Histomorphometric Analysis of Femoral and Sciatic Nerve Regeneration in a Rat Hindlimb Allotransplantation Model.

BACKGROUND: Peripheral nerve regeneration is essential for functional recovery after traumatic limb injury or limb transplantation. With the use of immunosuppression, it has the capacity to provide regeneration and function equal to that of an autograft. The purpose of this study was to determine whether there is any difference in regeneration and rejection response between peripheral femoral and sciatic nerve in rat hindlimb allotransplantation model. METHODS: The hindlimbs of recipient Fischer 344 rats were amputated at the mid-thigh and the donor and recipient femurs were joined by an intramedullary rod. The femoral and sciatic nerves were repaired with 10-O nylon end-to-end suture followed by vessel, muscle, and skin closure. The control group received auto-transplantation and the experimental group received allo-transplantation from Sprague-Dawley donor rats. The recipients were treated with an immunosuppressive agent, FK506 (2 mg/kg), for the observation period. Both sciatic and femoral nerves were harvested 10 weeks after operation and histomorphometric analysis was conducted between these 2 nerves and control group. RESULTS: The sciatic nerve showed better regeneration, with significantly increased percentage nerve, fiber count, and density (P < .05), but demonstrated more immune cell proliferation relative to femoral nerve. CONCLUSIONS: The data showed that there are some differences in axonal regeneration capacity and immune response between large peripheral nerves.

Induction of Endotoxin Tolerance Delays Acute Rejection in a Hindlimb Transplantation Model in Rats.

BACKGROUND: Acute rejection is seen in 85 percent of composite vascular allogeneic transplants despite long-term immunosuppression. Recently, it was reported that the induction of endotoxin tolerance prolonged heart allograft survival in mice. However, it produced side effects in all the animals secondary to the inflammatory reaction. Galactomannan has shown endotoxin tolerance without this side effect in vitro. The authors hypothesized that galactomannan-induced endotoxin tolerance delays acute rejection in vascular allogeneic transplantation without the side effects produced by lipopolysaccharide. METHODS: Twenty-four rat hindlimb transplants were divided into four groups according to the preconditioning received: control, lipopolysaccharide (0.16 ml/kg), galactomannan 72 hours before (galactomannan-72) (8 ml/kg), and galactomannan 24 hours before (galactomannan-24) (8 ml/kg). Median acute rejection time, weight loss, and diarrheal episodes were monitored. Blood samples were collected at 0, 7, 21, 30, 45, and 60 days. Plasma cytokines (i.e., tumor necrosis factor alpha, interferon gamma), peripheral chimerism, and lymphocyte percentages were analyzed. RESULTS: Median allograft survival was 40 days (range, 40 to 44 days) in the control group, 68 days (range, 61 to 71 days) in the lipopolysaccharide group, and 70 days (range, 69 to 73 days) in both galactomannan groups (p = 0.001). Weight loss was higher in the lipopolysaccharide group (p < 0.001), as was the 83.3 percent rate of diarrheal episodes (control, 0 percent, p = 0.015; galactomannan-72, 0 percent, p = 0.015; and galactomannan-24, 16.7 percent, p = 0.02). Preconditioned rats had higher peripheral blood chimerism (lipopolysaccharide, 2.30 ± 0.13 percent; galactomannan-72, 2.63 ±1.46 percent; and galactomannan-24, 2.47 ± 0.19 percent) compared to the control group (2.06 ± 0.36 percent) (lipopolysaccharide, p = 0.04; galactomannan-72, p = 0.002; and galactomannan-24, p = 0.002). CONCLUSIONS: Induction of endotoxin tolerance delays acute rejection in the rat hindlimb transplantation model. Galactomannan preconditioning has no lipopolysaccharide side effects and was equally effective in delaying acute rejection. CLINICAL RELEVANCE STATEMENT: The contributions of this experimental work are very incipient. Although the use of galactomannan in clinical practice requires more studies to assess its safety, there is no doubt that immunomodulation may be one of the responses that solve the problem of long-term immunosuppression.

Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

Generation of vascular chimerism within donor organs.

Whole organ perfusion decellularization has been proposed as a promising method to generate non-immunogenic organs from allogeneic and xenogeneic donors. However, the ability to recellularize organ scaffolds with multiple patient-specific cells in a spatially controlled manner remains challenging. Here, we propose that replacing donor endothelial cells alone, while keeping the rest of the organ viable and functional, is more technically feasible, and may offer a significant shortcut in the efforts to engineer transplantable organs. Vascular decellularization was achieved ex vivo, under controlled machine perfusion conditions, in various rat and porcine organs, including the kidneys, liver, lungs, heart, aorta, hind limbs, and pancreas. In addition, vascular decellularization of selected organs was performed in situ, within the donor body, achieving better control over the perfusion process. Human placenta-derived endothelial progenitor cells (EPCs) were used as immunologically-acceptable human cells to repopulate the luminal surface of de-endothelialized aorta (in vitro), kidneys, lungs and hind limbs (ex vivo). This study provides evidence that artificially generating vascular chimerism is feasible and could potentially pave the way for crossing the immunological barrier to xenotransplantation, as well as reducing the immunological burden of allogeneic grafts.

The Effects of Tacrolimus on Tissue-Specific, Protein-Level Inflammatory Networks in Vascularized Composite Allotransplantation.

Systems-level insights into inflammatory events after vascularized composite allotransplantation (VCA) are critical to the success of immunomodulatory strategies of these complex procedures. To date, the effects of tacrolimus (TAC) immunosuppression on inflammatory networks in VCA, such as in acute rejection (AR), have not been investigated. We used a systems biology approach to elucidate the effects of tacrolimus on dynamic networks and principal drivers of systemic inflammation in the context of dynamic tissue-specific immune responses following VCA. Lewis (LEW) rat recipients received orthotopic hind limb VCA from fully major histocompatibility complex-mismatched Brown Norway (BN) donors or matched LEW donors. Group 1 (syngeneic controls) received LEW limbs without TAC, and Group 2 (treatment group) received BN limbs with TAC. Time-dependent changes in 27 inflammatory mediators were analyzed in skin, muscle, and peripheral blood using Principal Component Analysis (PCA), Dynamic Bayesian Network (DyBN) inference, and Dynamic Network Analysis (DyNA) to define principal characteristics, central nodes, and putative feedback structures of systemic inflammation. Analyses were repeated on skin + muscle data to construct a "Virtual VCA", and in skin + muscle + peripheral blood data to construct a "Virtual Animal." PCA, DyBN, and DyNA results from individual tissues suggested important roles for leptin, VEGF, various chemokines, the NLRP3 inflammasome (IL-1ß, IL-18), and IL-6 after TAC treatment. The chemokines MCP-1, MIP-1α; and IP-10 were associated with AR in controls. Statistical analysis suggested that 24/27 inflammatory mediators were altered significantly between control and TAC-treated rats in peripheral blood, skin, and/or muscle over time. "Virtual VCA" and "Virtual Animal" analyses implicated the skin as a key control point of dynamic inflammatory networks, whose connectivity/complexity over time exhibited a U-shaped trajectory and was mirrored in the systemic circulation. Our study defines the effects of TAC on complex spatiotemporal evolution of dynamic inflammation networks in VCA. We also demonstrate the potential utility of computational analyses to elucidate nonlinear, cross-tissue interactions. These approaches may help define precision medicine approaches to better personalize TAC immunosuppression in VCA recipients.

Combination of a CD26 Inhibitor, G-CSF, and Short-term Immunosuppressants Modulates Allotransplant Survival and Immunoregulation in a Rodent Hindlimb Allotransplant Model.

