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

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.
Adolescent ; Adult ; Cord Blood Stem Cell Transplantation ; methods ; Cytokines ; metabolism ; Dendritic Cells ; metabolism ; Female ; Graft vs Host Disease ; immunology ; therapy ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Immunomodulation ; Killer Cells, Natural ; metabolism ; Male ; T-Lymphocyte Subsets ; metabolism ; Transplantation, Homologous ; adverse effects ; Young Adult

OBJECTIVETo explore the anti-tumor effect and its mechanism of Sendai virus Tianjin strain defective interfering particles (DIP) on mouse models of colon carcinoma.

METHODSCT26 cells (5×10(6)/0.1 ml) were subcutaneously injected into the back of Bal B/c mice to establish murine colon carcinoma model. After the tumors reached 5 mm in diameter, the mice were randomly divided into Tianjin strain DIP group and saline control group. The former was intratumorally injected with Tianjin strain DIP (0.1 ml) once a day on day 4, 7, 10 and 13 after CT26 cell inoculation. The latter was intratumorally injected with the same volume of saline. Tumor volume and survival rate of the mice were calculated to confirm the anti-tumor effect of DIP. Flow cytometry and ELISA were used to examine the maturation and release of cytokines IL-6, IFN-α and TNF-α from murine myeloid dendritic cells (DCs) induced by Tianjin strain DIP. Moreover, real-time RT-PCR and immunohistochemistry were performed to identify whether the Tianjin strain DIP could induce infiltration of CD11c(+) DCs, CD4(+) and CD8(+) T cells in the tumors.

RESULTSOn day 22 after CT26 cell inoculation, the average tumor volume of the Tianjin strain DIP group was (33.2 ± 2.0) mm(3), significantly smaller than that of the control group [(2 376.0 ± 130.8)mm(3), P < 0.01]. On day 50 after CT26 cell inoculation, the survival rate of mice was 90.0% in the Tianjin strain DIP group, much higher than that of the control group (30.0%, P < 0.01). Flow cytometry analysis showed that the expression of markers of DCs maturation, including CD40, CD80 and CD86, was dose-dependently increased by DIP or intact virus. No statistically significant difference was found betweent the DIP and intact virus groups. ELISA results showed that DIP could stimulate the secretion of IL-6, IFN-α and TNF-α from mouse DCs. The secretion of all of the cytokines was dose-dependently increased by DIP or intact virus. Real-time RT-PCR revealed that the expression of CD4, CD8 and CD11c mRNAs was increased in tumors treated with DIP compared with that of the saline group at all time points. Moreover, the expression level of all of them remained maximal at 120 h after the last treatment. Immunohistochemical staining revealed that the ratios of CD4(+), CD8(+) T cells or CD11c(+) DCs to total cells were (21.60 ± 1.49)%, (22.12 ± 2.84)% and (23.05 ± 2.91)%, respectively, in the DIP-treated tumors. In the tumors treated by saline, the ratios were (2.62 ± 0.60)%, (4.05 ± 0.12)% and (3.10 ± 0.09)%, respectively. The difference between experimental group and control group had statistical significance.

CONCLUSIONSTianjin strain DIP may exert anti-tumor effect on tumor-bearing mice. The mechanism is related with the antitumor immunity induced by DCs and T cells.

Animals ; Cell Line, Tumor ; Colonic Neoplasms ; metabolism ; pathology ; Cytokines ; metabolism ; Defective Viruses ; immunology ; Dendritic Cells ; metabolism ; Female ; Interferon-alpha ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Sendai virus ; immunology ; T-Lymphocytes ; metabolism ; Tumor Burden ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDAdvances in transplantation immunology show that the balance between dendritic cells (DCs) and their subsets can maintain stable immune status in the induction of tolerance after transplantation. The aim of this study was to investigate if DCs and DC subpopulations in recipient peripheral blood are effective diagnostic indicators of acute rejection following kidney transplantation.

METHODSImmunofluorescent flow cytometry was used to classify white blood cells (WBCs), the levels of mononuclear cells and DCs (including the dominant subpopulations, plasmacytoid DC (pDC) and myeloid DC (mDC)) in peripheral blood at 0, 1, 7, and 28 days and 1 year after kidney transplantation in 33 patients. In addition, the blood levels of interleukin-10 (IL-10) and IL-12 were monitored before and after surgery. Fifteen healthy volunteers served as normal controls. Patients were undertaking hemodialysis owing to uremia before surgery.

RESULTSThe total number of DCs, pDC, and mDC in peripheral blood and the pDC/mDC ratio were significantly lower in patients than controls (P < 0.05). Peripheral DCs suddenly decreased at the end of day 1, then gradually increased through day 28 but remained below normal levels. After 1 year, levels were higher than before surgery but lower than normal. The mDC levels were higher in patients with acute rejection before and 1 day after surgery (P < 0.005). There was no significant difference in IL-10 and IL-12 levels between patients with and without acute rejection.

