The effects of FTY720 on lymphocytes and monocytes in mouse intestinal transplantation

AIM：To test the effects of FTY720 on mouse intestinal allografts.METHODS：C3H mice（H-2k）were used as donor and C57BL/6 mice (H-2b) as recipients.FTY720 group,allogeneic control group and isogeneic control group were set up.6 and 14 days after transplantation,murine intestinal grafts were harvested for histologic assessment.Lymphocytes were collected from mesenteric lymph nodes (MLN),Peyer’s patch (PP),lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) in the graft,then were analyzed by cytometry.RESULTS：Rejection was inhibited in FTY720 group at the 6th post-transplant day,although not at the 14th day.Recipient CD4+ and CD8+ T cells,CD19+ B cells,as well as γδ TCR lymphocytes,were greatly reduced by FTY720 therapy.The similar action of FTY720 was also revealed in Gr1+CD11b+ monocytes.CONCLUSION：FTY720 is efficient on alleviating allo-immune response by reducing the infiltration of both lymphocytes and monocytes into the graft in a mouse intestinal transplantation model.     

Generation of hematopoietic stem/progenitor cells with property of strengthened cell mediated immunity from an embryonic stem cell line

AIM:To induce lymphoid stem cells and/or T-cell precursors to differentiate into functional mature T lymphocyte, and to increase the surface marker of T lymphocytes such as CD₃⁺, while embryonic stem(ES) cells differentiated into hematopoietic stem/progenitor cells(HSPCs) in vitro. When they were injected into lethally irradiated mice, these differentiated cells had the advantage in immune reconstitution. METHODS:Embryonic stem cells formed embryoid bodies(EBs) in the medium containing methycellulose, hematopoietic growth factors(HGFs) was added to the culture system on the 6th day, thymopeptide was added at the same time. Flow cytometry were performed to detect the surface marker CD₃₄⁺ and CD₃⁺ of the differentiated cells. Finally the differentiated cells were injected into lethally irradiated mice, 60 days later, the incidence rate of graft versus host disease(GVHD) was taken as the mark of cell mediated immunity, PCR was performed to detect the sex determining region of the Y-chromosome(Sry) in bone marrow cells and spleen cells of the survival host female mice. RESULTS:The percentage of CD₃⁺ T lymphocytes was 10.52% and the incidence rate of GVHD was 0% on the 13th day, but they respectively rose up to 22.93% and 100% if thymopeptide was added in the procedure of inducing ES cells to differentiate into HSPC in vitro. CONCLUSION:The quantity of CD₃⁺ T lymphocytes increased in medium containing thymopeptide when ES cells differentiated into CD₃₄⁺ HSPC.     

PDL1Ig gene-modified BMSCs induce immune tolerance in rat liver transplantation

AIM: To investigate the effects of bone marrow mesenchymal stem cells(BMSCs) modified by programed death ligand-1 immunoglobulin(PDL₁Ig) gene on immune rejection of orthotopic liver transplantation in rats. METHODS: Rat BMSCs were cultured and identified. The protein expression of PDL₁Ig in the BMSCs 72 h after infection with pAdEasy-1/PDL₁Ig was detected by Western blot. Mixed lymphocyte reaction was used to detect the inhibitory effect of BMSCs on the viability of T-lymphocytes in peripheral blood. The male Wistar rats were used as donors(n=40), and the male SD rats were used as recipients(n=40). The rat model of orthotopic liver transplantation was established by improved cuff method for observing acute rejection. The rats were randomly divided into control group, BMSCs treatment group, BMSCs/GFP treatment group and BMSCs/PDL₁Ig treatment group with 10 pairs each. Five rats were executed at the 7th day and the remains were used for measuring the survival time. RESULTS: The expression of PDL₁Ig in the BMSCs was detected after pAdEasy-1/PDL₁Ig infection. The effect of BMSCs/PDL₁Ig on the viability of the lymphocytes was stronger than that of BMSCs/GFP. The level of IL-4 in BMSCs/PDL₁Ig group was significantly higher than that in the other 3 groups, while the levels of IFN-γ and IL-2 were significantly decreased. The liver function in BMSCs/PDL₁Ig group was significantly improved and the levels of ALT, AST and TBil were almost recovered to normal at the 7th day after transplantation. Severe rejection reaction was observed in control group, and rejection reactions were decreased with different degrees in BMSCs treatment group and BMSCs/GFP treatment group. Much slighter rejection reaction and significantly longer survival time were showed in BMSCs/PDL₁Ig group than those in the other 3 groups. CONCLUSION: PDL₁Ig-modified BMSCs inhibit the rejection of liver transplantation in rats and induce the immune tolerance, and the effect is better than that of BMSCs alone.     

