Mimicking Clinical Rejection Patterns in a Rat Osteomyocutaneous Flap Model of Vascularized Composite Allotransplantation.

INTRODUCTION: Graft loss in vascularized composite allotransplantation (VCA) is more often associated with vasculopathy and chronic rejection (CR) than acute cellular rejection (ACR). We present a rat osteomyocutaneous flap model using titrated tacrolimus administration that mimics the graft rejection patterns in our clinical hand transplant program. Comparison of outcomes in these models support a role for ischemia reperfusion injury (IRI) and microvascular changes in CR of skin and large-vessel vasculopathy. The potential of the surgical models for investigating mechanisms of rejection and vasculopathy in VCA and treatment interventions is presented. MATERIALS AND METHODS: Four rodent groups were evaluated: syngeneic controls (Group 1), allogeneic transient immunosuppression (Group 2), allogeneic suboptimal immunosuppression (Group 3), and allogeneic standard immunosuppression (Group 4). Animals were monitored for ACR, vasculopathy, and CR of the skin. RESULTS: Transient immunosuppression resulted in severe ACR within 2 wk of tacrolimus discontinuation. Standard immunosuppression resulted in minimal rejection but subclinical microvascular changes, including capillary thrombosis and luminal narrowing in arterioles in the donor skin. Further reduction in tacrolimus dose led to femoral vasculopathy and CR of the skin. Surprisingly, femoral vasculopathy was also observed in the syngeneic control group. CONCLUSIONS: Titration of tacrolimus in the allogeneic VCA model resulted in presentations of rejection and vasculopathy similar to those in patients and suggests vasculopathy starts at the microvascular level. This adjustable experimental model will allow the study of variables and interventions, such as external trauma or complement blockade, that may initiate or mitigate vasculopathy and CR in VCA.

Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.

As clinical experience with surgical techniques and immunosuppression in vascularized composite allotransplantation recipients has accumulated, vascularized composite allotransplantation for hand and face have become standard of care in some countries for select patients who have experienced catastrophic tissue loss. Experience to date suggests that clinical vascularized composite allotransplantation grafts undergo the same processes of allograft rejection as solid organ grafts. Nonetheless, there are some distinct differences, especially with respect to the immunologic influence of the skin and how the graft is affected by environmental and traumatic insults. Understanding the mechanisms around these similarities and differences has the potential to not only improve vascularized composite allotransplantation outcomes but also outcomes for all types of transplants and to contribute to our understanding of how complex systems of immunity and function work together. A distinct disadvantage in the study of upper extremity vascularized composite allotransplantation recipients is the low number of clinical transplants performed each year. As upper extremity transplantation is a quality of life rather than a lifesaving transplant, these numbers are not likely to increase significantly until the risks of systemic immunosuppression can be reduced. As such, experimental models of vascularized composite allotransplantation are essential to test hypotheses regarding unique characteristics of graft rejection and acceptance of vascularized composite allotransplantation allografts. Rat hind limb vascularized composite allotransplantation models have been widely used to address these questions and provide essential proof-of-concept findings which can then be extended to other experimental models, including mice and large animal models, as new concepts are translated to the clinic. Here, we review the large body of rat hind limb vascularized composite allotransplantation models in the literature, with a focus on the various surgical models that have been developed, contrasting the characteristics of the specific model and how they have been applied. We hope that this review will assist other researchers in choosing the most appropriate rat hind limb transplantation model for their scientific interests.

Psychosocial factors and medication adherence among recipients of vascularized composite allografts.

