Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model

Our recent studies in an inbred swine model demonstrated that both peripheral and intra‐graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor‐matched kidney. Here, we have asked whether both peripheral and intra‐graft regulatory elements are required for adoptive transfer of tolerance when only a long‐term tolerant (LTT) kidney is transplanted. Nine highly‐inbred swine underwent a tolerance‐inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor‐matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance‐inducing and/or tolerance‐maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor‐matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.     

Neo-Angiogenesis,Transplant Viability,and Molecular Analyses of Vascularized Bone Allotransplantation Surgery in a Large Animal Model

Vascularized composite allotransplantation of bone is a possible alternative treatment for large osseous defects but requires life-long immunosuppression. Surgical induction of autogenous neo-angiogenic circulation maintains transplant viability without this requirement, providing encouraging results in small animal models [1–3]. A preliminary feasibility study in a swine tibia model demonstrated similar findings [4, 5]. This study in swine tibial allotransplantation tests its applicability in a pre-clinical large animal model. Previously, we have demonstrated bone vascularized composite allotransplantation (VCA) survival was not the result of induction of tolerance nor an incompetent immune system [1]. Fourteen tibia vascularized bone allotransplants were microsurgically transplanted orthotopically to reconstruct size-matched tibial defects in Yucatan miniature swine. Two weeks of immunosuppression was used to maintain allotransplant pedicle patency during angiogenesis from a simultaneously implanted autogenous arteriovenous bundle. The implanted arteriovenous bundle was patent in group 1 and ligated in group 2 (a neo-angiogenesis control). At twenty weeks, we quantified the neo-angiogenesis and correlated it with transplant viability, bone remodeling, and gene expression. All patent arteriovenous bundles maintained patency throughout the survival period. Micro-angiographic, osteocyte cell count and bone remodeling parameters were significantly higher than controls due to the formation of a neo-angiogenic autogenous circulation. Analysis of gene expression found maintained osteoblastic and osteoclastic activity as well as a significant increase in expression of endothelial growth factor-like 6 (EGFL-6) in the patent arteriovenous bundle group. Vascularized composite allotransplants of swine tibia maintained viability and actively remodeled over 20 weeks when short-term immunosuppression is combined with simultaneous autogenous neo-angiogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:288-296, 2020     

Dorsal Bipedicled Island Skin Flap: A New Flap Model in Mice

Mice are popular animals for biomedical studies, but few skin flap models have been reported in them. To investigate the ischaemia/reperfusion phenomenon in skin flaps, we first investigated the vascular anatomy of murine dorsal skin and then designed a suitable murine dorsal skin flap model. In 120 mice, six distinct vascular patterns were identified, one being seen in 111 mice (93%). Based on this finding, in Part 2 of the study, 15 mice had flaps (4 &#50 4 cm) raised based on the two caudal vascular pedicles of the left and right deep circumflex iliac vessels as a bipedicled flap in which the mean (SD) survival was 96 (5)%. In a further 10 mice, flaps were raised based on a single pedicle, the left deep circumflex iliac vessel, as a monopedicled flap, in which the mean (SD) survival was 71 (12)%. The bipedicled flap model was then used to study ischaemia/reperfusion injury. Twenty flaps were subjected to eight hours of ischaemia and subsequent reperfusion, and their mean (SD) survival was 43 (26)%. Histological assessments were also carried out using neutrophil and leucocyte counts, and significant differences between groups were observed.     

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.     

Establishment of a Long-Term Survival Swine Model for the Observation of Transplanted Islets: a Preliminary Step in an Allogeneic Transplant Experiment

BackgroundEvaluation of an experimental and preclinical islet transplantation (IsletTx) model to elucidate associated clinical problems is vital. This study aimed to introduce a simple methodology for producing a swine autologous IsletTx model as a preliminary step in an allogeneic transplant experiment.Methods and MaterialsTwenty-seven pigs were included in the study. Total pancreatectomy (TP) was performed in 8 pigs (TP group), TP with autologous IsletTx in 9 (TP + IsletTx group), and distal pancreatectomy (DP) with autologous IsletTx in 10 (DP + IsletTx group). An open biopsy was performed on all pigs during postoperative day 14 using an infrared imaging (IRI) system. Laboratory data and postoperative survival were analyzed and compared according to the procedures done.ResultsPostoperative survival rate was significantly higher in the pigs with autologous IsletTx than in those without (P = .026). There were no significant differences in survival between the TP + IsletTx and DP + IsletTx groups (P = .746). Significant hyperglycemia was not observed in both groups, but the DP + IsletTx group remained relatively stable throughout the postoperative course. There were no differences in serum creatinine, aspartate aminotransferase, and alanine aminotransferase levels between the 2 groups. By selective liver lobe transplantation and administration of the IRI system, localization of the transplanted islets via open biopsy was achieved.ConclusionsWe successfully developed an autologous IsletTx model and an open biopsy system using a swine model. This study will aid in the development of an allogeneic IsletTx experiment that may improve transplantation outcomes.     

Effects of Caffeine Consumption on Autologous Full-Thickness Skin Graft Healing in an Animal Model

Background There exists contradictory evidence that states both the beneficial and deleterious effects of caffeine on wound healing. The general population might unknowingly consume caffeine that negatively affects wound healing. The main objective of this study is to investigate the effect of daily caffeine consumption on wound healing, specifically full-thickness skin graft (FTSG). Methods Forty Sprague–Dawley rats were randomized into four groups of equal size: control-dose (CD), low-dose (LD), medium-dose (MD), and high-dose (HD) caffeine groups. After autologous FTSG, all subjects in the intervention group were given daily pure caffeine gavage. The FTSG was explanted 7 days posttransplant. The graft viability, secondary contraction, and adherence were evaluated macroscopically, while fibroblast and collagen deposition was analyzed microscopically with hematoxylin eosin stain. Results The least graft viability (72.8 ± 20.7%, clinical wound assessment scale [CWAS] 2.4), highest secondary contraction (11.4 ± 10.5%), and fibroblast count (331.8 ± 88.6 cells/5 high power fields) were observed in the MD group. More collagen synthesis was observed in subjects who consumed caffeine. The level of secondary contraction, fibroblast count as well as graft viability and collagen synthesis were positively correlated. Conclusions Daily consumption of caffeine impairs graft viability when given in medium dose and increases collagen synthesis, irrespective of dosage. This study was in experimental rats; the results are not directly translatable to humans.     

New Regenerative Vascular Grafts for Hemodialysis Access: Evaluation of a Preclinical Animal Model

The purpose of this study was to evaluate a suitable animal model for the in vivo evaluation of patency and vascular tissue regeneration in small intestinal submucosa (SIS) vascular grafts for hemodialysis access. First, a 4-mm U-shaped SIS vascular graft was implanted between the internal carotid artery (CA) and the external jugular vein (JV) in five sheep and six swine. The U-shape grafts remained functional for 53 ± 4 days in sheep and 32 ± 2 days in swine. The sheep model presented exaggerated inflammation, so the swine model was selected for the in vivo study. Based on these initial results, a 4-mm C-shape SIS vascular graft with SIS circumferential reinforcement was developed to mechanically improve the vascular graft and manage complications identified during surgery in both sheep and swine. The C-shape vascular graft was implanted in a swine model (n = 3) between the CA and JV. GORE-TEX® vascular grafts were used as controls in the contralateral side of the neck. C-shape grafts remained patent for 47 ± 4 days, whereas the GORE-TEX® grafts were patent for 30 ± 15 days. The C-shape vascular graft was easier to handle during surgery, and its circumferential reinforcement improved in vivo patency, avoiding kinks in the graft after implantation. Histological results showed neovascularization and some regeneration with the alignment of endothelial cells in the vascular wall of the grafts. The model developed may be helpful in other research involving in vivo studies of vascular grafts for hemodialysis access.     

Induction of Transplant Tolerance with Immunodominant Allopeptide-pulsed Host Lymphoid and Myeloid Dendritic Cells

We have studied the effects of adoptive transfer of host thymic dendritic cells pulsed with immunodominant WF Class I peptide 5 (residues 93-109) on cardiac allograft survival in the WF-to-ACI rat combination. Our results showed that, whereas intrathymic inoculation of WF peptide 5-pulsed ACI thymic dendritic cells alone on day -7 did not prolong graft survival, similar treatment combined with 0.5 mL antilymphocyte serum (ALS) led to 100% permanent acceptance (> 200d) of donor-specific cardiac allografts. Extension of our study to systemic administration of peptide 5-pulsed host thymic dendritic cells confirmed that intravenous injection of peptide 5-pulsed self thymic dendritic cells combined with ALS transient immunosuppression resulted in 100% permanent donor-specific graft survival (> 200d). These results were reproducible in a clinically relevant model using intravenous injection of peptide-pulsed host myeloid dendritic cells. In contrast, thymectomy prior to adoptive transfer of peptide-pulsed host dendritic cells resulted in acute graft rejection at times equivalent to rejection in thymectomized controls. The long-term unresponsive recipients challenged with second-set grafts accepted permanently (> 100d) donor-type (WF) but not third party (Lewis) cardiac allografts. This study suggests that intravenous administration of genetically engineered dendritic cells expressing donor MHC molecules has the potential of inducing transplant tolerance.     

游离股后侧皮瓣移植修复四肢大面积皮肤缺损

本文介绍采用游离股后侧皮瓣修复大片四肢皮肤缺损12例,均获成功。作者发现该皮瓣营养血管来自腘动脉直接皮支,其直径2.5～3.5mm,于膝上6～8cm发出。皮瓣大小可取到12～24cm,并在横径小于10cm时供区可直接关闭。该皮瓣皮支恒定,直径较粗易于吻合,皮瓣供区范围较大且较隐蔽,适于四肢皮肤软组织缺损的修复。     

Regulatory T Cells Are Critical to Tolerance Induction in Presensitized Mouse Transplant Recipients Through Targeting Memory T Cells

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40⁺CD44^hieffector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti‐OX40L monoclonal antibody (mAb) (α‐OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, α‐OX40L was added to the combined treatment protocol of LF15–0195 (LF) and anti‐CD45RB (α‐CD45RB) mAb—a protocol that induced heart allograft tolerance in non‐presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor‐specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs). Of note, CD25⁺ T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor‐primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.