This study was aimed to evaluate whether human mesenchymal stem cell (MSC) and the Stro-1 positive subgroup have inducing immune tolerance effect in mouse skin graft model. Human MSC were isolated and cultured from bone marrow-derived mononuclear cells of healthy adults, and Stro-1 positive cells were sorted out. Female C57BL/6 mice and female BALB/c mice were respectively used as donors and recipients in skin allogenic graft model. The recipients were divided randomly into 4 groups: (1) Stro-1(+) MSC group: 2×10(6) Stro-1(+) MSC were injected into the irradiated recipient mice before skin graft. (2) MSC group: 2×10(6) MSC were injected into the irradiated recipient mice before skin graft. (3) Irradiation control group: the recipient mice were just irradiated before skin allogenic graft. (4) Congenic control group: the irradiated BALB/c mice received the skin from the congenic mice. The survival time and pathologic changes of skin grafts were observed by macro- and microscopy with HE staining. The transforming growth factor β1 (TGF-β1) concentration in plasma of recipient mice was measured by ELISA before and after grafting. The results indicated that the survival time of skin grafts in the MSC group was (12.13 ± 3.34) d, which was not notably longer than the irradiation control group (11.38 ± 1.01) d. The survival time of skin grafts was significantly prolonged in the Stro-1(+) MSC group (30.68 ± 5.89) d, as compared with the irradiation control group and the MSC group, respectively; the pathologic examination of skin grafts showed a clear structure. After grafting, the TGF-β1 concentration in plasma of recipient mice was almost the same as before grafting in the irradiation control group and the congenic control group, but it significantly increased in the MSC group and the Stro-1(+) MSC group. It is concluded that the Stro-1(+) MSC induce greater immune tolerance than the unsorted MSC, and significantly prolong the survival time of skin grafts in vivo, while TGF-β1 does not contribute to the immune tolerance.
Animals ; Female ; Graft Survival ; immunology ; Humans ; Immune Tolerance ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; methods ; Transforming Growth Factor beta1 ; blood ; Transplantation, Homologous

The aim of this study was to explore the effect of hyperbaric oxygen (HBO) preconditioning on the rejection of skin allograft in mice and its molecular mechanism. BALB/c donor mice and C57BL/6 recipients received hyperbaric oxygen preconditioning once a day for 7 days. After skin transplantation, the recipients were treated with cyclosporine A (CsA) intraperitoneally. Immunofluorescent staining technique and flow cytometry were used to observe the influence HBO on percentage of spleen lymphocytes CD3+, CD4+, CD8+ and cell adhesion molecule LFA-1 (CD11a/CD18). The results showed that as compared with control, the numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+ CD18+, CD8+CD11a+, CD8+CD18+ lymphocytes of spleen decreased in HBO preconditioning groups and CsA group, and decreased markedly in HBO preconditioning combined with CsA group (p<0.05); the general state of recipient mice in HBO preconditioning combined with CsA group was better than that of recipient mice received HBO preconditioning or CsA only. It is concluded that the method of HBO preconditioning combined with traditional immunosuppressive agent CsA has remarkable advantage in inhibiting the rejection of skin graft. Its molecular protective mechanism is correlated with the expression of adhesive molecules on T cell subsets.
Animals ; Cell Adhesion ; Cell Adhesion Molecules ; pharmacology ; Cyclosporine ; pharmacology ; Female ; Graft Rejection ; prevention & control ; Graft Survival ; Hyperbaric Oxygenation ; Lymphocyte Count ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; Spleen ; cytology ; Transplantation Conditioning ; methods

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

In the early developing stage of burn surgery, severe burn patients with large and deep burn wound often died of complications because of shortage of auto-skin. The method of intermingled transplantation composed of a large sheet of partial thickness allo-skin with punched holes for in laying small pieces of partial thickness auto-skin was first advocated by Chinese doctors (Ruijin Hospital) in 1960's. This intermingled transplantation method has saved many severe burn patients with extensive full-thickness burn wound. The mortality rate of severe burn patients has decreased and the survival rate has increased remarkably since the intermingled transplantation treatment method used in the burn units. In this paper we review the process of formation of intermingled transplantation and the mechanisms of success of this Chinese method in repairing the large wound surface area after eschar excision. We will focus our discussion on the low systemic immunological reaction, the effect of auto-skin islet, local immunological tolerance induced by in layed auto skin, the balance of Th1 and Th2 cells and the effects of some cytokines such as IL-10 in local immunological tolerance and etc. after intermingled transplantation.
Burns ; immunology ; pathology ; surgery ; Humans ; Skin Transplantation ; immunology ; methods ; Wound Healing

The study was aimed to explore the roles of exosomes derived from regulatory dendritic cells of mice in the induction of immune tolerance. Immature DC (iDC) from mouse bone marrow cells and regulatory DCs (rDC) were induced by treating iDC with TGF-beta1 and IL-10. The phenotype of regulatory DCs and normal DCs were assayed by flow cytometry. Exosomes from immature DCs (iDex) and regulatory DCs (rDex) were isolated by ultracentrifugation and ultrafiltration. A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into PBS control group, iDex group (injection 10 microg iDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation), rDex group (injection 10 microg rDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation). The capacity of the donor mice and the unrelated allogeneic donor mice to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture (MLR). The results showed that TGF-beta1 and IL-10 could down-regulate the expressions of costimulatory molecules, including CD80, CD86 and CD40. The graft mean survival time (MST) in control group, iDex group and rDex group was 7.8, 10.7 and 18.8 days, respectively. There was significant difference in MST between iDex group and control group (p<0.05), and between rDex group and iDex group (p<0.01). The results of MLR assays indicated donor-specific hyporeactivity especially in rDex group, while the tolerant B/C mice were still immunocompetent to unrelated allogeneic DBA mouse. It is concluded that injection iDex or rDex of donor mice via tail vein before skin transplantation induces immunotolerance, and the effect of rDex is more significant.
Animals ; Dendritic Cells ; cytology ; immunology ; transplantation ; Exosomes ; immunology ; transplantation ; Female ; Graft Enhancement, Immunologic ; methods ; Graft Survival ; Immune Tolerance ; immunology ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Skin Transplantation ; Transplantation Immunology ; Transplantation, Homologous

OBJECTIVETo investigate the role of simultaneous blockade of CD40/CD40L and B7/CD28 pathways in the immune tolerance via co-expression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus.

METHODSAd-sCD40LIg-IRES(2)-CTLA4Ig, replication-defective adenovirus co-expressing sCD40LIg and CTLA4Ig, was constructed and identified. The co-expression of sCD40LIg and CTLA4Ig was evaluated with confocal laser scanning microscope and Western blotting. Skin transplantations of C57BL/6 to BALB/c mice were performed. PBS, Ad-Shuttle-CMV and Ad-sCD40LIg-IRES(2)-CTLA4Ig were administered. Skin graft survival was monitored and the mRNA expression of both genes was evaluated in the skin allografts.

RESULTSAd-sCD40LIg-IRES(2)-CTLA4Ig was constructed successfully and identified. The co-expression of sCD40LIg and CTLA4Ig was identified with confocal laser scanning microscopy and Western blotting. Compared to the skin graft mean survival time (MST) of non-treated group ((5.75+/-0.71) d) or Ad-Shuttle-CMV-treated group ((5.50+/-0.53) d), the skin graft MST was dramatically prolonged in the Ad-sCD40LIg-IRES(2)-CTLA4Ig-treated group ((16.38+/-1.19) d, P<0.001). The mRNA expression of both genes was detected.

CONCLUSIONAd-sCD40LIg-IRES(2)-CTLA4Ig, a replication-defective adenovirus carrying genes encoding sCD40LIg and CTLA4Ig, was constructed. Simultaneous blockade of CD40/CD40L and B7/CD28 costimulatory pathway mediated by replication-defective adenovirus significantly prolonged skin allograft survival in mice.