BACKGROUND: Recent studies have demonstrated that inhibition of CD26 potentiates stromal cell-derived factor-1α (SDF-1α), promotes tissue regeneration, and suppresses the rejection of organ transplants. This study investigated whether the combination of a CD26 inhibitor (CD26i) with granulocyte colony-stimulating factor (G-CSF) and short-term immunosuppressants modulates vascularized composite tissue allotransplant survival in a rodent orthotopic hindlimb allotransplant model. METHODS: The hindlimb allotransplantation from Brown-Norway to Lewis rats was divided into 4 groups. Group 1 (controls) did not receive any treatment. Group 2 was treated with short-term antilymphocyte serum (ALS) and cyclosporine-A (CsA). Group 3 was administrated CD26i and G-CSF. Group 4 received a combination of CD26i/G-CSF/ALS/CsA. Each subgroup comprised 10 rats. Peripheral blood and sampling of transplanted tissues were collected for immunological and histological analysis. RESULTS: The results revealed that allotransplant survival was found to be significantly prolonged in group 4 with CD26i/G-CSF/ALS/CsA treatment compared with those in the other groups. The interleukin-10 and transforming growth factor-ßl levels, the percentage of CD4+/CD25+/FoxP3+ T cells, as well as the levels of SDF-1α expressions were significantly increased in group 4 compared with those in the other groups. Group 4 revealed a statistical increase in the percentage of donor cells (RT1n) expression in the recipient peripheral blood, and the mixed lymphocyte reaction showed hyporesponsiveness of the T cells to donor alloantigens. CONCLUSION: The combination of CD26i/G-CSF and short-term immunosuppressants prolongs allotransplant survival by inducing immunoregulatory effects and enhancing the percentage of SDF-1α expression. This immunomodulatory approach has great potential as a strategy to increase vascularized composite allotransplantation survival.

Establishment of Heterotopic Hind Limb Transplantation Model in the Mouse.

BACKGROUND: The mouse is the most widely used animal for establishing in vivo models in transplant research. However, because of the advanced microsurgical skills required for these operations, the vascularized composite transplantation model in mouse has proven to be technically challenging. The purpose of this report is to describe novel modifications in surgical techniques to establish a consistent and reliable mouse model of hind limb transplantation. METHODS: Forty C57BL/6 male mice, half as donors and half as recipients, were used in this study. The donor hind limb was harvested and transplanted into the recipient's ipsilateral cervical region by anastomosing the donor femoral artery to the recipient common carotid artery with a modified sleeve technique. The donor femoral vein was mounted with a modified cuff and inserted into the recipient external jugular vein. The graft was evaluated at 2 weeks postoperatively. RESULTS: The modified cuff and modified sleeve technique facilitated anastomoses. The time spent on either of the donor operation and recipient operation was about 45 minutes. The graft survival rate was 80% (16 of 20) at 2 weeks after transplant. There was minimal blood loss and no infections were noted. CONCLUSIONS: Revised surgical techniques using a modified cuff proved to be a safe, reliable, and reproducible strategy in establishing a mouse model of hind limb heterotopic transplantation. The consistent graft survival in this syngeneic study demonstrates that this model can serve as a useful tool for further studies in vascularized composite transplantation.

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

Vascularized Composite Allotransplantation: A Functional Hind Limb Model in Mice.

BACKGROUND: Vascularized composite allograft has emerged as a reconstructive option for patients who have suffered severe tissue loss. Animal models are critical for understanding the unique mechanisms of rejection in vascularized composite allograft. We present a functional mouse model of orthotopic hind limb transplantation using end-to-side anastomoses of the donor aorta and inferior vena cava to the respective recipient vessels. To the best of our knowledge, this approach has not been reported in the scientific literature. MATERIALS AND METHODS: A single surgeon performed all transplants (J.W.). A total of 13 syngeneic and 10 fully mismatched allogeneic transplants were performed without immunosuppression. Skin samples from the grafts were collected at the time of euthanasia. RESULTS: Five syngeneic mice survived for more than 90 d after transplant. All allografts displayed clinical and histologic signs of acute rejection such as a rash at the time of graft excision. The overall technical success rate of all transplants in this study was 74% (17 of 23). CONCLUSIONS: We demonstrate the feasibility of end-to-side anastomoses of the donor aorta and inferior vena cava with functional recovery of the transplant in a mouse model of orthotopic hind limb transplantation.

Mechanical Irritation in Vascularized Composite Tissue Allotransplantation Triggers Localized Skin Rejection.

