Survival Time Of Skin Grafts Research Articles

Galectin-9 contributes to the survival of fully allogeneic skin grafts by modulating effector T cells and Tregs imbalance.

Allogeneic skin transplantation is an important clinical treatment for many diseases. Although Galectin-9 demonstrates multifaceted roles in modulating immune cell homeostasis and inflammation, its precise impact on balancing effector T cells and Tregs during allogeneic skin transplantation remains uncertain. This work was performed to evaluate the modulation of the survival time of allogeneic skin grafts by Gal-9 and to explore the critical mechanism involved in this process. Skin graft transplantation was conducted using C57BL/6 and BALB/c mice. Additionally, the levels of IL-2, IFN-γ, IL-4, and IL-17A were measured. Hematoxylin and eosin staining assay was performed to analyze the pathological conditions of skin grafts of experiment mice. The results revealed that Gal-9 noticeably prolonged the survival of the allogeneic skin graft and ameliorated the damage caused by acute immune rejection. Furthermore, Gal-9 resulted in selective reduction of effectors T cells such as Th1, and Th17. Simultaneously, Gal-9 didn't attenuate the protective function for allograft, which alleviated the immune rejection caused by abnormal immune imbalance. Gal-9 exhibited a therapeutic effect on the allogeneic skin graft by selectively reducing CD4+TIM-3+ T effector cells and inducing a shift from a Th1 to an anti-inflammatory Th2 response. Furthermore, Gal-9 did not attenuate the protective function. Our present study may represent a novel therapeutic candidate for modulating effector T cells and Tregs imbalance-based therapy of allograft transplantation.

Novel approach for enhancing skin allograft survival by bioadhesive nanoparticles loaded with rapamycin

Skin graft rejection is a significant challenge in skin allografts for skin defects, particularly in extensive burn injury patients when autografts are insufficient. Enhancing the survival duration of allogeneic skin grafts can improve the success rate of subsequent autologous skin grafting, thereby promoting the therapeutic efficacy for wound healing. Rapamycin (Rapa), a potent immunosuppressant with favorable efficacy in organ transplantation, is limited by its systemic administration-associated toxicity and side effects. Therefore, addressing the short survival time of allogeneic skin grafts and minimizing the toxicity related to systemic application of immunosuppressive agents is an urgent requirement. Here, we present a topical formulation based on bioadhesive poly (lactic acid)-hyperbranched polyglycerol nanoparticles (BNPs) with surface-modified encapsulation of Rapamycin (Rapa/BNPs), applied for local immunosuppression in a murine model of allogeneic skin grafts. Our Rapa/BNPs significantly prolong nanoparticle retention, reduce infiltration of T lymphocytes and macrophages, decrease the level of pro-inflammatory cytokines and ultimately extend skin allograft survival with little systemic toxicity compared to free Rapa or Rapamycin-loaded non-bioadhesive nanoparticles (Rapa/NNPs) administration. In conclusion, Rapa/BNPs effectively deliver local immunosuppression and demonstrate potential for enhancing skin allograft survival while minimizing localized inflammation, thus potentially increasing patient survival rates for various types of skin defects.

The Protective Effect of the Soluble Egg Antigen of Schistosoma japonicum in A Mouse Skin Transplantation Model.

BackgroundOrgan transplantation is currently an effective method for treating organ failure. Long-term use of immunosuppressive drugs has huge side effects, which severely restricts the long-term survival of patients. Schistosoma can affect the host’s immune system by synthesizing, secreting, or excreting a variety of immunomodulatory molecules, but its role in transplantation was not well defined. In order to explore whether Schistosoma-related products can suppress rejection and induce long-term survival of the transplant, we used soluble egg antigen (SEA) of Schistosoma japonicum in mouse skin transplantation models.Materials and methodsEach mouse was intraperitoneally injected with 100 μg of SEA three times a week for four consecutive weeks before allogenic skin transplant. Skin transplants were performed on day 0 to observe graft survival. Pathological examination of skin grafts was conducted 7 days post transplantation. The skin grafts were subjected to mRNA sequencing. Bioinformatics analysis was conducted and the expression of hub genes was verified by qPCR. Flow cytometry analysis was performed to evaluate the immune status and validate the results from bioinformatic analysis.ResultsThe mean survival time (MST) of mouse skin grafts in the SEA-treated group was 11.67 ± 0.69 days, while that of the control group was 8.00 ± 0.36 days. Pathological analysis showed that Sj SEA treatment led to reduced inflammatory infiltration within skin grafts 7 days after allogenic skin transplantation. Bioinformatics analysis identified 86 DEGs between the Sj SEA treatment group and the control group, including 39 upregulated genes and 47 downregulated genes. Further analysis revealed that Sj SEA mediated regulation on cellular response to interferon-γ, activation of IL-17 signaling and chemokine signaling pathways, as well as cytokine–cytokine receptor interaction. Flow cytometry analysis showed that SEA treatment led to higher percentages of CD4+IL-4+ T cells and CD4+Foxp3+ T cells and decreased CD4+IFN-γ+ T cells in skin transplantation.Conclusion Sj SEA treatment suppressed rejection and prolonged skin graft survival by regulating immune responses. Sj SEA treatment might be a potential new therapeutic strategy to facilitate anti-rejection therapy and even to induce tolerance.