OBJECTIVES: Psychosocial factors are important predictors of medication adherence, and subsequently graft survival, in solid organ transplantation. Early experiences suggest this may also be the case in vascularized composite allotransplantation. METHODS: Using validated tools, we surveyed upper extremity transplant recipients at two centers to assess depression (Patient Health Questionnaire-9), personality (Ten-Item Personality Inventory), anxiety (Generalized Anxiety Disorder 7-Item Scale), post-traumatic stress disorder (Primary Care Post-Traumatic Stress Disorder Screen for Diagnostic and Statistical Manual of Mental Disorders, 5th Edition), and social support (Multidimensional Scale of Perceived Social Support). Medication adherence among vascularized composite allotransplantation recipients at two centers was assessed by a member of the clinical research team using the recipients' medical records. RESULTS: Medication adherence was reported for 12 vascularized composite allotransplantation recipients, and 9 vascularized composite allotransplantation recipients completed psychosocial assessments. Most recipients were believed to be adherent to their immunosuppression, however, three recipients were believed to be non-adherent and a member of the clinical team had discussed non-adherence at least once with five recipients. Results from the psychosocial assessment (n = 9) indicated that eight participants had high levels of social support, and eight demonstrated high levels of conscientiousness which have been associated with better medication adherence in solid organ transplantation. However, three participants demonstrated mild anxiety, two demonstrated minimal symptoms of depression, and one demonstrated post-traumatic stress disorder which have been associated with worse medication adherence in solid organ transplantation. CONCLUSION: These findings lay the groundwork for future assessments of the role psychosocial factors play in facilitating medication adherence and broader transplant outcomes.

Defining chronic rejection in vascularized composite allotransplantation-The American Society of Reconstructive Transplantation and International Society of Vascularized Composite Allotransplantation chronic rejection working group: 2018 American Society of Reconstructive Transplantation meeting report and white paper Research goals in defining chronic rejection in vascularized composite allotransplantation.

OBJECTIVES: This report summarizes a collaborative effort between the American Society of Reconstructive Transplantation and the International Society of Vascularized Composite Allotransplantation to establish what is known about chronic rejection in recipients of vascularized composite allografts, with an emphasis on upper extremity and face transplants. As a picture of chronic rejection in hand and face vascularized composite allografts emerges, the results will be applied to other types of vascularized composite allografts, such as uterine transplantation. METHODS: The overall goal is to develop a definition of chronic rejection in vascularized composite allografts so that we can establish longitudinal correlates of factors such as acute rejection, immunosuppressive therapy, de novo donor-specific antibody and trauma/infection and other external factors on the development of chronic rejection. As Dr Kanitakis eloquently stated at the 2017 International Society of Vascularized Composite Allotransplantation meeting in Salzburg, "Before we can correlate causative factors of chronic rejection, we have to define what chronic rejection in VCA is." RESULTS: The first meeting report was presented at the sixth Biennial meeting of the American Society of Reconstructive Transplantation in November 2018. Based on collaborative efforts and descriptions of clinical cases of chronic rejection in vascularized composite allograft recipients, a working definition of chronic rejection in vascularized composite allografts with respect to overt functional decline, subclinical functional decline, histologic evidence without functional decline, and normal allograft function in the absence of histologic evidence of chronic rejection is proposed. CONCLUSIONS: It is the intent of this collaborative working group that these working definitions will help to focus ongoing research to define the incidence, risk factors and treatment regimens that will identify mechanisms of chronic rejection in vascularized composite allografts. As with all good research, our initial efforts have generated more questions than answers. We hope that this is the first of many updates.

Infrared imaging of lymphatic function in the upper extremity of normal controls and hand transplant recipients via subcutaneous indocyanine green injection.