BACKGROUND: Mechanical and thermal stress has been observed to trigger skin rejection in hand-transplanted patients. This study aims to investigate this phenomenon. METHODS: Syngeneic and allogeneic orthotopic hindlimb transplantations were performed using male rats (Brown Norway to Lewis). Using a specially designed device, standardized mechanical skin irritation at a force of 5 N was applied to the planta pedis of the transplanted limb for 10 days, 4 times daily for 10 minutes. Biopsies, taken on day 10 and after a 5-day observational period, were assessed for macroscopic alterations using a standardized scale, by histopathology and immunohistochemistry, and for inflammatory protein expression using Luminex technology. RESULTS: Allogeneic animals displayed significant aggravated macroscopic skin alterations compared with naive (P < 0.0001) and syngeneic controls (P = 0.0023). Histopathology showed a trend toward higher rejection/inflammation grades in allogeneic animals compared with syngeneic controls. Minor skin alterations in syngeneic limbs recovered quickly; however, in allogeneic limbs, macroscopic skin alterations were significantly more pronounced (P < 0.0001) 5 days after irritation. Interleukin-1b and interferon-γ levels were upregulated in skin of allogeneic limbs. CONCLUSIONS: Mechanical skin irritation in vascularized composite allotransplantation can trigger localized skin inflammation consistent with rejection.

Effect of Static Cold Storage on Skeletal Muscle after Vascularized Composite Tissue Allotransplantation.

BACKGROUND: Prolonged cold ischemia associated with static cold storage (SCS) results in higher incidence of acute and chronic allograft rejection in solid organ transplantations. Deleterious effects of SCS on vascularized composite tissue allograft were studied with limited data on muscle structure and function. The aim of this study is to evaluate the long-term impact of SCS on muscle metabolism, structure, and force generation using a syngeneic rat hindlimb transplantation model. METHODS: Sixty-five male Lewis rats (250 ± 25 g) were distributed into five groups, including naive control, sciatic nerve denervation/repair, immediate transplantation, transplantation following static warm storage for 6 hours at room temperature, and transplantation following SCS for 6 hours at 4°C. Sciatic nerves were repaired in all transplantations. Muscle samples were taken for histology and metabolomics analysis following electromyography and muscle force measurements at 12 weeks after transplantation. RESULTS: All cold-preserved limbs remained viable at 12 weeks, whereas animals receiving limbs preserved in room temperature had no survivors. The SCS transplantation group showed a 73% injury score, significantly higher than groups receiving immediate transplants without cold preservation (50%, p < 0.05). A significant decline in muscle contractile force was also demonstrated in comparison to the immediate transplantation group (p < 0.05). In the SCS group, muscle energy reserves remained relatively well preserved in surviving fibers. CONCLUSION: SCS extends allograft survival but fails to preserve muscle structure and force.

Self-organized formation of developing appendages from murine pluripotent stem cells.

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.

A Skin Rejection Grading System for Vascularized Composite Allotransplantation in a Preclinical Large Animal Model.

BACKGROUND: The Banff Criteria have been accepted as a system for grading histological rejection in graft skin in human vascularized composite allotransplantation (VCA). Preclinical swine hindlimb transplantation models have an important role in translational studies in VCA. However, unified grading criteria for rejection in swine skin have not yet been established. METHODS: Two hundred fourteen swine skin biopsy specimens were reviewed, including 88 native skin biopsies and 126 specimens from the skin component of heterotopic swine hindlimb transplants. Thorough review was performed in a blinded fashion by an expert veterinary pathologist with attention paid to the applicability of the Banff criteria as well as specific histologic characteristics and trends. Clinical and histopathologic rejection scores were then directly compared. RESULTS: Two hundred fourteen specimens reviewed showed significant similarities between swine and human skin, as previously published. Notable swine-specific characteristics, including paucicellular infiltration with rare epidermal cell infiltration or necrosis, were accounted for in a proposed grading system that parallels the Banff Criteria. CONCLUSIONS: This comprehensive grading system, based on the Banff Classification for skin rejection in VCA, provides a standardized system for more accurate comparison of rejection in preclinical swine VCA models.

Delivery of Rapamycin Using In Situ Forming Implants Promotes Immunoregulation and Vascularized Composite Allograft Survival.