PMA induces the differentiation of monocytes into immunosuppressive MDSCs.

The induction of immune tolerance without the use of immunosuppressive drugs is a crucial problem in organ transplantation. The use of myeloid-derived suppressor cells (MDSCs) as a cell-based adjuvant immunosuppressive therapy is a bright clinical prospect in organ transplantation. MDSCs with stable immunosuppressive activities can be used to treat immune-related diseases. In this study, macrophage colony-stimulating factor (M-CSF) was used to promote myeloid progenitor cell differentiation, and phorbol 12-myristate 13-acetate (PMA) was added to induce MDSCs at the later stage of induction in vitro. Cell phenotypes were detected by flow cytometry and mRNA was detected by real-time-polymerase chain reaction (RT-PCR). A mouse skin transplantation model was used to investigate the cell inhibitory function. The combination of PMA and M-CSF induced the differentiation of myeloid-derived monocytes into MDSCs. MDSCs were found to induce immune tolerance by inhibiting the proliferation and activation of T cells, promoting cytokine secretion and inducing T cell transformation to regulatory T cells (Treg ). PMA significantly up-regulated the expression of Arg-1 and the Arg-1 protein expression in MDSCs and arginase 1 (Arg-1) inhibitor nor-NOHA reversed the MDSC immunosuppressive activity, indicating the involvement of the Arg-1 pathway in MDSC-mediated immunosuppression. M-CSF+PMA-induced MDSCs also significantly prolonged the survival time of skin grafts in mice, showing that MDSCs exert immunosuppressive effects in vivo. We describe a novel scheme to induce immunosuppressive MDSCs in vitro. MDSCs induced by M-CSF with PMA showed stable immunosuppression. MDSCs induced by this protocol may benefit patients with organ transplantation through immune regulation.

Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice

Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice

Netrin-1 prolongs skin graft survival by inducing the transformation of mesenchymal stem cells from pro-rejection to immune-tolerant phenotype.

Mesenchymal stem cells (MSCs) induce allograft immune tolerance, but low efficacy severely limits their wide application. In this work, Netrin-1 was used to maintain MSC function in an IR environment to study its role in the immune tolerance induction of the allograft. The experiments were divided into three groups: the control group, the IR group and the Netrin-1 group (Netrin-1 was added to MSC medium and then cultured for 48 h). After digestion, MSCs were mixed with TLR4 and TLR3 antibodies (BD), incubated for 20 min, and washed with Phosphate-Buffered Saline (PBS) three times. The mean fluorescence intensity (MFI) of TLR4 and TLR3 was detected by flow cytometry. Isolated lymphocytes were divided into four groups: the control group (no treatment), the MSC group (lymphocytes were co-cultured with MSCs in the control group), the rejection group (lymphocytes were co-cultured with MSCs in the IR group), and the Netrin-1 group (MSCs in the IR group) was stimulated by Netrin-1 for 48h. Our study found that compared with control mice, toll-like receptor (TLR3) expression in bone marrow MSCs decreased as the expression of TLR4 increased, the secretion of transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) was reduced, while the secretion of IL-6 significantly increased in immune rejection (IR) mice. MSCs in IR mice promoted T-cell proliferation and reduced the ratio of Treg cells. Netrin-1 inhibited the pro-rejection effect of these MSCs, further inhibited T-cell proliferation and facilitated an increase in the ratio of Treg cells. The animal experiment results showed that MSC transplantation in the rejection group would shorten the mean survival time of the skin graft and induce the infiltration of lymphocytes. Netrin-1 prolonged the mean survival time of the skin graft by enhancing MSC function. The immunohistochemistry results showed that, compared with the rejection group, the T cell number in the skin graft significantly decreased in the Netrin-1 group. MSC can be divided into immune-tolerant and pro-rejection types in organ transplantation and Netrin-1 can induce the transformation of MSC from the pro-rejection to immune-tolerant type and markedly prolong the skin graft survival time.

