Skin Grafts In Mice Research Articles

Presentation of Allogeneic MHC On Cross-Dressed Recipient APCs vs. Passenger Leukocytes After Skin Transplantation.

We studied the trafficking of donor cells to recipient’s lymphoid organs and its relationship to the direct T cell alloresponse in mice transplanted with a fully allogeneic skin graft. First, we investigated the presence of donor passenger leukocytes in different lymph nodes and the spleen of BALB/c mice transplanted with a B6-GFP or B6-MHC class II-GFP skin allograft, using intra-vital microscopy. Very few donor cells were detected (MHC class II+, n < 5) in draining ipsilateral lymph nodes and only 5 days after transplantation. This did not reflect a lack of sensitivity of our method, given that many more passenger leukocytes were detected in all lymph nodes and spleen, as early as 1 day after transplantation of vascularized solid organs. Alternatively, in skin-grafted mice, imaging flow cytometry (Amnis ImageStream X Mark II) analysis revealed the presence of many recipient cells (B cells and dendritic cells, n > 1,000) displaying donor MHC class I and class II MHC molecules. These “cross-dressed” cells were found in several hosts’ lymphoid organs, as early as 36 hours after transplantation. Next, kinetics studies of direct T cell alloresponses were performed using an ELISPOT assay. Significant numbers of activated donor-specific activated T cells producing gIFN were observed starting at 48 hours post-transplantation. Altogether, these results suggest that, at least at an early time point, presentation of donor MHC molecules by cross-dressed recipient APCs represents the driving force behind direct T cell alloreactivity in skin transplantation.

Cell Tracking By Gadolinium Labelling and Mass Spectrometry:-A Tool for Evaluating The Persistence of Cellular Therapies in Transplantation.

Background:- Cellular therapy with defined populations of regulatory immune cells is entering Phase I/II clinical trials in solid organ transplantation. Safety is an important primary endpoint but this is only relevant if the cells are shown to persist. In the majority of cases, the cellular therapy will be autologous and thus invisible to FACS, histochemical or PCR based assays. Transduction with reporter proteins is unlikely to meet regulatory approval and so tracking presents a significant challenge. Aim:- An identification method is required that is sensitive, applicable to T cells without impairing function and likely to be given approval by regulatory agencies. We evaluated Gadolinium chelates widely used in clinical MRI for their ability to label mouse and human CD4+ T cells. Detection was by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). Results:- Mouse and human CD4+ T cells take up Gd-chelates (Omniscan and Dotarem) in normal tissue culture. Uptake is passive, time and dose dependent and efficient. Incubation for 16hr gives ≥108 Gd atoms/cell with no effects on viability or mitogen-driven proliferation. In subsequent culture for 10 days cells retained ˜50% of label cf. time-0. Mixing experiments (labelled + unlabelled) followed by blinded analysis detected Gd+ cells at a ratio of 0.7:100 (actual ratio 1:100). To assess the impact of Gd labelling on regulatory function, labelled and unlabelled mouse nTreg were compared for their ability to control mouse skin graft rejection in a well characterised immunodeficient-reconstitution skin graft model. As shown in Table 1, Gd labelling did not impair Treg function.Table: No Caption available.Conclusion:- Gadolinium labelling followed by LA-ICP-MS appears to offer a feasible method for assessing the persistence of Treg cellular therapies. Although Gd-MRI has been associated with Nephrogenic Systemic Fibrosis, it is important to note that at nTreg doses predicted for current clinical trials, labelling at 108 Gd atoms per cell equates to a total Gd burden of ˜80-200ng/kg, some 100,000-fold lower than loads typically experienced in MRI.

Abstract 135: improved engraftment of autologous skin grafts in diabetic mice with adipose-derived stem cells.

Abstract 135: improved engraftment of autologous skin grafts in diabetic mice with adipose-derived stem cells.

Development of Antibody-Tagged Nanoparticles for Detection of Transplant Rejection Using Biomagnetic Sensors

Organ transplantation is a life-saving procedure and the preferred method of treatment for a growing number of disease states. The advent of new immunosuppressants and improved care has led to great advances in both patient and graft survival. However, acute T-cell-mediated graft rejection occurs in a significant quantity of recipients and remains a life-threatening condition. Acute rejection is associated with decrease in long-term graft survival, demonstrating a need to carefully monitor transplant patients. Current diagnostic criteria for transplant rejection rely on invasive tissue biopsies or relatively nonspecific clinical features. A noninvasive way is needed to detect, localize, and monitor transplant rejection. Capitalizing on advances in targeted contrast agents and magnetic-based detection technology, we developed anti-CD3 antibody-tagged nanoparticles. T cells were found to bind preferentially to antibody-tagged nanoparticles, as identified through light microscopy, transmission electron microscopy, and confocal microscopy. Using mouse skin graft models, we were also able to demonstrate in vivo vascular delivery of T-cell targeted nanoparticles. We conclude that targeting lymphocytes with magnetic nanoparticles is conducive to developing a novel, noninvasive strategy for identifying transplant rejection.