OBJECTIVES: The goal of this study was to define the parameters of movement of indocyanine green in the upper extremity of normal control and hand transplant recipients. The purpose was to establish a non-invasive method of determining the level of lymphatic function in hand transplant recipients. In hand transplantation (and replantation), the deep lymphatic vessels are rarely repaired, resulting in altered lymphatic connections. In most cases, the relatively rapid inosculation of superficial lymphatic networks and drainage via the venous systems results in sufficient interstitial fluid and lymph drainage of the graft to prevent edema. However, our group and others have determined that some transplant recipients demonstrate chronic edema which is associated with lymphatic stasis. In one case, a patient with chronic edema has developed chronic rejection characterized by thinning of the skin, loss of adnexal structures, and fibrosis and contracture of the hand. METHODS: Lymphatic function was evaluated by intradermal administration of near-infrared fluorescent dye, indocyanine green, and dynamic imaging with an infrared camera system (LUNA). To date, the assessment of lymphatic drainage in the upper extremity by clearance of indocyanine green dye has been studied primarily in oncology patients with abnormal lymphatic function, making assessment of normal drainage problematic. To establish normal parameters, indocyanine green lymphatic clearance functional tests were performed in a series of normal controls, and subsequently compared with indocyanine green clearance in hand transplant recipients. RESULTS: The results demonstrate varied patterns of lymphatic drainage in the hand transplant patients that partially mimic normal hand lymphatic drainage, but also share characteristics of lymphedema patients defined in other studies. The study revealed significant deceleration of the dye drainage in the allograft of a patient with suspected chronic rejection and edema of the graft. Analysis of other hand transplant recipients revealed differing levels of dye deceleration, often localized at the level of surgical anastomosis. CONCLUSION: These studies suggest intradermal injection of indocyanine green and near-infrared imaging may be a useful clinical tool to assess adequacy of lymphatic function in hand transplant recipients.

Current Status of Vascularized Composite Allotransplantation.

The field of vascularized composite allotransplantation (VCA) has moved from a highly experimental procedure to, at least for some patients, one of the best treatment alternatives for catastrophic tissue loss or dysfunction. Although the worldwide experience is still limited, progress has been made in translation to the clinic, and hand transplantation was recently designated standard of care and is now covered in full by the British Health System. This progress is tempered by the long-term challenges of systemic immunosuppression, and the rapidly evolving indications for VCA such as urogenital transplantation. This update will cover the state of and recent changes in the field, and an update of the Louisville VCA program as our initial recipient, the first person to receive a hand transplant in the United States celebrates the 20th anniversary of his transplant. The achievements and complications encountered over the last two decades will be reviewed. In addition, potential directions for research and collaboration as well as practical issues of how third party payers and funding are affecting growth of the field are presented.

Microstructural integrity of corticospinal and medial lemniscus tracts: insights from diffusion tensor tractography of right-hand amputees.

Reductions in sensory and motor activity following unilateral upper limb amputation during adulthood are associated with widespread, activity-dependent reorganization of the gray matter and white matter through the central nervous system. Likewise, in cases of congenital limb absence there is evidence that limited afferent or efferent activity affects the structural integrity of white matter pathways serving the affected side. Evidence that the structural integrity of mature sensory and motor tracts controlling the lost upper limb exhibits similar activity dependence is, however, sparse and inconsistent. Here we used diffusion tensor tractography to test whether amputation of the dominant right hand during adulthood (n = 16) alters the microstructural integrity of the major sensory (medial lemniscus, ML) and motor (corticospinal tract, CST) pathways controlling missing hand function. Consistent with prior findings, healthy control subjects (n = 27) exhibited higher fractional anisotropy (FA), an index of white matter microstructural integrity, within dominant left CST and nondominant right ML. Critically, in contrast to what might be expected if the microstructural organization of these tracts is activity dependent, these asymmetries persisted in amputees. Moreover, we failed to detect any differences in dominant left ML or CST between healthy control subjects and amputees. Our results are consistent with these white matter tracts being robust to changes in activity once mature or that continued use of the residual limb (in a compensatory fashion or with prosthesis) provides stimulation sufficient to maintain tract integrity. NEW & NOTEWORTHY We report that unilateral hand amputation in adults has no significant effects on the structure of major sensory or motor pathways contralateral to the amputation. Our results are consistent with the organization of these white matter tracts being robust to changes in activity once mature or that continued use of the residual limb (with or without a prosthesis) provides stimulation sufficient to maintain tract integrity.

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.

The role of B cell immunity in VCA graft rejection and acceptance.