Role of B cells in CD45RB antibody-induced transplantation immune tolerance

AIM: To investigate the role of B cells in CD45RB antibody-induced transplantation immune tolerance. METHODS: Single cell suspension was made from the spleen of BALB/c nude mice disposed by CD45RB antibody, then mixed cultured with T cells of BALB/c mice and spleen cells of C57BL/6 mice. The Th1, Th2, Treg and Tm cells were monitored by flow cytometry during the culture process. The skin graft model was set up with B6.μMT^-/- mice as receptors and BALB/c mice as donors. CD45RB antibody was intraperitoneally injected into the receptors after transplantation and then CD3⁺CD45RB^hi cells were detected by flow cytometry. In another mixed lymphocyte culture, CD45RB antibody was added, and then B cells were isolated and injected into B6.μMT^-/- mice through the tail vein. The heart transplantation model was established with B6.μMT^-/- mice as receptors and BALB/c mice as donors, and then the survival and the migration of B cells to the thymus were observed. RESULTS: When T lymphocytes were co-cultured with B lymphocytes treated with anti-CD45RB monoclonal antibody(mAb) in vivo, the percentages of Th2 and Treg cells were up-regulated and Th1 cells were down-regulated, but Tm cells were not altered as compared with the control. In vivo without B lymphocytes, anti-CD45RB mAb also down-regulated the expression of CD45RB in T lymphocytes. The reduction was faster and the percentage of CD3⁺CD45RB^hi T cells was not altered as compared with the control. The B lymphocytes treated with anti-CD45RB mAb in vitro prolonged the lifetime of receptor in heart transplantation model but failed to induce complete tolerance. After recieving B cells treated with anti-CD45RB mAb and allogeneic heart transplantation, B cells migrated to the thymus in B6.μMT^-/- mice. CONCLUSION: B lymphocytes play a definite role in the transplantation immune tolerance induced by anti-CD45RB mAb through their affection on T-cell subgroups and also in the central tolerance. However, the induction of immune tolerance can not only rely on B cells.     

Effects of deep-frozen treatment on the immune response of bone-patellar tendon-bone xenograft rejection

AIM: To investigate the effects of deep-frozen treatment on the immune response of bone-patellar tendon-bone (BPB) xenograft rejection. METHODS: A muscle pocket model was used to study the immune response of rat to deep-frozen treated BPB xenograft from guinea pig, autograft and fresh xenograft served as controls. The expression of T-cell surface activation antigen CD25 in the peripheral blood and the morphological changes of the implants were used to measure the immune response. RESULTS: The expression of CD25 in CD4+, CD8+ cells greatly increased 3 d after fresh BPB xenograft, the obvious difference to that of autograft (P<0.05) was observed. It reached a peak level 14 d after fresh BPB xenograft and didnt decrease 35 d after transplantation. The expression of CD25 in CD4+, CD8+ cells was greatly inhibited in deep-frozen treated BPB xenograft. Its expression was delayed and just had a small increase 14 d after transplantation. The P values were all less than 0.05 compared to those of fresh xenograft at every time-point after transplantation. Histologic examination showed that there were a few lymphocytes surrounding the bone and tendon tissue in deep-frozen treated BPB xenograft, but the lymphocytes hadnt invaded into the tissues. No tissue necrosis, but a clear tendon structure and regular fibrogen arrangement were observed. There were large amount of fragmentary lymphocytes infiltrating into or surrounding the bone and fibrogen tissues after fresh xenograft, the bone was broken into pieces, the tendon structure was unclear and the fibrogen arrangement was irregular, quite a few of inflammatory cells such as acidophilic granulocytes and macropolycytes were found in some slices. CONCLUSION: Deep-frozen treatment markedly reduces BPB xenograft antigenicity and inhibits the immune rejection.     

Effect of pioglitazone on pre-adipocytes differentiation and GILZ expression

AIM: To investigate the roles of pioglitazone on differentiation and expression of GILZ in 3T3-L1 pre-adipocytes. METHODS: The morphological changes during 3T3-L1 cell differentiation were observed. The cells were treated with pioglitazone at concentrations of 1×10^-4～1×10^-2 mmol/L for 48 h, then the relative content of triglyceride were analyzed by oil red O staining at 2nd, 4th and 6th day during adipogenesis. The mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) was measured by real-time PCR. GILZ protein expression was determined by Western blot after the cells were treated with pioglitazone at concentrations of 1×10^-4 ～1×10^-2 mmol/L for 48 h.RESULTS: Oil-red O staining showed that the relative contents of triglyceride in adipocytes were increased with the increase in the pioglitazone concentration. Compared with the control, the relative contents of triglyceride in group 1×10^-3 mmol/L and group 1×10^-2 mmol/L were significantly increased (P < 0.05). The mRNA expression of PPARγ2 and LPL was also increased with the increase in the pioglitazone concentration. When pioglitazone concentration was more than 1×10^-3 mmol/L, compared with the control, the mRNA expression of PPARγ2 and LPL significantly increased (P < 0.01). The protein expression of GILZ was decreased with the increase in the pioglitazone concentration.CONCLUSION: Pioglitazone down-regulates GILZ expression, and up-regulate PPARγ2 expression and the downstream functional factor such as LPL.     

Study on animal intracorneal implantation model with xenogeneic swine corneal stroma

AIM: To study the immunogenicity and biocompatibility of xenogeneic swine corneal stroma as biological carrier for cornea reconstruction and to reconstruct corneal endothelial tissue with this carrier in vitro. METHODS: (1) The lymphocytes from the peripheral blood of F344 rats were immunologically labeled by anti-rat CD25-FITC and anti-rat CD4/CD8-PE, then determined by flow cytometry (FCM) at 12th， 90th day after intracorneal implantation with fresh and dehydrated swine corneal stroma. (2) The fresh and dehydrated grafts made of swine corneal stroma were implanted intralamellarly in corneas of New Zealand rabbits. Clinical examinations were performed monthly and histological examinations were made at 14th， 30th， 60th， 120th and 240th day. (3) The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology of reconstructed tissue was tested by microscope. RESULTS: (1) Compared to isograft group and negative control, the expression CD4＋CD25＋， CD8＋CD25＋ and CD4＋/CD8＋ of xenograft rat group implantation with swine corneal stroma did not appear significantly different in statistics (P>0.05). (2) In the total 12 rabbits, all the cornea grafts survived without rejection reaction， corneal haze or corneal neovascularization. The fresh grafts got transparent after 2 months, and the dehydrated grafts got transparent after 6 months. Histological study demonstrated both fresh and dehydrated stroma grafts had fused with rabbits'corneal stroma very well without lymphocytes infiltrating. (3) As shown in histological observations, the reconstructed cat corneal endothelial tissue was similar to the nature tissue. Cultured cat endothelial cells connected tightly to each other and attached to the Descemet's membrane closely. CONCLUSION: Swine corneal stroma has low immunogenicity and satisfying biocompatibility,it is an ideal biological carrier for cornea reconstruction in vitro.     

Effect of human β-NGF gene-modified bone marrow-derived mesenchymal stem cells on rotational behavior of rats with Parkinson disease

AIM：To investigate the effect of human β-nerve growth factor (β-NGF) gene-modified bone marrow-derived mesenchymal stem cells (MSCs) transplantation on the rotational behavior improvement in a rat model of Parkinson disease (PD). METHODS：The rat model of PD was established successfully and the animals were divided into 4 groups:β-NGF-MSCs group (transplanted with 5×105 β-NGF-engineered MSCs), MSCs group (transplanted with 5×105 MSCs), DMEM/F12 group (5 μL transplantation medium was injected in the right striatum of the rats) and PD model group (without transplantation). The rotational scores were assessed 2 weeks, 4 weeks and 6 weeks after transplantation. At different time points after transplantation, the rats were tested for apomorphine (APO)-induced rotational behavior and the brains of the PD model rats were examined by fluorescence microscopy and immunohistochemical staining. RESULTS：Transplantation of human β-NGF gene-modified MSCs effectively improved the behavioral performance in the rats. At the 2nd, 4th and 6th weeks after cell transplantation, the rotational frequencies after injection of APO decreased significantly in β-NGF-MSCs group compared with MSCs group and PD group (P<0.05). Both β-NGF gene-modified MSCs and MSCs survived in the brains of PD model rats, had good compatibility with the host cells, and showed no signs of destroying the host and the glial cicatrisation. The β-NGF gene-modified MSCs expressed β-NGF stablely in the brains of PD model rats, and showed obvious improvement of the rotational behavior in the PD model rats induced by APO compared with MSCs group. CONCLUSION:The behavior of the rats with PD is significantly improved by transplanting β-NGF-modified MSCs in right striatum, and β-NGF gene therapy has potential clinical value．     

Role of NK cells in mouse allogeneic bone marrow transplantation

AIM: To study the effect of natural killer(NK)-cells on graft-versus-host disease(GVHD), graft rejection, engraftment and reconstituting of hematopoiesis in mouse allogeneic bone marrow transplantation. METHODS: Lethally irradiated BALB/c(H-2^d) mice were transplanted with C57/6j(H-2^b) bone marrow containing donor peripheral T cells and/or NK cells. Recipients CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were detected by flow cytometry, peripheral white blood cell(WBC) was detected by auto-cytometry, and the recipients survival rates, GVHD, engraftment and hematopoiesis recovery were then observed. RESULTS: In the group of transplanted with NK cells infusion, the incidence of GVHD was evidently lessened, the survival rates, WBC and CD₃₄⁺ cell counts and the expression of H-2K^b＋ cell were significantly high than that without NK cells infusion. CONCLUSION: In mouse allogeneic bone marrow transplantation, alloreactivity NK cells prevents GVHD, reduces graft rejection, and promotes engraftment and reconstituting of hematopoiesis.     

Effect of cornus officinalis glycosides ophthalmic solution on the corneal allograft rejection

AIM:To observe the effect of cornus of ficinalis glycosides(COG) ophthalmic solution on the corneal allograft rejection by topical instillation.METHODS:The corneal transplantation model on the closed colony rats was established. The rejection time of all animals was recorded and compared by slit-lamp microscope. The pathologic changes were measured by immunohistochemistry and scanning electron microscope.RESULTS:The histopathological and immunohistochemistry findings showed that the lymphocytes, neovascularity and the expression of ICAM-1 in COG-treated group were significantly fewer than that in control group at 15 d after operation.CONCLUSION:COG ophthalmic solution prevents and suppresses the corneal allograft rejection.     

Immunosupressive mechanism of cornus officinalis glycosides on the corneal allograft rejection in rats

AIM: To investigate the influence of the cornus officinalis glycosides (COG) on immunological function of corneal transplantation model of rats, and to clarify the immunosuppressive mechanism of COG through observing the activation of lymphocytes in blood. METHODS: Wister rats were used as recipients and SD rats were used as corneal graft donors, then the corneal allografts transplantation model on the closed colony rats were set up. Splenocytes proliferation and mixed lymphocyte reaction of Wister rats activated by ConA were observed. The phenotype change of CD4, CD8, CD25 in blood in different time postoperatively were observed by the di-sign flow cytometry, and the rate of CD4/CD8 was calculated. RESULTS: 1. The COG suppressed the proliferation of T lymphocytes and one-way mixed lymphocyte reaction on the corneal allografting. 2. The phenotype change of lymphocytes in boold was as follows: there was no significant difference between the different time of the CD4, CD8 expression and the CD4/CD8 rate in blood of the control group. The CD4 positive cells expressed CD25 postoperatively increased obviously. The CD4/CD8 rate of medicine group had the tendency to decrease. The CD4 positive cells expressed CD25 postoperation in the medicine group were less than that in the control group obviously. CONCLUSION: The suppression of the T lymphocyte proliferation, mixed lymphocyte reaction, CD molecule expressed by the activated T lymphocytes and the IL-2 receptor expression may be the main immunosuppressive mechanisms of Cornus officinalis glycosides on the cell-mediated immunity.     

Iron accumulation in acute cardiac allograft rejection in mice

AIM: To investigate the significance of iron in acute cardiac allograft rejection in mice. METHODS: The model of cervical heterotopic heart transplantation in mice was established. Two groups were studied, including control group (C57BL/6→C57BL/6) and experimental group (BALB/c→C57BL/6). Beating of the grafted heart was Palpated everyday. At day 1, 3, 5, 7 after transplantation, the grafted hearts were obtained. The grade of acute rejection was estimated by observing the histological sections with HE staining. Prussian blue staining was employed to detect the deposits of iron in myocardium. The expression of HO-1 proteins in myocardium was measured by immunohistochemistry technique. RESULTS: Prussian blue staining of allograft tissue revealed an accumulation of iron primarily in infiltrating macrophages. Iron was increased in cardiac allografts with acute rejection in mice. Graft cellular infiltrates were identified as the principal source of HO-1 expression in allograft by immunohistochemistry. CONCLUSIONS: These data suggest that iron may play an important role in acute cardiac allograft rejection. Determination of iron deposits may help estimate the grade of acute cardiac allograft rejection.     

Effect of granulocyte colony-stimulating-factor on acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in a murine model

AIM: To explore the impact of granulocyte colony-stimulating factor (G-CSF) on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model and its possible mechanisms. METHODS: Male C57BL/6 (H-2^b) and BALB/c (H-2^d) mice were used as the allogeneic and syngeneic donor mice, respectively. Moreover, female BALB/c mice were used as recipient mice. The recipient mice were conditioned by a single dose (8 Gy) of total body irradiation (TBI). The recipient mice were randomly divided into 7 groups: TBI group, Syn-BMST control group, post-Syn-BMST G-CSF administration (Syn-BMST+G-CSF)group, allo-BMT control group, post-allo-BMT G-CSF administration (allo-BMT+G-CSF)group, allo-BMST control group and post-allo-BMST G-CSF administration (allo-BMST+G-CSF) group. The mice in control groups and G-CSF administration groups were subcutaneous injected with 0.1 mL normal saline (NS) and 0.1 mL NS containing 2 μg G-CSF per day from 1st day, respectively. The effect of G-CSF on aGVHD was evaluated by clinical manifestations and pathological changes, as well as survival time of the mice in different groups. The serum levels of IL-2, IL-4, IFN-γ and TNF-α in allo-BMST and allo-BMST+G-CSF groups were detected by ELISA at 10th day. Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10th day. RESULTS: The mice in TBI group were all died for hematologic failure on 9~15 d after TBI. No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed. The mean survival days in allo-BMST group and allo-BMST+G-CSF group were (34.8±4.5) d and (19.8±6.1) d'respectively (P<0.01). Moreover, post-transplant administration of G-CSF increased the spleen total nucleated cells count (SpTNC), NK cells subset, and DC1/DC2 ratio in the spleen with over 99% of donor chimerism rate at 10th day. No difference in the levels of serum IL-2, IL-4, IFN-γ and TNF-α between the 2 group at 10th day was found. CONCLUSION: The administration of G-CSF after allo-BMST significantly aggravates mouse aGVHD. The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells. These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clinical allo-HSCT.     

Effect of 1α, 25-dihydroxyvitamin D3 on T helper cell 17 and expression of related cytokines in penetrating keratoplasty in mice

AIM: To evaluate the effects of 1α, 25-dihydroxyvitamin D3 on T helper cell 17 (Th17 cells) and its related cytokines in a mouse model of corneal allograft transplantation. METHODS: C57BL/6 mice were transplanted with corneal grafts from BALB/c mice and treated intraperitoneally with 1.0 μg 1α, 25-dihydroxyvitamin D3 or soybean oil every other day after operation. The transparency of the corneal grafts was evaluated for potential rejection signs by slit lamp biomicroscopy and histopathology. The expression levels of IL-17, RORγt and IFN-γ in the spleen were measured by real-time PCR. Moreover, the protein expression of RORγt and IL-17 in the peripheral blood was analyzed by Western blotting. IL-17 and IFN-γ in peripheral blood were measured by flow cytometry. RESULTS: 1α, 25-dihydroxyvitamin D3 significantly inhibited the rejection of the corneal allograft and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen, 1α, 25-dihydroxyvitamin D3 treatment reduced the expression levels of IL-17, RORγt and IFN-γ. In the peripheral blood, 1α, 25-dihydroxyvitamin D3 treatment downregulated the expression levels of RORγt, IL-17 and IFN-γ. CONCLUSION: The effects of 1α, 25-dihydroxyvitamin D3 on suppressing corneal transplantation-induced allograft rejection in mice are closely associated with its modulation on IL-17 and related cytokine RORγt.     

Study on histocompatibility of pinealocyte microencapsules

AIM: To explore the histocompatibility of pinealocyte microencapsules in vivo. METHODS: The pineal glands of neonatal rats were removed under operating microscope and pinealocytes were isolated through collagenase and trypsin digestion. Pinealocytes were cultured for one week in vitro and collected immediately after digesting was encapsulated in APA microencapsules. The cells and empty capsules were transplanted into abdominal cavity or intermuscular space respectively and retrieved at the 15th or 30th day after operation. Morphological observation, HE staining, cell counting, and HPLC technique were used to analyze the shape, proliferation and function, the degree of inflammation fibrosis of retrieved microencapsules. RESULTS: The retrieve rate of cell capsule from abdominal cavity was about 85%. The retrieved capsules had integrated profile mostly although some were damaged. The amount of macrophages attached to capsule wall and the thickness of wall increased gradually following the period of transplantation. However, the retrieve rate, wall thickness had no difference between retrieved cell and empty capsules at the same time. Secretion ability of pinealocytes in capsule retrieved at 15th day after operation decreased rapidly and those retrieved at 30th day after operation lossed secretion function. CONCLUSION: APA microencapsules had histocompatibility relatively in vivo and protected pinealocytes in capsule from immunologic rejection of the host. The survival time was about 20 days. During this period cells in capsule maintained activity and MT secretion ability.     