Vascularized composite allotransplantation (VCA), such as hand and face transplantation, is emerging as a potential solution in patients that suffered severe injuries. However, adverse effects of chronic high-dose immunosuppression regimens strongly limit the access to these procedures. In this study, we developed an in situ forming implant (ISFI) loaded with rapamycin to promote VCA acceptance. We hypothesized that the sustained delivery of low-dose rapamycin in proximity to the graft may promote graft survival and induce an immunoregulatory microenvironment, boosting the expansion of T regulatory cells (Treg). In vitro and in vivo analysis of rapamycin-loaded ISFI (Rapa-ISFI) showed sustained drug release with subtherapeutic systemic levels and persistent tissue levels. A single injection of Rapa-ISFI in the groin on the same side as a transplanted limb significantly prolonged VCA survival. Moreover, treatment with Rapa-ISFI increased the levels of multilineage mixed chimerism and the frequency of Treg both in the circulation and VCA-skin. Our study shows that Rapa-ISFI therapy represents a promising approach for minimizing immunosuppression, decreasing toxicity and increasing patient compliance. Importantly, the use of such a delivery system may favor the reprogramming of allogeneic responses towards a regulatory function in VCA and, potentially, in other transplants and inflammatory conditions.

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Functional Equivalency in Human Somatic Cell Nuclear Transfer-Derived Endothelial Cells.

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.

Relative antigenicity of components in vascularized composite allotransplants: An experimental study of microRNAs expression in rat hind limb transplantation model.

BACKGROUND: Skin is considered to be the most antigenic component of all vascularized composite allotransplantation tissues. However, no studies have used methods other than histological assessment to analyze the relative antigenicity of various components. In this study, we analyzed gene expression to investigate the relative antigenicity of each component in the transplanted limb. METHODS: Seven Brown Norway rats and 31 Lewis rats were assigned to two groups: an allograft group and a syngeneic (control) group. Brown Norway rats were used as the allogeneic donors, and Lewis rats were used as the syngeneic donors and recipients. About 13 recipients in the allograft group and 12 recipients in the control group were analyzed. Histological assessment was performed in 5 of the recipients in each group, and microRNA expression was analyzed in the remaining recipients, except for 1 recipient in the syngeneic group. RESULTS: In the allograft group, the relative microRNA-146a expression was significantly higher in skin (2.34 ± 0.44) than in muscle (1.25 ± 0.22; p = .034) and bone (1; p = .0081). In the allograft group, microRNA-155 expression was significantly higher in skin (1.91 ± 0.18) than in bone (1; p = .010). Histological assessment showed that some skin tissue in the allograft group showed evidence of severe acute rejection. CONCLUSIONS: The microRNA-146a and microRNA-155 seemed to reflect the relative antigenicity during acute rejection of transplanted limbs. Skin seemed to be more antigenic than muscle and bone in both the histological assessment and gene expression analysis.

Estudio sobre quimerismo reverso y el efecto de la movilización de células madre de médula ósea sobre la expresión de citoquinas en un modelo experimental de trasplante de tejidos compuestos / Study on reverse chimerism and the effect of mobilization of bone marrow stem cells on the expression of cytokines in an experimental model of composite tissue transplantation

Introducción y Objetivo: Los trasplantes de tejidos compuestos sufren rechazo crónico modulado entre otros factores por citoquinas. El quimerismo reverso o quimerismo del aloinjerto se define como la repoblación del tejido trasplantado por células circulantes del receptor. Plerixafor produce la movilización de células madre de médula ósea CD34+ hacia la sangre periférica. El objetivo del estudio fue conocer los mecanismos moleculares que intervienen en el rechazo crónico y el quimerismo reverso tras la administración de plerixafor. Material y Método: Realizamos 16 trasplantes osteomusculares heterotópicos de pata posterior entre ratas Brown-Norway hembra y Wistar Lewis macho bajo inmunosupresión subterapéutica con tacrólimus. Establecimos 2 grupos de estudio según la administración postoperatoria de plerixafor. Transcurridas 9 semanas estudiamos la expresión de citoquinas y el infiltrado leucocitario en distintas localizaciones musculares, así como el grado de rechazo crónico y porcentaje de quimerismo reverso en diferentes tejidos del aloinjerto. Resultados: Encontramos diferencias estadísticas en la expresión de factor estimulante de colonias granulocíticas e interleucina 12 a nivel de los tercios medio y distal del aloinjerto, y de interleucina 6 a nivel del tercio medio del aloinjerto. La intensidad del infiltrado leucocitario fue mayor en el grupo que no recibió plerixafor. Ambos grupos desarrollaron rechazo crónico y pudimos observar la aparición de quimerismo reverso. Sin embargo no observamos diferencias significativas en el infiltrado leucocitario, el rechazo crónico ni el quimerismo reverso. Conclusiones: La movilización de células madre de médula ósea CD34+ se asoció con una menor expresión de factor estimulante de colonias granulocíticas, interleucina 6 e interleucina 12. Estos hallazgos contribuyen a elucidar los mecanismos moleculares que podrían conducir a la creación de quimeras en el aloinjerto