SAT0052 TNFγ adherent BM-DC efficiently inhibit collagen-induced- arthritis, and delayed skin graft rejection in mouse

BackgroundRheumatoid Arthritis (RA) is a chronic inflammatory disease, initiated by a break in self-tolerance. Tolerogenic dendritic cells (Tol-DC) are involved in the maintenance of tolerance to antigen, but are deficient...

Human endometrial mesenchymal stem cells induced FOXP3+ CD25+ double positive T cells and suppressed immunogenicity of mouse blood cells in the rats.

Presence of mesenchymal stem cells (MSC) were reported from the almost every body parts studies and are believed to be involved in the homeostasis of the organism. Due to this reason, MSCs were considered to be a prime candidate for the cell therapies to replace the damaged or degenerated, thereby, non-functional tissues. Recently, however, transplantation studies revealed that the duration of mouse skin graft to baboon was extended when cord blood MSCs were co-transplanted. To understand the spectrum of MSCs and the mechanism involved in the immunomodulation, several MSCs were isolated from the human endometrium (EnMSC). The established EnMSCs were able to suppress conconavalin A (con A)-stimulated proliferation of rat peripheral blood mononucleocytes (rPBMNC). A similar suppression of rPBMNC was observed when mitomycin C-exposed mouse PBMNC was used as antigen in the mixed lymphocyte reaction experiments. Flow cytometric analyses confirmed that the ratio of FoxP3+ CD25+ double positive cells were increased from 1.7% to 6.5% when EnMSCs were present at the expense of CD4+ FoxP3- T cells (from 22.9% to 8.9%), suggesting the induction of Treg cells and the suppression of Teff. Furthermore, the number of rats and the amount of anti-mouse IgG and IgM produced by the rats were drastically suppressed when EnMSC was co-transplanted with mitomycin C-exposed mouse PBMNC. RT-PCR confirmed the involvement of EnMSC in the immunosuppression: IL-1β being suppressed and IL-10 and TGF-β being induced. Taken together, human EnMSC, although extensive studies are needed, may be considered as a promising candidate to replace the toxic pharmacological immunosuppressants in the clinical transplantations.

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.

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.

Stevens-Johnson syndrome/toxic epidermal necrolysis mouse model generated by using PBMCs and the skin of patients

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening cutaneous reactions caused by drugs or infections and exhibiting widespread epidermal necrosis. Currently, there is no animal model that reproduces SJS/TEN symptoms. We sought to develop a novel mouse model of SJS/TEN by using PBMCs and skin from patients who had recovered from SJS/TEN. For our mouse model, patients' PBMCs were injected intravenously into immunocompromised NOD/Shi-scid, IL-2Rγ(null) (NOG) mice, followed by oral administration of a causative drug. Subsequently, to replace human skin, unaffected skin specimens obtained from patients who had recovered from SJS/TEN were grafted onto NOG mice, after which patient-derived PBMCs and the causative drug were applied. Mice injected with PBMCs from patients with SJS/TEN and given the causative drug showed marked conjunctival congestion and numerous cell death of conjunctival epithelium, whereas there were no symptoms in mice injected with PBMCs from patients with ordinary drug skin reactions. CD8(+) T lymphocyte-depleted PBMCs from patients with SJS/TEN did not elicit these symptoms. In addition, skin-grafted mice showed darkening of the skin-grafted areas. Cleaved caspase-3 staining showed that dead keratinocytes were more numerous in the skin-grafted mice than in the healthy control animals. We have established a novel human-oriented SJS/TEN mouse model and proved the importance of CD8(+) T lymphocytes in SJS/TEN pathogenesis. The mouse model promises to promote diagnostic and therapeutic approaches.