Role of regulatory T cells in CD47/donor-specific transfusion-induced immune tolerance in skin-heart transplantation mice.

To explore the role of regulatory T (Treg ) cells in the establishment of immune tolerance induced by donor-specific transfusion (DST) in mice with skin-heart transplantation. C57BL/6 mice received DST of splenocytes from CD47+/+ or CD47-/- H-2bm1 mice or no DST 7days before skin-heart transplantation from major histocompatibility complex class I-mismatched H-2bm1 donors. The number and proportion of Treg cells in graft and lymphoid organs were measured by flow cytometry (FACS) and immunohistochemistry (IHC). The inhibitory function of Treg cells and anti-donor T-cell responses were assessed by mixed lymphocyte reaction. We observed that mean survival time (MST) of skin or heart graft was significantly longer in C57BL/6 mice which received DST from CD47+/+ H-2bm1 mice than from CD47-/- H-2bm1 mice. By FACS, we found that the number of Treg cells in spleen was increased significantly in mice which received CD47-/- DST compared to mice which received CD47+/+ DST. However, the percentages of Treg cells in total splenocytes and lymph node cells were significantly higher in mice that received CD47+/+ DST than mice which received CD47-/- DST. Immunohistochemistry showed an increased heart grafts infiltration of Treg cells in the recipients with CD47-/- DST, but not CD47+/+ DST. Supporting this, we found that donor T-cell proliferation was significantly suppressed in mice which received CD47+/+ DST compared to mice which received CD47-/- DST. There was no difference of inhibitory function of Treg cells between these two groups. Our results indicated that CD47 expression on DST cells plays an important role in the induction of immune tolerance in mice with skin-heart transplantation. Increased percentage of Treg cells may contribute to immune tolerance induced by CD47+/+ DST.

Transforming Growth Factor-β Partially Reversed the Immunosuppressive Effect of Mesenchymal Stem Cells in Mice

BackgroundMesenchymal stem cells (MSCs) possess powerful immunosuppression capacity. Transforming growth factor-β (TGF-β) is a well-known anti-inflammatory cytokine and plays an important role in various inflammatory processes. We hypothesized that TGF-β could synergize with MSCs in suppressing immune responses, and therefore established a mouse skin graft model to evaluate the effect of MSCs and MSCs combined with TGF-β on transplantation immunity in vivo. MethodsBalb/c and C57BL/6 mice were used to establish the skin graft model. The recipients were divided into 3 groups and received intravenous bone marrow mesenchymal stem cells (BMSCs), BMSCs pretreated with TGF-β, and 0.9% saline solution, respectively. Skin graft survival time, pathological detection, the ratio of CD4+CD25+Foxp3+Treg cell of spleens, and the level of IFN-γ, IL-2, IL-10, and TGF-β expression were tested. ResultsThe survival time of skin grafts were prolonged in both BMSC (12.5 ± 1.35 days) and BMSC-TGF-β (10.6 ± 1.90 days) recipients compared to the blank control recipients (8.0 ± 1.05 days). The ratio of CD4+CD25+Foxp3+Treg cell of spleens from BMSC and BMSC-TGF-β recipients was higher than that of the blank control, and the upregulated proliferation in the BMSC group occurred earlier and was prolonged compared to the BMSC-TGF-β group. The expression of IFN-γ and IL-2 was inhibited in both the BMSC and BMSC-TGF-β groups compared to the blank, while the expression of IL-10 and TGF-β was boosted. In contrast to the BMSC group, the BMSC-TGF-β group exhibited a weaker effect on the expression of cytokines. ConclusionTGF-β partially reversed the immunosuppressive effect of MSCs in vivo. This immunoregulatory feature may have potential applications for treating transplant rejection.