CONCLUSIONThe changes in DCs and DC subpopulations during the acute rejection period may serve as effective markers and referral indices for monitoring the immune state, and predicting rejection and reasonably adjusting immunosuppressants.

Adolescent ; Adult ; Dendritic Cells ; immunology ; Graft Rejection ; immunology ; Humans ; Kidney Transplantation ; adverse effects ; Middle Aged ; Myeloid Cells ; immunology ; Young Adult

PURPOSE: Cancer stem cells have recently been thought to be closely related to tumor development and reoccurrence. It may be a promising way to cure malignant glioma by using glioma stem cell-targeted dendritic cells as a tumor vaccine. In this study, we explored whether pulsing dendritic cells with antigens of glioma stem cells was a potent way to induce specific cytotoxic T lymphocytes and anti-tumor immunity. MATERIALS AND METHODS: Cancer stem cells were cultured from glioma cell line U251. Lysate of glioma stem cells was obtained by the repeated freezing and thawing method. Dendritic cells (DCs) were induced and cultured from the murine bone marrow cells, the biological characteristics were detected by electron microscope and flow cytometry. The DC vaccine was obtained by mixing DCs with lysate of glioma stem cells. The DC vaccine was charactirizated through the mixed lymphocyte responses and cell killing experiment in vitro. Level of interferon-gamma (IFN-gamma) in the supernatant was checked by ELISA. RESULTS: After stimulation of lysate of glioma stem cell, expression of surface molecules of DC was up-regulated, including CD80, CD86, CD11C and MHC-II. DCs pulsed with lysate of glioma stem cells were more effective than the control group in stimulating original glioma cells-specific cytotoxic T lymphocytes responses, killing glioma cells and boosting the secretion of IFN-gamma in vitro. CONCLUSION: The results demonstrated DCs loaded with antigens derived from glioma stem cells can effectively stimulate naive T cells to form specific cytotoxic T cells, kill glioma cells cultured in vitro.
Animals ; Antigens, Neoplasm/immunology ; Apoptosis ; Brain Neoplasms/*therapy ; Cancer Vaccines/*therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Dendritic Cells/*cytology ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Glioma/*therapy ; Humans ; Interferon-gamma/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Neoplastic Stem Cells/*cytology ; T-Lymphocytes, Cytotoxic/immunology

OBJECTIVETo explore the biological characteristic of third-party-derived tolerogenic DC(tDC) and the influence of third-party-derived tDC on acute graft-versus-host-disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) in mice.

METHODStDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-β1D1. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4(+)T cells proliferation in vitro. Different doses of D1-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Th1/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control.

RESULTStDC expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86 and CD40, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-β, Fas Ligand, indoleamine 2, 3-dioxygenase (IDO) and arginase. In the allogeneic MLR, third-party tDC suppressed allo-CD4(+)T cells proliferation, which was relative to the dose of tDC. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 10(4) third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 10(3) cells, only 20% of mice survived day 60, and when increased tDC to 10(5), all of the mice died within day 37 after allo-BMT. The cytokine levels in serum indicated that 10(4) tDC-treated mice secreted in vivo high level of IL-10 21d after BMT (P < 0.05), the levels of IL-10 in 10(3), 10(4) and 10(5) tDC-treated mice were (114.23 ± 7.78), (646.18 ± 212.02), (121.97 ± 10.47) ng/L, respectively.

CONCLUSIONThird-party tDC could suppress allo-CD4(+)T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-β1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.

Animals ; Bone Marrow Transplantation ; adverse effects ; CD4-Positive T-Lymphocytes ; cytology ; Cell Proliferation ; Dendritic Cells ; cytology ; immunology ; metabolism ; Graft vs Host Disease ; prevention & control ; Interleukin-10 ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Transforming Growth Factor beta1 ; immunology ; Transplantation, Homologous

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

BACKGROUNDAllogeneic transplant rejection is currently a major problem encountered during organ transplantation. The dendritic cell (DC) is the most effective powerful known professional antigen-presenting cell, and recent studies have found that DCs can also induce immune tolerance, and avoid or reduce the degree of transplant rejection. The aim of this study was to evaluate the effect of transfused immature CD4(+) DCs on renal allografts in the rat model.

METHODSIn this study, we induced CD4(+) immature DCs from rat bone marrow cells by a cytokine cocktail. The immature CD4(+) DCs were identified by morphological analysis and then the suppressive activity of these cells conditioned with donor kidney antigen was evaluated in vitro and in vivo.

RESULTSImmature CD4(+) DCs conditioned with donor kidney antigen possessed immunosuppressive activity in vitro and they were able to prolong renal transplant survival in an allograft rat model in vivo.