Background and Objective: Vascularized composite allotransplantation suffer chronic rejection modulated by cytokines. Reverse chimerism or allograft chimerism is defined as the repopulation of the transplanted tissue by circulating cells of the recipient. Plerixafor mobilizes CD34+ bone marrow stem cells to the peripheral blood. The aim of the study was to know the molecular mechanisms involved in chronic rejection and reverse chimerism after plerixafor administration. Methods: Sixteen heterotopic osteomuscular hindlimb transplants were performed between female Brown-Norway rats as donors and male Wistar Lewis rats as recipients under subtherapeutic immunosuppression with tacrolimus. Two groups were established according to the postoperative administration of plerixafor. After 9 weeks, expression of cytokines and leukocyte infiltration were studied in different muscle locations, as well as the degree of chronic rejection and percentage of reverse chimerism in different tissues of the allograft. Results: Statistical differences were found in granulocyte colony stimulating factor and interleukin 12 expression at middle and distal allograft thirds, and interleukin 6 expression at middle allograft third. The intensity of leukocyte infiltration was greater in the group that did not receive plerixafor. Both groups developed chronic rejection and the appearance of reverse chimerism could be observed. However, no significant differences were observed in leukocyte infiltration, chronic rejection or reverse chimerism. Conclusions: The mobilization of CD34+ bone marrow stem cells was associated with a lower expression of granulocytic colony stimulating factor, interleukin 6 and interleukin 12. These findings contribute to elucidate the molecular mechanisms that could lead to the creation of chimeras in the allograft

Local Injections of Tacrolimus-loaded Hydrogel Reduce Systemic Immunosuppression-related Toxicity in Vascularized Composite Allotransplantation.

BACKGROUND: Routine application of vascularized composite allotransplantation is hampered by immunosuppression-related health comorbidities. To mitigate these, we developed an inflammation-responsive hydrogel for local immunosuppression. Here, we report on its long-term effect on graft survival, immunological, and toxicological impact. METHODS: Brown Norway-to-Lewis rat hindlimb transplantations were treated either systemically with daily injections of 1 mg/kg tacrolimus (TAC) or with subcutaneous intragraft injections of hydrogel containing 7 mg TAC, every 70 days. Animals were monitored for rejection or other pathology for 280 days. Systemic and graft TAC levels, regulatory T cells, and donor cell chimerism were measured periodically. At endpoint, markers for kidney, liver, and metabolic state were compared to naive age-matched rats. RESULTS: Both daily systemic TAC and subcutaneous intragraft TAC hydrogel at 70-day intervals were able to sustain graft survival longer than 280 days in 5 of 6 recipients. In the hydrogel group, 1 graft progressed to grade 3 rejection at postoperative day 149. In systemic TAC group, 1 animal was euthanized due to lymphoma on postoperative day 275. Hydrogel treatment provided stable graft and reduced systemic TAC levels, and a 4 times smaller total TAC dose compared with systemic immunosuppression. Hydrogel-treated animals showed preserved kidney function, absence of malignancies or opportunistic infections and increased hematopoietic chimerism compared with systemic immunosuppression. CONCLUSIONS: Our findings demonstrate that localized immunosuppression with TAC hydrogel is a long-term safe and reliable treatment. It may reduce the burden of systemic immunosuppression in vascularized composite allotransplantation, potentially boosting the clinical application of this surgical intervention.

Subcutaneous administration of a neutralizing IL-1ß antibody prolongs limb allograft survival.

Cytokine-expression profiles revealed IL-1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL-1ß antibody in limb transplantation. Following allogenic hind-limb transplantation, Lewis rats were either left untreated or treated with anti-lymphocyte serum + tacrolimus (baseline); baseline immunosuppression + anti-IL-1ß (1 mg/kg once/week, 6-8 subcutaneous injections) into the transplanted or contralateral limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti-IL-1ß injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL-1ß, IL-4, IL-13, IP-10, MCP-1, and MCP-3 in long-term-survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti-IL-1ß. Inhibition of IL-1ß with short-term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.