Interleukin 17 (IL-17)-Induced Mesenchymal Stem Cells Prolong the Survival of Allogeneic Skin Grafts.

BackgroundMesenchymal stem cells (MSCs) have the potential of self-renewal and multi-differentiation and have a wide application prospect in organ transplantation for the effect of inducing immune tolerance. It has found that interleukin 17 (IL-17) could enhance the inhibition effect of MSCs on T cell proliferation and increase the immunosuppressive effect of MSCs. In this study, we aimed to investigate the effect of IL-17-induced MSCs on allograft survival time after transplantation.Material/MethodsBMSCs were characterized by differential staining. The allogenic skin transplantations were performed and the BMSCs pre-treated by IL-17 were injected. To assess the immunosuppressive function of IL-17-induced BMSCs, the morphology of the grafts, the homing ability of the BMSCs, and the survival time of the grafts were analyzed.ResultsBMSCs from BALB/c have multidirectional differentiation potential to differentiate into osteogenic, chondrogenic, and adipogenic lineage cells. IL-17-induced BMSCs prolonged the survival time of allogeneic skin grafts dramatically. We found that there were more labeled MSCs in the skin grafts, and the Treg subpopulations percentage, IL-10, and TGF-β were significantly increased, while the IFN-γ level was decreased compared to the control group and MSCs group. In conclusion, IL-17 can enhance the homing ability of MSCs and regulate the immunosuppressive function of MSC.ConclusionsOur data demonstrate that IL-17 plays the crucial role in MSC homing behaviors and promotes immunosuppression of MSCs during transplantation procedures, suggesting that IL-17-pre-treated MSCs have potential to prolong graft survival and reduce transplant rejection.

Immune Tolerance Induced by Decitabine Combined With Multiple Allogeneic Bone Marrow Mononuclear Cell Infusion

ObjectiveThe aim of our study was to investigate the strategy of immune tolerance induced by the demethylating drug decitabine (DAC) combined with multiple allogeneic bone marrow mononuclear cell (BMMNC) infusions. MethodsAn animal model with C57BL/6 mice as recipients and a BALB/c mouse as the donor was established. C57BL/6 mice were randomly divided into the following groups: DAC, BMMNC, DAC+BMMNC, and the control groups. Mice in the DAC and DAC+BMMNC groups were given DAC (5 μg/kg) on days 1, 2, 7, 8, 13, and 14. On experimental days 3, 9, and 15, BMMNCs (5 × 106/mouse/time) from BALB/c mice were infused into mice of the BMMNC and DAC+BMMNC groups. One-way mixed lymphocyte reaction (MLR), interleukin-2 reverse test, and in vitro adoptive transfer experiments were performed. The ratio of regulatory T cells in splenocytes, chimera detection, and median survival time of skin grafts were recorded. ResultsMLR showed that the stimulation index of the DAC+BMMNC group was significantly lower compared with the other 3 groups (P < .01), but still showed a strong proliferative response to the third-party unrelated donor KM mice, with no significant difference compared with the other 3 groups. The MLR low reactivity of the DAC+BMMNC group could be reversed by exogenous interleukin-2. After adding an equal amount of splenocytes from a C57BL/6 mouse in the DAC+BMMNC group, MLR of control group significantly decreased (P < .05). Flow cytometry showed the ratio of regulatory T cells in splenocytes from DAC and DAC+BMMNC groups was significantly higher than that from the BMMNC and control groups (P < .05). Chimeric rate of the DAC+BMMNC (60%) group was significantly higher than that of the BMMNC group (30%). Moreover, survival time of skin grafts in the DAC+BMMNC group was significantly longer than that in the other 3 groups (P < .001). No death or graft-vs-host disease was observed. ConclusionDAC combined with multiple allogeneic BMMNC infusion could successfully induce specific immune tolerance in mice, which may provide some new strategies to improve immune tolerance after organ transplant.