Analysis of Human Biologics With a Mouse Skin Transplant Model in Humanized Mice

Preclinical testing of human therapeutic monoclonal antibodies has been limited in murine models due to species differences in pharmacokinetics and biologic responses. To overcome these constraints we developed a murine skin transplant model in humanized mice and used it to test human monoclonal antibody therapy. Neonatal NOD/SCID/IL2Rγc(null) mice (NSG) were reconstituted with human CD34(+) hematopoietic stem cells (hNSG). When adult, these mice rejected MHC mismatched murine C57BL/6J skin grafts. Rejection required adequate reconstitution with human cells. There was diffuse infiltration of the epidermis and dermis with hCD8 and hCD4 cells in rejected grafts by immunohistochemistry. Studies with B6/MHC class I and II knockout mice donors indicated that neither is required for rejection. Graft rejection was associated with the development of effector and central memory T cells and an increase in serum immunoglobulins. We also tested the effects of teplizumab (anti-CD3 mAb) and found it could delay skin graft rejection, whereas ipilimumab (anti-CTLA-4 [cytotoxic T-lymphocyte antigen-4] mAb) treatment accelerated rejection. These findings demonstrate that hNSG mice reliably and predictably reject a xenogenic mouse skin graft by a human T cell mediated mechanism. The model can be utilized to investigate the ability of human immunotherapies to enhance or suppress functional human immune responses.

CCR4 dependent migration of Foxp3+ Treg cells to skin grafts and draining lymph nodes is implicated in enhanced graft survival in CD200tg recipients

We have previously reported that transgenic overexpression of CD200 in either mouse skin graft donors or recipients significantly enhances skin allograft survival. By focused microarray analysis we showed this enhanced graft survival is associated with increased expression of Foxp3, GITR, CTLA-4 and CCR4 mRNA, all genes related to Treg cell induction/function, and of Gata3, IL-4, IL-5, IL-13, and somewhat surprisingly, of T-bet, INF-γ and granzyme b. Gene-specific real-time PCR and immunohistochemistry analysis confirmed an increase in Foxp3+ Treg cells in both the skin grafts and draining lymph nodes (DLNs) of CD200tg recipient mice at both 7/14 days post engraftment, as well as providing evidence for increased expression of the ligands for CCR4, CCL17 and CCL22 in both locations. Following lentivirus-mediated shRNA treatment of Dox-treated CD200tg mice to attenuate expression of CCR4 mRNA, the increased localization of Treg cells in skin/DLN of CD200tg recipients was abolished, and the enhanced graft survival similarly reversed. We conclude that enhanced CCR4 dependent migration of Foxp3+ Treg to grafted tissue and DLNs is an essential step in the graft prolongation afforded by overexpression of CD200.

Alternatively Expressed Genes Identified in the CD4+ T Cells of Allograft Rejection Mice

Allograft rejection is a leading cause for the failure of allotransplantation. CD4(+) T cells play critical roles in this process. The identification of genes that alternatively expressed in CD4(+) T cells during allograft rejection will provide critical information for studying the mechanism of allograft rejection, finding specific gene markers for monitoring, predicting allograft rejection, and opening new ways to regulate and prevent allograft rejection. Here, we established allograft and isograft transplantation models by adoptively transferring wild-type BALB/c mouse CD4(+) T cells into severe combined immunodeficient (SCID) mice with a C57BL/6 or BALB/c mouse skin graft. Using the whole transcriptome sequencing-based serial analysis of gene expression (SAGE) technology, we identified 97 increasingly and 88 decreasingly expressed genes that may play important roles in allograft rejection and tolerance. Functional classification of these genes shows that apoptosis, transcription regulation, cell growth and maintenance, and signal transduction are among the frequently changed functional groups. This study provides a genome-wide view for the candidate genes of CD4(+) T cells related to allotransplantation, and this report is a good resource for further microarray studies and for identifying the specific markers that are associated with clinical organ transplantations.

Mice lacking C1q or C3 show accelerated rejection of minor H disparate skin grafts and resistance to induction of tolerance.

Complement activation is known to have deleterious effects on organ transplantation. On the other hand, the complement system is also known to have an important role in regulating immune responses. The balance between these two opposing effects is critical in the context of transplantation. Here, we report that female mice deficient in C1q (C1qa−/−) or C3 (C3−/−) reject male syngeneic grafts (HY incompatible) at an accelerated rate compared with WT mice. Intranasal HY peptide administration, which induces tolerance to syngeneic male grafts in WT mice, fails to induce tolerance in C1qa−/− or C3−/− mice. The rejection of the male grafts correlated with the presence of HY DbUty-specific CD8+ T cells. Consistent with this, peptide-treated C1qa−/− and C3−/− female mice rejecting male grafts exhibited more antigen-specific CD8+IFN-γ+ and CD8+IL-10+ cells compared with WT females. This suggests that accumulation of IFN-γ- and IL-10-producing T cells may play a key role in mediating the ongoing inflammatory process and graft rejection. Interestingly, within the tolerized male skin grafts of peptide-treated WT mice, IFN-γ, C1q and C3 mRNA levels were higher compared to control female grafts. These results suggest that C1q and C3 facilitate the induction of intranasal tolerance.