CONCLUSIONSOur study provides new information on efficacious renal transplantation, which might be useful for understanding the function of immature CD4(+) DCs in modulating renal transplant rejection and improving clinical outcome in future studies.

Animals ; Antigens ; immunology ; CD4 Antigens ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Graft Survival ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-4 ; metabolism ; Kidney Transplantation ; immunology ; Male ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Homologous ; immunology

OBJECTIVETo explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice.

METHODSB16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-β were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay.

RESULTSCisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01).

CONCLUSIONCisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; B7-2 Antigen ; metabolism ; CD8-Positive T-Lymphocytes ; pathology ; Cancer Vaccines ; pharmacology ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Dendritic Cells ; immunology ; metabolism ; Female ; Genes, MHC Class II ; HMGB1 Protein ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Melanoma, Experimental ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; T-Lymphocytes, Regulatory ; pathology ; Tumor Burden ; drug effects

OBJECTIVETo investigate the mechanism of immune hyporesponsiveness induced by donor-antigen- unloaded recipient-derived immature dendritic cell (imDC) of liver grafts in rats.

METHODSForty Sprague-Dawley rats (donor) and forty male Wistar rats (recipient) were randomly divided into 4 groups: control, cyclosporine A (CsA), mature DC (mDC), and imDC groups respectively, with 10 donor rats and 10 recipient rats in each group. Recipient rats in CsA group were treated with 10 mg•kg⁻¹•d⁻¹ CsA starting day 2 after the transplantation. Recipients in the mDC or imDC groups were given Wistar rat derived mDCs (1 × 10⁶/rat) or imDCs (1 × 10⁶/rat) via dorsal vein of the penis respectively 1 day before the transplantation. In each group, 5 recipients were kept for determination of survival time and the other 5 rats were executed at day 10 after transplantation. Blood samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBIL), interleukin 2 (IL-2), interferon gamma (IFN-γ), IL-4, and IL-10 levels. Liver tissue was harvested for HE staining and acute rejection evaluation. Expression levels of Fas-L/Fas in the grafts were detected by immunohistochemical staining; and Western blot was used to detect the expression level of Scurfin.

RESULTSThe survival time of CsA and imDC groups was significantly longer than that of control and mDC groups (all P < 0.05). The levels of serum ALT and TBIL in the control group (2072.20 ± 217.93 IU/L and 147.42 ± 22.02 µmol/L) and mDC group (2117.00 ± 285.13 IU/L and 141.58 ± 20.82 µmol/L) were significantly higher than those in the CsA group (59.68 ± 13.48 IU/L and 15.40 ± 2.13 µmol/L) or imDC group (50.80 ± 9.63 IU/L and 14.44 ± 3.49 µmol/L) (all P < 0.05). In the CsA and imDC groups, the levels of IL-2 (22.52 ± 3.75 pg/mL and 22.12 ± 3.90 pg/mL) and IFN-γ (309.20 ± 25.19 pg/mL and 321.00 ± 21.64 pg/mL) were significantly lower, but the levels of IL-4 (297.60 ± 25.07 pg/mL and 277.00 ± 22.47 pg/mL) and IL-10 (1226.00 ± 140.49 pg/mL and 1423.00 ± 106.39 pg/mL) were higher than those of the control (IL-2: 147.78 ± 12.80 pg/mL, IFN-γ: 1758.60 ± 106.22 pg/mL, IL-4: 17.40 ± 4.77 pg/mL, IL-10: 81.00 ± 9.47 pg/mL) and mDC groups (IL-2: 142.34 ± 9.29 pg/mL, IFN-γ: 1835.00 ± 82.63 pg/mL, IL-4: 15.60 ± 3.96 pg/mL, IL-10: 68.80 ± 11.23 pg/mL) (all P < 0.01). The expression level of Scurfin protein on CD4+ CD25+ T cells of the imDC group (1.34 ± 0.29) was significantly higher than that in the control (0.72 ± 0.13), CsA (0.37 ± 0.11), and mDC groups (0.78 ± 0.17) (all P < 0.05).

CONCLUSIONDonor-antigen-unloaded recipient-derived imDC is an effective treatment in inducing immune hyporesponsiveness through induction of T cell apoptosis, shift in Thl/Th2 balance, and proliferation of regulatory T cell.

Animals ; Antigens ; immunology ; Cytokines ; immunology ; Dendritic Cells ; cytology ; immunology ; transplantation ; Fas Ligand Protein ; immunology ; Forkhead Transcription Factors ; metabolism ; Graft Rejection ; immunology ; Graft Survival ; immunology ; Humans ; Immunity ; physiology ; Liver ; cytology ; immunology ; pathology ; Liver Transplantation ; immunology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; fas Receptor ; immunology