Abatacept ; Adenoviridae ; genetics ; Animals ; Cytopathogenic Effect, Viral ; Graft Survival ; immunology ; Immunoconjugates ; genetics ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Skin Transplantation ; immunology ; methods ; Transfection

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.
Skin Transplantation/*immunology/methods ; Mice, Inbred BALB C ; Mice ; Male ; Interleukin-2/*immunology ; Immunoconjugates/*administration & dosage ; Graft Survival/*immunology ; Drug Combinations ; CD40 Ligand/*immunology ; Bone Marrow Transplantation/*immunology/methods ; Antibodies/*administration & dosage/immunology ; Animals

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.
Skin Transplantation/*immunology/methods ; Mice, Inbred BALB C ; Mice ; Male ; Interleukin-2/*immunology ; Immunoconjugates/*administration & dosage ; Graft Survival/*immunology ; Drug Combinations ; CD40 Ligand/*immunology ; Bone Marrow Transplantation/*immunology/methods ; Antibodies/*administration & dosage/immunology ; Animals

OBJECTIVETo explore a feasible method to repair full-thickness skin defects utilizing tissue engineered techniques.

METHODSThe Changfeng hybrid swines were used and the skin specimens were cut from the posterior limb girdle region, from which the keratinocytes and fibroblasts were isolated and harvested by trypsin, EDTA, and type II collagenase. The cells were seeded in Petri dishes for primary culture. When the cells were in logarithmic growth phase, they were treated with trypsin to separate them from the floor of the tissue culture dishes. A biodegradable material, Pluronic F-127, was prefabricated and mixed with these cells, and then the cell-Pluronic compounds were seeded evenly into a polyglycolic acid (PGA). Then the constructs were replanted to the autologous animals to repair the full-thickness skin defects. Histology and immunohistochemistry of the neotissue were observed in 1, 2, 4, and 8 postoperative weeks.

RESULTSThe cell-Pluronic F-127-PGA compounds repaired autologous full-thickness skin defects 1 week after implantation. Histologically, the tissue engineered skin was similar to the normal skin with stratified epidermis overlying a moderately thick collageneous dermis. Three of the structural proteins in the epidermal basement membrane zone, type IV collagen, laminin, and type VII collagen were detected using immunohistochemical methods.

CONCLUSIONSBy studying the histology and immunohistochemistry of the neotissue, the bioengineered skin graft holds great promise for improving healing of the skin defects.

Animals ; Disease Models, Animal ; Epidermis ; pathology ; Skin ; immunology ; injuries ; pathology ; Skin Transplantation ; methods ; Swine ; Tissue Engineering ; methods ; Transplantation, Autologous ; Transplants ; Treatment Outcome ; Wounds and Injuries ; surgery

OBJECTIVETo investigate the immunologic reaction difference between xenogeneic and allogeneic acellular dermal matrix (ADM) grafting.

METHODSSplit thick skin samples harvested from healthy piglets and human volunteers who underwent losing-weight operation were processed to be xeno-ADM and allo-ADM. The ADMs overlapped with ultrathin auto-skin were employed to immediately cover the wound after escharectomy in deep burn patients. The patients were correspondingly set to be Xeno (26 cases) and Allo (10 cases) groups. Another 8 cases with deep burn wounds were grafted with only split thick autoskin (TTS) after escharectomy as control group. The tissue samples from grafted area were observed by immunohistochemistry after the grafting. The typing of immune cells in peripheral blood and grafted tissue was determined.

RESULTS(1) The CD4(+), CD45RO(+) and CD4(+)/CD8(+) cell ratios in peripheral blood in Xeno group increased slightly after the skin grafting when comparing to those in control group (P > 0.05). (2) There existed lasting inflammatory and immunological reaction in the local site of grafts in Xeno group. In addition, more than 80% of the inflammatory cells could be found to be CD3(+)/CD4(+), CD45RO(+). But CD8(+), Vs8C(+) plasmocytes and CD57(+) NK cells were found less. Furthermore, eosinophil and CD68(+)/CD4(+) foreign body megalocyte reactions could also be identified, especially in Xeno-ADM before rejection (P < 0.05 - 0.001). There was only mild inflammatory and immunological reaction during early grafting stage (within 8 post-operational weeks) in Allo-group.

CONCLUSIONThe specific immunologic reaction of human host to ADM might be participated by mononuclear cells and macrophages and presented mainly as cellular immune reaction induced by CD4(+) T lymphocytes. Furthermore, the foreign body megalocyte constructed by help T cell and macrophage might play important roles in the reaction.

Animals ; Burns ; immunology ; surgery ; Dermis ; transplantation ; Graft Rejection ; Humans ; Skin Transplantation ; immunology ; methods ; Swine ; Transplantation, Heterologous ; Transplantation, Homologous