Vascularized composite allotransplantation (VCA) has emerged as the most recent field of transplantation to offer an alternative treatment for those patients that have failed or are not suitable candidates for conventional therapy. Most of the current clinical experience in this field is with recipients of skin containing grafts such as the face, upper extremity and abdominal wall transplants. Like solid organ recipients, VCA recipients require lifelong systematic immunosuppression to maintain their grafts. To date, the most successful immunosuppressant regimens are calcineurin inhibitor based and have been targeted to the control of T cells. While these regimens have resulted in excellent short term graft survival in solid organ transplantation, achieving significant improvements in long term survival has been more challenging. The reasons are multi-factorial, but a role for B cells and humoral immunity has been proposed. Antibody mediated rejection leading to chronic rejection has been cited as the leading cause of renal graft loss. While the number of VCA transplants performed is still small, evidence to date suggests that antibody mediated rejection may occur less frequently than seen in solid organ transplants. Here we will discuss the role of B cell immunity in solid organ transplantation as it pertains and contrasts to the field of VCA and present some examples of possible sequela of B cell immunity in a series of hand transplant recipients.

Clinical Significance of Alloantibodies in Hand Transplantation: A Multicenter Study.

BACKGROUND: Donor-specific antibodies (DSAs) have a strong negative correlation with long-term survival in solid organ transplantation. Although the clinical significance of DSA and antibody-mediated rejection (AMR) in upper extremity transplantation (UET) remains to be established, a growing number of single-center reports indicate their presence and potential clinical impact. METHODS: We present a multicenter study assessing the occurrence and significance of alloantibodies in UET in reference to immunological parameters and functional outcome. RESULTS: Our study revealed a high prevalence and early development of de novo DSA and non-DSA (43%, the majority detected within the first 3 postoperative y). HLA class II mismatch correlated with antibody development, which in turn significantly correlated with the incidence of acute cellular rejection. Cellular rejections preceded antibody development in almost all cases. A strong correlation between DSA and graft survival or function cannot be statistically established at this early stage but a correlation with a lesser outcome seems to emerge. CONCLUSIONS: While the phenotype and true clinical effect of AMR remain to be better defined, the high prevalence of DSA and the correlation with acute rejection highlight the need for optimizing immunosuppression, close monitoring, and the relevance of an HLA class II match in UET recipients.

Adipose-derived cellular therapies in solid organ and vascularized-composite allotransplantation.

PURPOSE OF REVIEW: Controlling acute allograft rejection following vascularized composite allotransplantation requires strict adherence to courses of systemic immunosuppression. Discovering new methods to modulate the alloreactive immune response is essential for widespread application of vascularized composite allotransplantation. Here, we discuss how adipose-derived cellular therapies represent novel treatment options for immune modulation and tolerance induction in vascularized composite allotransplantation. RECENT FINDINGS: Adipose-derived mesenchymal stromal cells are cultured from autologous or allogeneic adipose tissue and possess immunomodulatory qualities capable of prolonging allograft survival in animal models of vascularized composite allotransplantation. Similar immunosuppressive and immunomodulatory effects have been observed with noncultured adipose stromal-vascular-fraction-derived therapies, albeit publication of in-vivo stromal vascular fraction cell modulation in transplantation models is lacking. However, both stromal vascular fraction and adipose derived mesenchymal stem cell therapies have the potential to effectively modulate acute allograft rejection via recruitment and induction of regulatory immune cells. SUMMARY: To date, most reports focus on adipose derived mesenchymal stem cells for immune modulation in transplantation despite their phenotypic plasticity and reliance upon culture expansion. Along with the capacity for immune modulation, the supplemental wound healing and vasculogenic properties of stromal vascular fraction, which are not shared by adipose derived mesenchymal stem cells, hint at the profound therapeutic impact stromal vascular fraction-derived treatments could have on controlling acute allograft rejection and tolerance induction in vascularized composite allotransplantation. Ongoing projects in the next few years will help design the best applications of these well tolerated and effective treatments that should reduce the risk/benefit ratio and allow more patients access to vascularized composite allotransplantation therapy.

Accommodation and related conditions in vascularized composite allografts.