Blockade of GITR-GITRL interaction maintains Treg function to prolong allograft survival.

Involvement of Treg in transplant tolerance has been demonstrated in multiple models. During the active process of graft rejection, these regulatory cells are themselves regulated and inactivated, a process termed counter-regulation. We hypothesize that ligation of the costimulatory molecule glucocorticoid-induced TNF receptor-related protein (GITR) on Treg inhibits their ability to promote graft survival, and by blocking GITR ligation graft survival can be prolonged. To this aim, we have designed a soluble GITR fusion protein (GITR-Fc), which binds GITR ligand and inhibits activation of GITR. Here, we show that GITR-Fc prolonged mouse skin graft survival, and this prolongation is dependent on Treg. In a full MHC-mismatched skin graft setting, GITR-Fc significantly improved graft survival when used in combination with MR1, anti-CD40L, while GITR-Fc alone did not demonstrate graft prolongation. These results demonstrate that disruption of binding of GITR with GITR ligand may be an important strategy in prolonging allograft survival.

Tetrandrine Attenuates Dendritic Cell-mediated Alloimmune Responses and Prolongs Graft Survival in Mice

Tetrandrine, a bisbenzylisoquinoline alkaloid, has significant immunosuppressive effects; however, the effects of tetrandrine on dendritic cells (DCs) and the associated immune reactions are unclear. In this study, we investigated the effects of tetrandrine on DCs and the effects of the tetrandrine-treated DCs on alloimmune reactions in vitro and graft survival in vivo. Tetrandrine significantly downregulated the expression of CD80 and CD86 of DCs and increased their secretion of IL-10 (p = 0.0001). Mixed leukocyte reaction showed that tetrandrine inhibited dendritic-cell allo-stimulatory activity, which was reversed by the anti-IL-10 treatment. An in vivo study demonstrated that tetrandrine-treated DCs prolonged the survival time of skin grafts in mice compared to control (p = 0.005) and decreased cellular infiltration of the graft in the histopathological study. The data suggest that tetrandrine-treated DCs cause immunosuppression and protect skin grafts from rejection. The tetrandrine-induced immunosuppression seems to be partially due to increased IL-10 secretion.

147 Involvement of the Human Antimicrobial Peptide LL-37 in Wound Repair

Antimicrobial peptides are major components of the innate defense system that present microbicidal activity against gram positive and negative bacteria, yeast, and some enveloped viruses. The human cathelicidin LL-37 is expressed in skin upon wounding and, in addition to its role in antimicrobial defense, it has also been previously demonstrated that this peptide might be involved in reepithelization. We have generated an adenoviral vector containing the complete sequence of LL-37 along with an Ires-GFP expression cassette. The efficacy of this vector was probed by testing the conditioned medium from adenoviral transduced keratinocytes in preventing bacterial growth. We have studied the in vitro effects of LL-37 on HK migration and proliferation. In addition, we have efficiently transduced human skin grafts (in immunodeficient mice) using an in vivo gene transfer approach and we are currently testing the involvement of this peptide in wound repair.

Is There an Ideal Animal Model to Study Hypertrophic Scarring?

Wound healing in hypertrophic scarring and keloid animal models presents significant differences when compared with humans. A brief review is presented about hypertrophic scarring in animal models during the last 5 years. Models were described by animals and scientific artifices to cause hypertrophic scarring. They were divided into 1) heterologous hypertrophic scarring or keloid implants in immunodeficient animals (athymic mice and rats); 2) heterologous hypertrophic scarring or keloid implant in immune privileged site (hamster cheek pouch); 3) hypertrophic scarring or keloid induction via chemically mediated injury (guinea pigs); 4) hypertrophic scarring or keloid induction in anatomic specific site (rabbit ear); and the 5) porcine model. The ideal model would allow to research pathophysiology, histology, and molecular events during time and to test prophylactic and therapeutic treatments for humans. Some of these animals were useful to study specific steps of the scarring process and better understand abnormal wound healing, but none of them have a widespread use. Most recently, the female red Duroc pigs were validated as a new model, demonstrating its similarity to human conditions in different ways. Full-thickness human skin grafts in nude mice also represent improvement in the search of an ideal hypertrophic scarring animal model.

