD in group III was modest and the survival rate was higher than that in other groups (P <0.05). The peak time of IL-2, IFN-gamma, IL-4 and IL-10 in groups III and IV were later than that in group I. The levels of IL-4 and IL-10 in groups III and IV were higher than that in group I and for the levels of IL-2 and IFN-gamma were on the contrary (P < 0.05). The level of IL-6 in all groups peaked on day 5 to day 10 after TBI and was higher in group I than in others (P <0.05). In group IV the mean value of donor H-2b cells was (98.1 +/- 1.1)% on day 60 and WBC counts recovered normal on day 20.

CONCLUSIONSDelayed sequential transplantation can reduce the morbidity of aGVHD ,improve the survival rate and not affect the engraftment and reconstitution of hematopoiesis in mouse allo-BMT. The mechanism of aGVHD prevention may be related to the reducing of type 1 cytokines of T lymphocyte and the increasing of type 2 cytokines.

Animals ; Bone Marrow Transplantation ; immunology ; methods ; Disease Models, Animal ; Female ; Graft vs Host Disease ; immunology ; prevention & control ; Interleukin-2 ; blood ; Interleukin-4 ; blood ; Interleukin-6 ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

OBJECTIVETo study the effects of small interfering RNA (siRNA)-mediated silencing of CD158b expression on the efficiency of the natural killer (NK) cells in killing allogeneic dendritic cells.

METHODSAfter the knockdown of CD158b by CD158b -SiRNA, the CD158b mRNA expression in natural killer cells was examined by qRT-PCR and the CD158b protein expression by flow cytometer. The cytotoxic activity of RNAi-NK cells and normal NK cells against the allogeneic dendritic cells was detected by LDH release assay.

RESULTSThe CD158b mRNA expression and its protein expression were decreased significantly in the NK cells by CD158b siRNA (P/0.05). The cytotoxic activities of alloreactive NK cells generated by RNAi CD158b expression against allogeneic dendritic cells were increased significantly.

CONCLUSIONSilencing CD158b gene can inhibit the NK cell CD158B mRNA and protein expression. Alloreactive NK cells generated by RNAi CD158b expression have the potential for use in interventions of GVHD.

Bone Marrow Transplantation ; immunology ; methods ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; Flow Cytometry ; Gene Silencing ; Graft vs Host Disease ; immunology ; Humans ; Killer Cells, Natural ; cytology ; immunology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Receptors, KIR ; metabolism ; Receptors, KIR2DL3 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction