Skin Graft Survival Research Articles

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.

Signaling through C5a receptor and C3a receptor diminishes function of murine natural regulatory T cells

Thymus-derived (natural) CD4(+) FoxP3(+) regulatory T cells (nT reg cells) are required for immune homeostasis and self-tolerance, but must be stringently controlled to permit expansion of protective immunity. Previous findings linking signals transmitted through T cell-expressed C5a receptor (C5aR) and C3a receptor (C3aR) to activation, differentiation, and expansion of conventional CD4(+)CD25(-) T cells (T conv cells), raised the possibility that C3aR/C5aR signaling on nT reg cells could physiologically modulate nT reg cell function and thereby further impact the induced strength of T cell immune responses. In this study, we demonstrate that nT reg cells express C3aR and C5aR, and that signaling through these receptors inhibits nT reg cell function. Genetic and pharmacological blockade of C3aR/C5aR signal transduction in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival. Mechanisms involve C3a/C5a-induced phosphorylation of AKT and, as a consequence, phosphorylation of the transcription factor Foxo1, which results in lowered nT reg cell Foxp3 expression. The documentation that C3a/C3aR and C5a/C5aR modulate nT reg cell function via controlling Foxp3 expression suggests targeting this pathway could be exploited to manipulate pathogenic or protective T cell responses.

Skin graft survival on subcutaneous hinge flaps: An algorithm for nasal reconstruction

To assess the survival of full-thickness skin grafts and perichondrial cutaneous grafts when placed on subcutaneous soft tissue flaps used in nasal reconstruction. Retrospective case series from a secondary-care cutaneous cancer practice. Twenty-eight patients with nasal defects secondary to basal cell carcinoma excision were included. Clinical information, including case notes and photography, was obtained and analyzed. Graft survival was assessed in relation to type of graft and subcutaneous soft tissue flap employed for the reconstruction. Overall graft survival was 79%, with 89% and 74% for perichondrial cutaneous graft and full-thickness skin grafts, respectively. Anecdotally, procerus and nasalis flaps were found to yield higher graft survival than cheek fat flaps. The combination of subcutaneous soft tissue flap and skin graft cover offers a valuable addition to the treatment algorithm for nasal reconstruction following cutaneous malignancy excision.

The Effect of Atorvastatin (Lipitor) on the Duration of Survival of Allogeneic Skin Graft and the Growth of B16F10 Melanoma Cells in Mice

Aims: To evaluate the immunomodulatory effect of using non-cholesterol lowering dose of atorvastatin (AS) on skin allograft survival and on tumor growth in mice. Study Design: Experimental Study. Place and Duration of Study: Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut; 2011-2012. Methodology: BALB/c mice were transplanted with skin allografts from C57BL/6 mice and given either AS alone or in combination with immunosuppressive agents. Average survival days of skin allografts were recorded and serum levels of interleukin-1β (IL-1β) and interferon-γ (IFN-γ) were quantified. BALB/c mice and C57BL/6 mice were challenged intraperitoneally with B16F10 melanoma cancer cells (cancer cell line syngeneic to C57BL/6 mice) and were then treated with AS. They were observed Research Article British Journal of Medicine & Medical Research, 3(4): 1938-1951, 2013 1939 regularly for tumor growth. Results: The results indicated that in transplant mice AS given alone or in combination with immunosuppressive agents prolonged allograft survival time through noncholesterol lowering mechanisms in spite of a non-significant change in serum cytokine levels. Furthermore, AS treatment enhanced tumor growth in C57BL/6 mice and promoted tumor growth in BALB/C mice. Conclusion: It can be speculated that AS down expresses TLR and modifies MHC presentation resulting in hindering the generation of an innate and adaptive immune response.

Blockade of T-Cell Homeostasis by Anti-Interleukin-7 Receptor Monoclonal Antibody Allows Indefinite Islet and Long-Term Skin Allograft Survival

Background: T cell depletion is followed by T cell homeostasis with increase in memory T cells which are more potent in mediating rejection. We demonstrate that IL-7R blockade with or without prior T cell depletion inhibits T cell homeostasis and prolongs graft survival. Methods: Tail skin from C57BL/6 was transplanted to Balb/c mice with 3 treatment protocols: protocol 1 (no treatment, n=10), 2 (T cell depletion by combining 2 depleting mAbs: anti-CD4 and anti-CD8 on day -3 and -1, n=10), and 3 (same as protocol 2, followed by a blocking anti-IL-7Rα mAb qod, n=11). Islets from C57BL/6 were transplanted to streptozotocin-induced diabetic Balb/c mice, divided into 3 groups: group A (no treatment, n=10), B (anti-IL-7Rα mAb qod from day 0 to rejection, n=5), and C (anti-IL-7Rα mAb qod from day -21 to PTD90, n=6). Results: Median skin graft survival with protocol 1, 2, and 3 was 9.5, 30, and 58 days, respectively (protocol 3 vs 2, p< 0.0001). IL-7R blockade following T cell depletion profoundly inhibited lymphocyte reconstitution. At PTD35, the mean absolute numbers of CD3+, CD3+CD4+, CD3+CD8+, and CD19+ cells in the spleen were 6.7, 4.1, 0.8, and 119 in protocol 3 vs 57.5, 43.6, 7.3, and 276 ×105 cells in protocol 2, respectively (all p< 0.05). Importantly, the mean absolute number of CD44hiCD62Llomemory T cells was 4-fold lower in protocol 3 vs protocol 2: 3.8 vs 14.3 ×105 cells, and the percentage of CD4+CD25+FOXP3+ Treg to total CD4+ T cells was slightly increased: 13 vs 10% (all p< 0.05). Similar results were found in the lymph node and in the blood. Moreover, thymic cellularity was markedly reduced in protocol 3 vs 2: 0.7 vs 142 ×105 cells, p< 0.05. IL-7R blockade abrogated cellular immune responses as shown by direct IFNγ ELISPOT (41.3 vs 193.5 spots/105 T cells) and by MLR-3H thymidine (238 vs 10510 CPM) and inhibited humoral immune responses as shown by a reduction in DSA (310 vs 996 MFI) (protocol 3 vs 2, all p< 0.05). Histology revealed abundant subcutaneous and dermal leukocyte infiltrates at PTD21 in protocol 2, whereas few infiltrates were seen in protocol 3 at PTD21 and PTD35. The addition of low-dose tacrolimus 0.5 mg/kg/d to protocol 2 did not improve median graft survival (32.5 days, n=6, p=NS), whereas the same dose of tacrolimus added to protocol 3 further prolonged skin graft survival to >90 days in 4 of 6 recipients (p< 0.05). Median islet graft survival was not significantly prolonged when anti-IL-7Rα was given from day 0 (group B vs A: 29 vs 21 days, p=NS). However, when treatment was started 21 days before islet graft, 5 of 6 recipients had indefinite graft survival >180 days (group C vs A, p< 0.005), associated with an abrogation of DSA (group C vs A: 260 vs 976 MFI, p< 0.01). Conclusion: IL-7R blockade following with T cell depletion is a potent immunosuppressive regimen which abrogates both cellular and humoral allo-immune responses and may be used synergistically with low-dose tacrolimus to avoid nephrotoxity.

Skin Graft Survival in Genetically Identical Bone Marrow Mesenchymal Stem Cells Cloned Pigs with Different Mitochondrial Haplotypes

Skin Graft Survival in Genetically Identical Bone Marrow Mesenchymal Stem Cells Cloned Pigs with Different Mitochondrial Haplotypes

Prolonged Survival of Skin Graft by Dendritic Cells Derived Donor-Specific ILT3 Lentivirus-Transduced Human CD34+ Cells in hu-NOD/SCID Mice

Prolonged Survival of Skin Graft by Dendritic Cells Derived Donor-Specific ILT3 Lentivirus-Transduced Human CD34+ Cells in hu-NOD/SCID Mice

T2 B Cells Are Expanded During Experimental Tolerance Protocol and Contribute to Skin Graft Survival

We have recently reported that, in humans, tolerance to organ transplantation is associated with alterations in the transcription of B cell related genes and changes in the phenotypes of the circulating B cells, suggesting a “B cell signature” for transplant tolerance. Whether these B cells have a role in maintaining tolerance in humans is as yet unclear. Here we use an experimental mouse model of tolerance to investigate the contribution of B cells to the maintenance of tolerance. We demonstrate that B cells are required for long-term tolerance to MHC I mismatched skin grafts and that adoptive transfer of B cells, but not non-B cells, from tolerised mice can prolong skin allograft survival in naïve transplant recipients. Further, as in humans, tolerance is associated with an increase in Transitional-2 (T2) B cells, a subset previously reported to have regulatory capacity. T2 B cells from tolerised mice, but not from naïve animals, are capable of prolonging skin allograft survival following adoptive transfer to naïve recipients and suppressing T cell activation in vitro. The mechanisms involved in the regulatory function of T2 B cells will be discussed. These results suggest that the increase in T2 B cells in tolerance represents an increase in regulatory B cells that actively contribute to the maintenance of tolerance.

Impact of Interleukin-2–expanded Regulatory T Cells in Various Allogeneic Combinations on Mouse Skin Graft Survival

The impact of in vivo regulatory T cells (Treg) expansion using short-term injections of interleukin-2 (IL-2) coupled to a specific anti–IL-2 antibody was examined in various allogeneic combinations of murine skin transplantations. In a model of a single major histocompatibility complex (MHC) class II disparity, the IL-2–expanded Tregs infiltrated the transplanted skin, inhibited Th1 alloreactivity, and prevented acute graft rejection. However, in the presence of increased load of CD4-recognized alloantigens, exogenous IL-2 only moderately prolonged graft survival as attested by CD8 T cell-depletion in full minor plus major mismatched recipients treated with IL-2. If direct CD8 alloreactivity remained intact, the IL-2/anti-IL-2–mediated Tregs expansion failed to delay allograft rejection. This observation was confirmed by the inability of expanded Tregs to delay rejection of multiple minor disparate (MHC matched) skin allografts. Altogether, these results warn that cross-reactive CD8+ T cells represent an important hurdle to Treg-based tolerance induction.

DNAM-1 - a New Player in Renal Allograft Rejection

Despite effective treatment protocols to prevent rejection many renal allografts are lost due to the toxicity of immunosuppressants. Thus, more specific and less toxic immunosuppression is needed. DNAM-1 (CD226) on T cells has been shown to play an important role for allogeneic graft-versus-host responses. DNAM-1 has two ligands: the adhesion molecules CD155 and CD112. Both are expressed on dendritic, epithelial, and endothelial cells. The importance of DNAM-1and its ligands for renal allograft rejection is unknown. Primary cultures of murine renal tubular epithelial cells (rTECs) were prestimulated with IFN-β and IFN-γ to induce high surface expression of MHC. Surface expression of CD155 and CD112 was tested by FACS. Responder T cells were stimulated in vitro with allogeneic fully MHC-mismatched splenocytes. From these stimulation cocultures we measured proliferation (thymidine incorporation), cytokine production (ELISA) and cytotoxicity against IFN-stimulated rTECs from WT or CD155−/− mice (51Cr-release-assay). To test for a role of this pathway in vivo, we performed fully MHC-mismatched skin and kidney grafts from WT and CD155−/− donors. CD155 and CD112 were both highly expressed on rTECs and could be further increased by IFN-stimulation. Also, in acutely rejected murine renal allografts an upregulation of both molecules was detected. However, when CD155 was missing on targets in an allospecific chromium-release-assay, no difference in cytotoxicity was measured compared to WT targets. Also, allospecific T cell proliferation and IFN-γ secretion were not altered, when stimulator cells lacked CD155. When testing for the role of this pathway for allograft rejection in vivo, no difference between skin graft survival of WT and CD155−/−was observed. Renal allografts from CD155−/− donors show similar amounts of infiltrates and interestingly higher incidence of infarcts compared to WT donors. The reason for this is under investigation. In contrast to these results, allospecific T cell proliferation was be reduced with the use of a blocking antibody against DNAM-1. Also allospecific cytotoxicity was reduced, when DNAM-1 was blocked during stimulation. Experiments blocking DNAM1 or CD112 in vivo are ongoing. Thus, DNAM-1 is an important costimulatory receptor for alloreactive T cells. However, the important ligand might be CD112 rather than CD155. Thus, blocking DNAM-1 or CD112 might be a therapeutically interesting option to prevent renal allograft rejection.

The aryl hydrocarbon receptor influences transplant outcomes in response to environmental signals

The aryl hydrocarbon receptor (AHR) is a cytosolic transcription factor with numerous endogenous and xenobiotic ligands, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent data suggest that TCDD may induce regulatory T cells, while a second AHR ligand, FICZ, promotes Th17 differentiation. The aim was to examine whether the injection of recipient mice with either TCDD or FICZ altered skin allograft rejection in a fully mismatched model. TCDD or FICZ was given to recipient C57BL/6 mice intraperitoneally (IP). Twenty-four hours later, donor skin was grafted from BALB/c mice. An additional dose of FICZ was given on day 3. Treatment with TCDD delayed graft rejection for more than 4 weeks while FICZ treatment accelerated the rejection by 1–2 days. In vivo exposure with TCDD led to a rise in the frequency of FoxP3+ CD4+ T cells in the spleen, while FICZ increased IL-17 secretion by splenocytes from the treated animals. Activation of the AHR receptor by different AHR ligands in vivo resulted in opposing the effects on skin graft survival. AHR serves as a sensor to environmental signals, with effects on the acquired immune system that may alter outcomes after organ transplantation. This model will be useful to further delineate direct effects of the environment on the immune system and outcomes of organ transplantation.

Transcutaneous Gene Gun Delivery of hNC16A Induces BPAG2-Specific Tolerance

Immune recognition and rejection of tissues expressing transfected genes is a major complication of gene replacement therapy for inherited genetic disorders. Owing to the high immunogenicity of human bullous pemphigoid antigen 2 (hBPAG2), the induction and maintenance of tolerance to this neo-antigen is essential to deliver the gene product to patients with epidermolysis bullosa junctionalis. In a skin grafting mouse model, we used gene gun transfection with a construct encoding hNC16A, the immunodominant domain of hBPAG2, to induce antigen-specific immune tolerance. Eighty percent of wild-type mice transfected with hNC16A showed long-term survival of skin grafts expressing hBPAG2. Tolerance was stable and transferable by T cells but not by B cells of tolerant mice to naive hosts. A dense Foxp3(+) regulatory T-cell (T(reg)) infiltrate was noticed in grafts of tolerant mice and depletion of these cells resulted in a loss of tolerance. Taken together, we show that long-lasting hBPAG2-specific tolerance was induced with gene gun delivery of hNC16A through a T(reg)-dependent mechanism. This is of relevance to patients undergoing gene therapy and has broader implications for the treatment of antigen-specific autoimmune diseases.

Targeting of the hNC16A collagen domain to dendritic cells induces tolerance to human type XVII collagen

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.