Mesenchymal stem cells increase skin graft survival time and up-regulate PD-L1 expression in splenocytes of mice

Recently, mesenchymal stem cells (MSCs) have gained considerable interests as hopeful therapeutic cells in transplantation due to their immunoregulatory functions. But exact mechanisms underlying MSCs immunoregulatory function is not fully understood. Herein, in addition to investigate the ability of MSCs to prolong graft survival time, the effects of them on the expression of PD-L1 and IDO immunomodulatory molecules in splenocytes of skin graft recipient mice was clarified. To achieve this goal, full-thickness skins were transplanted from C57BL/6 to BALB/c mice. MSCs were isolated from bone marrow of BALB/c mice and injected to the recipient mice. Skin graft survival was monitored daily to determine graft rejection time. On days 2, 5 and 10 post skin transplantation, serum cytokine levels and expression of PD-L1 and IDO mRNA and protein in the splenocytes of recipient mice were evaluated. The results showed that administration of MSCs prolonged skin graft survival time from 11 to 14 days. On days 2 and 5 post transplantation, splenocytes PD-L1 expression and IL-10 serum level in MSCs treated mice were higher than those in the controls, while IL-2 and IFN-γ levels were lower. Rejection in MSCs treated mice was accompanied by an increase in IL-2 and IFN-γ, and decrease in PD-L1 expression and IL-10 level. No difference in the expression of IDO between MSCs treated mice and controls was observed. In conclusion, we found that one of the mechanisms underlying MSCs immunomodulatory function could be up-regulating PD-L1 expression.

The Effect of Traditional Chinese Formula Danchaiheji on the Differentiation of Regulatory Dendritic Cells.

Recently, regulatory dendritic cells (DCregs), a newly described dendritic cell subset with potent immunomodulatory function, have attracted increased attention for their utility in treating immune response-related diseases, such as graft-versus-host disease, hypersensitivity, and autoimmune diseases. Danchaiheji (DCHJ) is a traditional Chinese formula that has been used for many years in the clinic. However, whether DCHJ can program dendritic cells towards a regulatory phenotype and the underlying mechanism behind this process remain unknown. Herein, we investigate the effects of traditional Chinese DCHJ on DCregs differentiation and a mouse model of skin transplantation. The current study demonstrates that DCHJ can induce dendritic cells to differentiate into DCregs, which are represented by high CD11b and low CD86 and HLA-DR expression as well as the secretion of IL-10 and TGF-β. In addition, DCHJ inhibited DC migration and T cell proliferation, which correlated with increased IDO expression. Furthermore, DCHJ significantly prolonged skin graft survival time in a mouse model of skin transplantation without any liver or kidney toxicity. The traditional Chinese formula DCHJ has the potential to be a potent immunosuppressive agent with high efficiency and nontoxicity.

Microvascular integrity plays an important role for graft survival after experimental skin transplantation

BackgroundEvery transplanted organ relies on a reliable and sound vascular system. Therefore, our study focused on the investigation if platelet inhibition alone or combined with mTOR-inhibition has a beneficial effect on the microvascular integrity in allogeneic murine skin grafts. MethodsSkin transplantation was performed from fully MHC-mismatched C57BL/6 (H-2b) donors to CBA/J (H-2k) recipient mice. Skin allograft recipients were assigned to several experimental groups and either treated with clopidogrel alone, everolimus alone or a combination of both. Graft survival was evaluated and transplants were harvested after 8days and analyzed for CD31 and C4d by immunohistochemistry. ResultsUntreated allografts showed a reduced amount of CD31 on postoperative day 8 as well as an increase in C4d compared to isografts. All treated animals showed a significant improvement regarding CD31 [1577.7±200.4 (clopidogrel)/1702.8±151.1 (clopidogrel+everolimus) vs. 479.7±184.2 (control), n=8, p<0.05] and C4d [420.9±70.9 (clopidogrel)/324.5±77.3 (clopidogrel+everolimus) vs. 772.4±159.7 (control), n=8, p<0.05]. In addition, skin grafts of animals treated with clopidogrel and everolimus survived significantly longer compared to untreated controls [19.2±4.2 d vs. 12.8±2.4 d, n=10, p<0.05]. ConclusionIn this study we could show that clopidogrel alone and in combination with everolimus substantially improved microvascular integrity and resulted in increased survival time of skin grafts.