PURPOSE OF REVIEW: The outcome of vascularized composite allografts (VCA) often appear unrelated to the presence of donor-specific antibodies (DSA) in blood of the recipient or deposition of complement in the graft. The attenuation of injury and the absence of rejection in other types of grafts despite manifest donor-specific immunity have been explained by accommodation (acquired resistance to immune-mediated injury), adaptation (loss of graft antigen) and/or enhancement (antibody-mediated antigen blockade). Whether and how accommodation, adaptation and/or enhancement impact on the outcome of VCA is unknown. Here we consider how recent observations concerning accommodation in organ transplants might advance understanding and resolve uncertainties about the clinical course of VCA. RECENT FINDINGS: Investigation of the mechanisms through which kidney allografts avert antibody-mediated injury and rejection provide insights potentially applicable to VCA. Interaction of DSA can facilitate replacement of donor by recipient endothelial cells, modulate or decrease synthesis of antigen, mobilize antigen that in turn blocks further immune recognition and limit the amount of bound antibody, allowing accommodation to ensue. These processes also can explain the apparent dissociation between the presence and levels of DSA in blood, deposition of C4d in grafts and antibody-mediated rejection. Over time the processes might also explain the inception of chronic graft changes. SUMMARY: The disrupted tissue in VCA and potential for repopulation by endothelial cells of the recipient establish conditions that potentially decrease susceptibility to acute antibody-mediated rejection. These conditions include clonal suppression of donor-specific B cells, and adaptation, enhancement and accommodation. This setting also potentially highlights heretofore unrecognized interactions between these 'protective' processes.

Magnetic resonance spectroscopy of current hand amputees reveals evidence for neuronal-level changes in former sensorimotor cortex.

Deafferentation is accompanied by large-scale functional reorganization of maps in the primary sensory and motor areas of the hemisphere contralateral to injury. Animal models of deafferentation suggest a variety of cellular-level changes including depression of neuronal metabolism and even neuronal death. Whether similar neuronal changes contribute to patterns of reorganization within the contralateral sensorimotor cortex of chronic human amputees is uncertain. We used functional MRI-guided proton magnetic resonance spectroscopy to test the hypothesis that unilateral deafferentation is associated with lower levels of N-acetylaspartate (NAA, a putative marker of neuronal integrity) in the sensorimotor hand territory located contralateral to the missing hand in chronic amputees (n = 19) compared with the analogous hand territory of age- and sex-matched healthy controls (n = 28). We also tested whether former amputees [i.e., recipients of replanted (n = 3) or transplanted (n = 2) hands] exhibit NAA levels that are indistinguishable from controls, possible evidence for reversal of the effects of deafferentation. As predicted, relative to controls, current amputees exhibited lower levels of NAA that were negatively and significantly correlated with the time after amputation. Contrary to our prediction, NAA levels in both replanted and transplanted patients fell within the range of the current amputees. We suggest that lower levels of NAA in current amputees reflects altered neuronal integrity consequent to chronic deafferentation. Thus local changes in NAA levels may provide a means of assessing neuroplastic changes in deafferented cortex. Results from former amputees suggest that these changes may not be readily reversible through reafferentation.NEW & NOTEWORTHY This study is the first to use functional magnetic resonance-guided magnetic resonance spectroscopy to examine neurochemical mechanisms underlying functional reorganization in the primary somatosensory and motor cortices consequent to upper extremity amputation and its potential reversal through hand replantation or transplantation. We provide evidence for selective alteration of cortical neuronal integrity associated with amputation-related deafferentation that may not be reversible.

Immunobiology in VCA.

Transplantation of vascularized composite tissue is a relatively new field that is an amalgamation of experience in solid organ transplantation and reconstructive plastic and orthopedic surgery. What is novel about the immunobiology of VCA is the addition of tissues with unique immunologic characteristics such as skin and vascularized bone, and the nature of VCA grafts, with direct exposure to the environment, and external forces of trauma. VCAs are distinguished from solid organ transplants by the requirement of rigorous physical therapy for optimal outcomes and the fact that these procedures are not lifesaving in most cases. In this review, we will discuss the immunobiology of these systems and how the interplay can result in pathology unique to VCA as well as provide potential targets for therapy.