Effect of IFN-γ inhalation on the cellular immunity of the lungs

AIM:To study the effect of IFN-γ inhalation on the anti-infection ability of the lungs in the immunocompromised host. METHODS:The immunological factors in the immunocompromised rats and the immunocompromised rats administrated IFN-γ via aerosol were investigated after 1, 3, 7 days when they were injected Candida albicans via tracheal. The Canidda albicans count of the left lung was also determined after 7 days when injecting pathogen. RESULTS:The Canidda albicans count of the left lung in IFN-γ group was significantly less than that of control group. The phagocyting and bactericidal percentages, Ia antigen expression percentages, the levels of TNF-α, IL-1β and IL-6 in the culture supernatant of the AM, the activity of IFN-γ and TNF-α in BALF (except the TNF-α on 7 th day) in IFN-γ group were markedly higher than those in control group. The expression of IFN-γ and IL-1β pulmonary tissues in IFN-γ group was higher than that in control group. The expression of TNF-α in IFN-γ group was less than that in control group. The expression of IL-6 was no changes between two groups. The levels of IFN-γ, IL-1β and IL-6 in the blood (except IL-1β on 3 rd day), and the killing ability of the lymphocytes in blood had no difference between two groups. CONCLUSION:Administration of IFN-γ via aerosol obviously enhanced the anti-infection ability of the lungs in the immunocompromised host, but has no influence on the whole body cellular immunity.     

The prevention of acute graft versus host disease by Rhodamine 123-mediated photodynamic therapy

AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.     

In vitro perfusion method during cardiac graft preservation and its application in adenovirus mediated gene transfection

AIM: To establish a method of in vitro donor heart perfusion in murine cardiac transplantation during preservation and apply it in adenovirus mediated gene transfection for donor heart. METHODS: Donor heart was transfected with recombinant adenovirus and stored for 2 hours after harvest, then it was transplanted heterotopically in abdomen. The grafts were appraisal by palpitation. Marked gene products were determined by X-Gal staining, and T cell infiltration was determined by immunohistochemistry. The activation markers of recipients' lymphocytes were examed by cytometry. RESULTS: The grafts survival rate is 100% after perfusion and cold storage. The LacZ staining became strong 1 week after transplantation. The grafts remained an intact structure and no apparent T cell infiltration. The activation status of recipients' lymphocytes were not enhanced by transfected cardiac graft. CONCLUSION: In vitro perfusion during graft cold preservation is feasible for adenovirus mediated gene transfection.     

NK cells from donor liver inhibit NF-κB activity and IL-15 expression after liver transplantation in rats

AIM:To investigate the mechanism that donor liver natural killer (NK) cells alleviate acute rejection after liver transplantation by observing the secretion level of interleukin 15 (IL-15) in peripheral blood, the protein expression of IL-15 in transplanted liver tissues and the activity of NF-κB in spleen tissues in rat acute liver graft rejection model. METHODS:An acute rejection model of liver transplantation in rats was established by the modified two-cuff method, in which Lewis rats were used as donors and BN rats as recipients. The donor leukocytes were depleted by whole body irradiation of ［60Co］ source and the donor liver immunity was reconstituted by transfusion of liver NK cells from the same type of donor (donor type liver NK cells, dtlNKs) via portal vein immediately after grafting the irradiated liver. The rats were divided into the following groups: group A, acute rejection group; group B, BN rats receiving the liver of Lewis rats with ［60Co］ irradiation; group C, BN rats receiving the liver of ［60Co］-irradiated Lewis rats and treated with dtlNKs via the portal vein. The recipients were sacrificed at 1 d, 3 d and 7 d after transplantation. IL-15 level in peripheral blood was detected by ELISA. The expression of IL-15 in the liver grafts was determined by Western blotting. NF-κB activity in the spleen tissues of recipient rats was identified by electrophoretic mobility shift assay. The survival quality and living time in crude survival subgroup were observed. RESULTS:Acute rejection in group B was severer than that in group A and group C. The rats in group B showed significantly shorter average survival time compared with group A and group C. At 3 d and 7 d after transplantation, the IL-15 content in peripheral blood was significantly higher in group B than that in group A and group C. The expression of IL-15 in transplanted liver tissues was significantly higher in group B than that in group A and group C. The activity of NF-κB in the spleen tissues was higher in group B. CONCLUSION: IL-15 might be a significant indicator for monitoring acute rejection after liver transplantation. The donor liver NK cells modulate the immunity of liver transplantation by inhibiting the expression of IL-15 via the suppression of NF-κB activity.