Role of dentritic epidermal T lymphocytes in immune rejection of skin allograft in mice and its mechanism

To explore the role of dentritic epidermal T lymphocytes ( DETCs) in immune rejection of skin allograft in mice and its related mechanism. Methods (1) Full-thickness skin was harvested from back of one male wild type (WT) C57BL/6 mouse. Epithelial cells were isolated for detection of the expression of DETCs and their phenotype with flow cytometer. Another male WT C57BL/6 mouse was used to harvest full-thickness skin from the back. Epidermis was isolated for observation of the morphological characteristics of DETCs with immunofluorescence technology. (2) Four male green fluorescence protein (GFP)-marked C57BL/6 mice, 7 female WT C57BL/6 mice (group WT), and 7 female ybT lymphocytes 8 gene knock-out (GK) C57BL/6 mice (group GK) were used. Full-thickness skin in the size of 1.4 cm x 1.4 cm on the back of mice in groups WT and GK were excised, and the wounds were transplanted with full-thickness skin in the size of 1.2 cm x 1.2 cm obtained from male GFP-marked C57BL/6 mice. The survival time of skin grafts was affirmed with small animal in vivo imager and naked eyes and recorded. (3) Two male WT C57BL/6 mice were used to isolate epithelial cells. Cells were inoculated into 48-well plate and divided into activation group (A) and control group (C) according to the random number table, with 4 wells in each group. Cells in group A were treated with 10 pL concanavalin A in the concentration of 2 microg/mL for 24 hours, while those in group C with PBS in the same volume as that in group A. The expression of interferon y in DETCs was detected with flow cytometer. (4) Four male GFP-marked C57BL/6 mice were used as donors. Fourteen female WT C57BL/6 mice were used as receptors and divided into interferon gamma neutralizing group (IN) and control group (C) according to the random number table, with 7 mice in each group. The skin transplantation model of C57BL/6 male to C57BL/6 female was established as in part (2). Before surgery and 72 hours after, mice in group IN were intraperitoneally injected with 200 pL interferon y neutralizing antibody in the concentration of 1 mg/mL, and those in group C with normal saline in the same volume as that in group IN. The survival time of skin grafts was observed and recorded using the methods in part (2), and the result of group IN was compared with that of group GK in part (2). The survival curve of skin grafts was processed with Log-rank ( Mantel-Cox) test. Results (1) The positive expression rate of DETCs in epithelial cells of skin in mouse was 7.27%, and they were all CD3 cells. DETCs were found to be scattered in the epidermis of skin in mouse with dendritic morphology. (2) The survival time of skin grafts of mice in group GK was 22-35 d, obviously longer than that in group WT (12-16 d, y2 = 14. 10 , P < 0.001). (3) Expression of interferon gamma was detected in 22. 70% DETCs in group A, which was obviously higher than that in group C (0.51%). (4) The survival time of skin grafts of mice in group IN was 19-24 d, which was obviously longer than that in group C (12-16 d, chi 2 = 13.60, P < 0.001) but close to that in group GK as in part (2) (chi2 = 0.06, P = 0.810). Conclusions DETCs are involved in promotion of immune rejection of skin allograft probably by secretinf interferon gamma.

The roles of IL-2 and IL-10 enhance anti-CD45RBmAb immune inhibition in allograft skin.

As a new type of immune tolerance inducer, anti-CD45RB monoclonal antibodies (anti-CD45RBmAb) can prolong the graft survival time of animal organs or cell transplantation as well as induce stable immune tolerance. Both interleukin (IL)-2 and IL-10 have important roles in the induction and maintenance of immunological tolerance. However, whether these cytokines combined with anti-CD45RBmAb can promote immune tolerance is poorly understood. Therefore, we investigated the effect of IL-2 and IL-10 in vitro and in vivo on the tolerance induction by anti-CD45RBmAb. The changes of Treg and Th17 cells and Th1/Th2 cytokines in anti-CD45RBmAb induced prolongation of skin allograft survival in mice. The finding of a role for IL-2 is novel, of interest, IL-2 promoted anti-CD45RBmAb-induced CD4(+) T cell differentiation into Treg and Th2 cells and suppressed Th17 and Th1 cells. IL-2 enhanced the induction of immune tolerance by anti-CD45RBmAb and significantly prolonged skin graft survival time in vivo. In contrast, this effect should be demonstrated experimentally by neutralizing IL-2 and inhibition of the effect of anti-CD45RBmAb, and neutralizing IL-10 showed no effect for anti-CD45RBmAb-induced tolerance. These data reveal that IL-2 significantly enhances anti-CD45RBmAb-induced immune tolerance via up-regulated T regulatory (Treg) cells and the balance of Th1/Th2 shifts. Conversely, IL-10 showed no effect on anti-CD45RBmAb-induced tolerance.