Retrospective study of negative pressure wound therapy promoting survival of the skin grafts

Objective To observe the effects of negative pressure wound therapy (NPWT) on survival of the blade-thickness free skin grafts. Methods Sixty-five patients with skin defects were divided into NPWT treatment group ( Group Ⅰ,n =35) and conventional treatment group ( Group Ⅱ,n =30) according to different postoperative fixation methods.The patients in Group Ⅰ were fixed with the aid of NPWT after blade-thickness free skin grafting,and the patients in Group Ⅱ were fixed with tie-over bolster dressing. Results The survival rate of the skin grafts of Group Ⅰ was significantly higher than that of Group Ⅱ at day5 postoperatively [ (80.59 ±10.30)％ vs (71.46 ±10.68)％,P＜0.05].The survival period for the skin grafts of Group Ⅰ was shorter than that of Group Ⅱ[ (5.34 ± 0.87) days vs ( 11.20 ± 1.65) days,P ＜ 0.01].The duration of postoperative hospital stay of Group Ⅰ was obviously shorter than that of Group Ⅱ (P ＜ 0.01 ).The cost of antibiotics of Group Ⅰ was less than that of Group Ⅱ [ ( 1 765.71 ± 164.39) RMB yuan vs (2 700.00 ±221.28) RMB yuan,P ＜0.01].The dressing frequency and cost of group Ⅰ were less than those of group Ⅱ [(3.11 ± 0.32) times,(249.14 ±25.82) RMB yuan vs (4.53 ±0.68) times,(362.67 ±54.52) RMB yuan,P＜0.01]. Conclusions The application of NPWT to the postoperative period of skin grafting can promote the survival of the skin grafts,shorten the duration of hospitalization and reduce antibiotics use and dressing frequency. Key words: Surgical flaps; Transplantation; Negative pressure wound therapy

Chimeric acceleration by donor CD4+CD25+T-reg depleted fraction in splenocyte transplantation

BackgroundWe have established a splenocytic chimera model that can induce donor-specific tolerance and reconstitute the recipient immune system by donor splenocytes. AimTo accelerate such reconstitution, we investigated the role of donor-derived CD4+CD25+ regulatory T-cells (T-reg). MethodsWe established C3H/B6D2F1 mixed bone marrow chimeras in lethally irradiated C3H mice. We transplanted skin grafts from C57BL/6 mice 30 d later. After an additional 30 d, we transplanted the following types of splenocytes from B6C3F1 mice: total splenocytes (group A), CD4+CD25+ T-reg depleted splenocytes (group B), CD8+-depleted splenocytes (group C), and CD4+-depleted splenocytes (group D). We assessed class I major histocompatibility complex, percentage of chimeric cells in peripheral blood, and survival of skin grafts in each group. ResultsGroup A and B mice switched to splenocytic chimeras, permitting the long-term survival of skin grafts. The proportions of H-2Kb+H-2Kk− cells in group B were significantly lower than those in group A on day 14 (0.47% ± 0.68% versus 9.49% ± 8.30%; P = .01) and day 21 (0.16% ± 0.25% versus 3.35% ± 2.78%; P = .01). The initial increase in the proportion of H-2Kb+H-2Kk+ double-positive cells in group B was faster than that in group A (from 0.33% ± 0.10% versus. 0.39% ± 0.14% before splenocyte injection to 39.03% ± 30.50% versus 10.73% ± 11.54% on day 7; P = .02). The initial increase in the proportion of CD8+ T-cells was faster in group B than in group A (from 2.72% ± 0.52% versus 2.49% ± 1.07% before splenocyte injection to 29.61% ± 26.72% versus 4.92% ± 1.56% on day 7; P = .04). ConclusionsThe depletion of CD4+CD25+ T-reg fraction in donor splenocytes can accelerate switching to splenocytic chimera.