Expression of MHC-I mRNA in Peripheral Blood Lymphocytes as an Early Marker of Acute Rejection Following Skin Transplantation in Mice

Early prediction of acute rejection (AR) is important in clinical practice of organ and tissue transplantation. The aims of this study were to investigate the expression of major histocompatibility complex (MHC)-I and MHC-II genes in peripheral blood lymphocytes (PBLs) following skin grafting and whether their expression can be used as early markers for AR. Skin-grafted mice were selected as an animal model and PBL samples were collected daily for up to 2 weeks post-transplant. Full-thickness skin from the backs of C57BL/6 mice (H-2b) was transplanted onto that of BALB/c mice (H-2d) in allograft group (H-2b to H-2d) and in syngeneic graft group (H-2d to H-2d). The expression levels of MHC-I (H-2K, H-2D) and MHC-II (H-2Ia, H-2Ie) mRNAs were examined using real-time PCR. The histopathological changes of graft biopsies were also analyzed with hematoxylin-eosin staining. The real-time PCR analysis showed that MHC-I and MHC-II mRNA levels were increased in a bimodal distribution pattern during AR in allograft group, whereas no significant changes were detected in syngeneic graft group. The level of H-2K mRNA was significantly increased at day 5 post-transplants compared with those pre-transplant controls (p < 0.01). This increase was detected 5-6 days earlier before graft rejection observed macroscopically. H-2K mRNA level was increased significantly in 93.8% of mice (61/65) in allograft group. These results indicate that the expression of MHC-I and MHC-II mRNAs is up-regulated in PBLs during AR. Especially, the expression of H-2K mRNA can be used as an early marker for AR.

Cartilage Oligometric Matrix Protein-Angiopoietin-1 Promotes Revascularization Through Increased Survivin Expression in Dermal Endothelial Cells of Skin Grafts in Mice

The present study examined the effects of cartilage oligometric matrix protein angiopoietin-1 (COMP-Ang1) on the revascularization of mice skin grafts. Full-thickness skin grafts were autotransferred into BALB/c mice. The donor grafts were soaked in COMP-Ang1 protein (50 mug/ml, n = 10) or in bovine serum albumin (BSA) (50 mug/ml, n = 10) dissolved in 1 ml of sterile, phosphate-buffered saline for 5 minutes before transfer. Revascularization of the grafts was monitored using an intravital microscope on postoperative days 3, 4, and 5. Morphological and immunohistochemical analyses were performed to evaluate platelet-endothelial cell adhesion molecule-1 and survivin expression and apoptotic signal in the transplanted grafts. Grafts soaked in COMP-Ang1 (COMP-Ang1 group) showed significantly increased revascularization compared with grafts soaked in BSA (BSA group) on intravital microscopy and platelet-endothelial cell adhesion molecule-1 staining. The COMP-Ang1 group showed a significant increase of survivin expression in the endothelial cells and a reduction of apoptotic signal in comparison to the BSA group. Therefore, we believe that COMP-Ang1 provides the therapeutic benefit of enhancing the survival of vascular endothelial cells during transplantation of skin graft.

Skin tolerance: in search of the Holy Grail

In 1943, Gibson and Medawar opened the modern era of transplantation research with a paper on the problem of skin allograft rejection. Ten years later Billingham, Brent and Medawar demonstrated that it was possible to induce selective immune acceptance of skin grafts in mice, a state of tolerance. After over six decades, however, the precise mechanism of skin allograft rejection remains still ill-defined. Furthermore, it has not been possible to achieve reliably clinical tolerance allowing the widespread application of skin allotransplantation techniques. The first successful applications of skin allotransplantation have included the hand and face. However, complications from the chronic immunosuppression regimens limit the application of these techniques. Induction of tolerance to skin (and the other tissues in the allograft) would be the most effective way to overcome all these difficulties, but this is yet to be achieved reliably, stimulating some to look for other ways to surmount the current limitations. This paper summarizes alternatives to enlarge the scope of skin allotransplantation techniques, current understanding of mechanisms of skin rejection, and the utility and limitations of animal models used to study skin rejection and tolerance induction. Finally, manipulation strategies to achieve skin tolerance are outlined.