SAT0187 Immunomodulatory effects of human umbilical cord wharton jelly-derived mesenchymal stem cells (HUCMS) on circulating T-cell subsets in patients with sjögren’s syndrome

BackgroundhUCMS are adult stem cells easy to retrieve in bulk. Their immunomodulatory and pro-differentiation properties has been demonstrated. Indeed they may suppress lymphocyte activation, thereby prolonging skin graft survival time....

Stro-1–Positive Human Mesenchymal Stem Cells Prolong Skin Graft Survival in Mice

BackgroundHuman mesenchymal stem cells (MSCs) and its stromal cell antigen 1 Stro-1 positive (Stro-1+) subgroup possess immunosuppressive properties. Our objective was to evaluate the in vivo inhibitory effect of MSCs and the Stro-1 subset. MethodsIsolated human MSCs from bone marrow-derived mononuclear cells of healthy adults, and Stro-1+ cells were cultured before sorting. Female C57BL/6 mice and female BALB/c mice were used as donors and recipients in an allogeneic skin graft model, respectively. The recipients were divided randomly into 4 groups: (1) The Stro-1+ MSCs group received 2 × 106 Stro-1+ MSCs injected into irradiated recipients before skin grafting. (2) The MSC group (2 × 106) injected into the irradiated recipient mice before skin grafting. (3) The irradiated control group just irradiated before skin grafting. (4) The syngenic control group included irradiated BALB/c mice that received skin from syngenic mice. The main data included skin graft survival times, histologic changes on hematoxylin and eosin (HE) staining and plasma transforming growth factor (TGF)-β concentrations in recipients measured by enzyme-linked immunosorbent assay (ELISA) before and after transplantation. ResultsThe skin graft survival time in the MSCs group (12.13 ± 3.34 days) was not significantly prolonged versus the irradiated controls (11.38 ± 1.01 days), but it was notably prolonged among the Stro-1+ MSCs group (30.68 ± 5.89 days) compared with the irradiated control and the MSCs groups, respectively. The histology of skin grafts among the stro-1+ group showed a clear structure. After grafting, plasma TGF-β concentrations were almost the same as before transplantation among the irradiated and the syngenic controls but significantly increased in the MSCs and Stro-1+ MSCs groups. ConclusionsStro-1+ MSCs induced greater prolongation of skin grafts in mice than unsorted MSCs; however, TGF-β expression did not contribute to this effect.

Chronic Transplantation Immunity in Newts: Temperature Susceptibility of an Effector Phase in Allo-Skin Graft Rejection

Urodele amphibians are unique due to their greatly reduced immune responsiveness compared to bony fishes, which show acute immune responsiveness. In newts, the mean survival time of allogenic skin grafts in the transplantation immunity was 48.8 ± 8.3 days at 25°C, suggesting that it occurs in a chronic manner. The graft rejection process was categorized into three stages: a latent stage with frequent blood circulation, or the immune induction phase; a vascular stoppage stage with dominant infiltrating cells of T cells; and a rejection stage showing the change of the dominant cells to monocytes/macrophages, probably as effector cells, tetntatively referred to as the immune effector phase. The immune induction phase is susceptible to the cyclophosphamide (CY) mitosis inhibitor, but not to a temperature shift from 18 to 27°C, while the immune effector phase is susceptible to temperature shifts, but not CY-treatment, although the temperature shift failed to shorten the graft survival time to less than 25 days, which nearly equals that of the secondary set of grafts where the lack of complete blood circulation is remarkable and graft rejection is resistant to CY-treatment. In contrast, a very low temperature (5-10°C) completely prevented effector generation in newts; in frogs, however, it is reported that such low temperatures did not prevent the generation of effectors. Taken together, these data suggest that chronic responses in newts are due to effector cells other than cytotoxic T cells; possible effector cells are discussed.