Smoking and diabetes mellitus type 2 reduce skin graft take; the use of fibrin glue might restore graft take to optimal levels

Efficacy has been demonstrated in some uses of fibrin glue associated with graft loss. Smoking and hyperglycemia significantly decrease the success of skin graft survival in specific injuries. This retrospective study aimed to verify the association with decreased skin graft survival and whether fibrin glue is useful in reversing the influence of these factors. This bicentric, retrospective, cross sectional case control study was carried out on 1881 medical patients. Patients who met inclusion criteria were admitted to the Plastic Surgery Service of Reina Sofia University Hospital (Spain) and the Trauma/Burn intensive Care Unit of UAB Hospital at Birmingham (USA) between January 2000 and December 2009. The successful graft take for each group and its control were analyzed by a Chi-square test; the confidence interval was 95%. Smoking and DM type 2 decrease skin graft survival when compared with their control groups. There was a statistically significant improvement in skin graft take when fibrin glue was used. The percentage improvement in the control groups was approximately 10%, whereas in the study groups it was 2-3 times higher. We conclude that graft loss is associated with smoking and DM type 2, but fibrin glue might restore graft adherence to almost normal levels.

A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16–21 and ubiquitin 52–57 fragments, and second designed to mimic the ubiquitin 5–13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.

Improvement of skin-graft survival after autologous transplantation of adipose-derived stem cells in rats

Skin grafts are frequently used for a variety of indications in plastic and reconstructive surgery. Their necrosis is a common complication, while different therapies have been proposed. Currently, adipose-derived stem cells (ASCs) hold great promise for their angiogenic potential and role during tissue repair. In this study, autologous transplantation of ASCs was used in skin grafts in rats to determine if it increases angiogenesis, skin-graft survival and wound healing. ASCs were isolated, cultured, labelled with fluorescent dye and injected under full-thickness skin grafts in 10 rats (group 1), while 10 others served as controls (group 2). Skin grafts were analysed after 1 week. Collagen's framework was assessed with Masson's trichrome stain and angiogenesis with von Willebrand factor (vWF) immunohistochemistry. In addition, immunohistochemical staining intensity of vascular endothelial growth factor (VEGF) and transforming growth factor b3 (TGFb3) was assessed in all grafts. Mean area of graft necrosis was significantly less in group 1 than in group 2 (6.12% vs. 32.62%, p<0.01). Statistically significant increase of microvessel density, collagen density, VEGF and TGFb3 expression was noted in group 1 compared with group 2 (all: p<0.01). These findings suggest that autologous ASCs transplantation increases full-thickness skin-graft survival and shows promise for use in skin-graft surgery. This might be both due to in situ differentiation of ASCs into endothelial cells and increased secretion by ASCs of growth factors, such as VEGF and TGFb3 that enhance angiogenesis and wound healing.

Graft-infiltrating cells expressing a CD200 transgene prolong allogeneic skin graft survival in association with local increases in Foxp3+Treg and mast cells

Expression of the molecule CD200 has been reported to increase allograft survival by suppression of inflammation and acquired immunity. In previous studies we have shown that increased skin and cardiac allograft survival in transgenic mice over-expressing CD200 (CD200tg) occurs in association with increased intra-graft expression of mRNAs for genes associated with altered T cell subset differentiation.We investigated changes in graft-infiltrating cells, Treg and mast cells in skin grafts post transplantation into control or CD200tg mice, using focused gene array and real-time PCR to assess altered gene expression, and FACS, immunohistology and MLC to determine numbers/function of those cells. Graft-infiltrating cells isolated from CD200tg recipients suppressed induction of CTL from control lymph node cells in vitro, and contained increased numbers of infiltrating, non-degranulating, mast cells and Foxp3+Treg. Mast cells were also evident in graft tissue of control animals, but there these cells showed evidence for degranulation, and fewer Foxp3+Treg were present than was the case of CD200tg mice. The infusion of a competitive inhibitor of CD200:CD200R interactions, CD200tr, at high concentrations (50μg/mouse iv) caused rapid rejection of grafts in CD200tg mice, mast cell degranulation within graft tissue, and a decrease in Treg infiltrates. These effects were attenuated by simultaneous infusion of the mast cell stabilizer, sodium cromoglycate.We conclude that CD200 expression contributes to graft prolongation through local suppression of mast cell degranulation, attraction/expansion of Treg, and attenuation of T cell effector activation.

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.