Study of immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in allogenic transplantation

To study the immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) in the allogeneic transplantation. Forty female C57BL/6 mice and forty male BALB/C mice were respectively used as donors and recipients in skin allogenic graft model. Forty male BALB/C mice were divided randomly into 4 groups: blank control group, CP group, BMSCs group , CP + BMSCs group, with 10 mice in each group. Before skin graft, high-dose abdominal injection of cyclophosphamide (200 mg/kg, 2 d, q. d.) was performed in recipient mice in CP and CP + BMSCs groups. On the transplantation day, a bonus of 2 x 10(6) BMSCs from the SD rat (SD-BMSCs) were injected through the tail vein in the BMSCs and CP + BMSCs groups. The observation and HE staining of skin grafts were used. The expressions of CD29, CD34, CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify BMSCs. The CD4+, CD25+, Foxp3 and Treg cells of spleen were detected by flow cytometry. Cytokine in peripheral blood of recipient mice were measured by ELISA, including TGF-beta, IL-10 and IFN-gamma. T cells were co-cultured with 60Co-irradiated bone marrow MSCs from different individuals. The proliferative activity of T cells were evaluated with MTT assay. The skin graft survival time was significantly prolonged in the CP + BMSCs group, as compared with that in the blank control group, the CP group, the BMSCs group, respectively. Cells cultured by whole bone marrow adherent cultivation showed CD29 (99.7%), CD44+ (96.7%), CD34- (1.6%), CD45- (1.3%). Compared with the control group and CP group, the ratio of the CD4+, CD25+, Foxp3+ and Treg cells significantly increased in the SD-BMSCs group and CP + BMSCs group (P < 0.05). Analysis of peripheral blood by ELISA showed significant high level of TGF-beta, IL-10 and low level of IFN-gamma in BMSCs group and CP group,compared with that in control group. When co-cultured with BMSCs from different individuals, T- lymphocytes proliferation decreased apparently in SD-BMSCs group and C57-BMSCs group (P < 0.05), but there was no significant difference between SD-BMSCs group and C57-BMSCs group (P > 0.05). The immunotolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in the allogeneic transplantation might be associated with its effect on the proliferation of Treg cells and increasing expression of TGF-beta and IL-10, decreasing expression of IFN-gamma.

The inhibitory effect of decorin on lymphocyte proliferation and immunological rejection in skin transplantation

Objective To study the inhibitory effect of decorin (DCN) on immunological rejection in skin transplantation.Methods Spleen lymphocytes were obtained from C57BL/6J mice,inoculated in a 96-well plate,and every well was added with 25 μg CoA.These cells were divided into 5 groups and cultured with DCN in different concentrations (0,25,50,75,100 μg/L) for different time (24,48,72,96 and 120 h).Negative control group (lymphocytes only) and blank control group (culture medium only)were set up.Cell proliferation rate was measured by methylthiazol tetrazolium (MTT) assay,and the percentage of cell apoptosis was analyzed by flow cytometry.Twenty-four healthy BALB/C mice served as donors.Thirty C57BL/6J mice served as recipients and divided into 5 groups.Full-thickness-skin was transplanted through back to back from donor BALB/C to recipient C57BL/6J.Postoperatively,the recipients in group A (negative control) were intraperitoneally injected with 0.9% NaCl,and those in groups B,C and D with DCN (50,100 and 150 μg/L respectively).In group F skin transplantation was performed between two C57BL/6J mice.After skin transplantation,the survival time of skin allografts and the time of acute rejection were observed.Skin specimens were collected from one recipient of every group 7 days after operation and examined pathologically.Results DCN obviously depressed mice spleen lymphocyte proliferation in vitro and inducef lymphocyte apoptosis in vitro.In 100 μg/L DCN group ( group C ),there were most significant inhibitory effects,and the apoptosis rate was 77.6%.There were significant difference between group C and other groups ( P＜0.05 ).DCN could inhibit immunological rejection of skin transplantation and prolong the survival time of skin allografts.Among all the groups,survival time in group D was longest with the average survival time being ( 16.6 ±2.4) days ( P ＜0.05 ).The inhibitory effect of DCN was increased and the acute rejection was alleviated with the increase of DCN dosage.Conclusion DCN has the inhibitory effect.It can induce lymphocyte apoptosis and inhibit acute immunological rejection in skin transplantation.It can prolong the survival time of skin grafts. Key words: Skin transplantation; Decorin; Apoptosis; Immunological rejection