Vascularized composite allotransplantation (VCA) has emerged over the last two decades as a promising therapeutic option for persons who have suffered the loss of limbs or who have suffered major facial disfigurement. Despite the clear advantages of facial and upper extremity VCA in terms of function and cosmesis, VCA has elicited a great deal of ethical concern. Much of that concern is centered around whether or not persons should be exposed to the toxic side effects and possible shortening of life associated with immunosuppression as part of treatment for conditions which are not life-threatening. Ethical concern has also been raised about the vulnerability, dignity and autonomy of VCA candidates and about the justice of allocating the necessary resources to research a treatment that seems unlikely to become widely available in the foreseeable future. <br>While this dissertation will demonstrate familiarity with the technical aspects of VCA, and with the ethical issues just mentioned, its focus will be on the implications of this new therapeutic option for the manner in which consent is understood. In particular, it will argue that the nature and duration of the treatment involved in upper extremity and face transplants makes necessary some modification to the theory and practice of consent. The concept of covenant will be put forward as a resource for this modification. Covenants, as agreements which establish and maintain on-going personal relationships of mutual obligation, are both durable and flexible. Covenants, by engaging persons affectively, promote commitment and encourage the formation of strong therapeutic alliances. Such alliances are especially fitting in light of the lengthy and demanding course of VCA, from screening through surgery and years of physiotherapy, maintenance of immunosuppression and self-monitoring for signs of rejection. <br>Covenant consent is needed for VCA because it more adequately describes what is being asked of recipients and what is necessary for the treatment to succeed. It is also needed because it appropriately honors the recipients by understanding them as active partners rather than as passive patients, and as people assuming major burdens and risks while contributing meaningfully to the development of the field. The employment of covenant consent significantly strengthens the ethical justification for vascularized composite allotransplantation of faces and upper extremities by acknowledging what is actually required of patients and by creating a structure through which they are supported in carrying out their commitment through the long, arduous period of rehabilitation and beyond. / McAnulty College and Graduate School of Liberal Arts; / Health Care Ethics / PhD; / Dissertation;

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Vascularized composite allotransplantation has developed as a specialty at the interface of reconstructive and transplant surgery, offering restoration of function and form in scenarios where options for autologous reconstruction are limited, and for which the burden of donor-site morbidity may be high. Over the past 15 years some 28 patients have received face, and 85 patients upper extremity transplants. Results have been encouraging, with good functional outcomes, and the majority of patients reporting return to independence, employment and improved quality of life. However, the immunological management of these patients remains a significant challenge. While conventional immunosuppressive regimens have proven effective in preventing graft loss to rejection, they have failed to protect patients from acute rejection episodes, which have been reported in 85% during the first year post-transplant. When considered alongside the burden of comorbidity associated with life-long immunosuppression, the impetus for development of novel approaches to the prevention of rejection is clear. Induction of hematopoietic mixed chimerism has successfully achieved transplant tolerance, defined as specific unresponsiveness to donor antigens permitting life-long acceptance of transplanted tissues without maintenance immunosuppression, in numerous animal models and recently, of renal allografts in clinical trials. The work presented in this thesis investigates mixed chimerism for induction of tolerance of vascularized composite allografts (VCAs) across class I and II major histocompatibility (MHC) barriers in the Massachusetts General Hospital miniature swine model; a large animal model with defined MHC immunogenetics, and skin closely analogous to that of humans. The data presented demonstrate development of a reproducible model of VCA tolerance and stable hematopoietic mixed chimerism in a preclinical model. Importantly, tolerance extended to all components of VCAs including the epidermis and dermis, a previously un-reproducible finding. In vitro analysis demonstrated no evidence for either IL-2 reversible anergy or cellular regulation as mechanisms of donor-specific unresponsiveness, suggesting that at a systemic level, tolerance in this model may be primarily mediated by clonal deletion. In contrast, characterization of the cutaneous immune system demonstrated rapid infiltration of VCAs with recipient T cells and Langerhans’ cells, which in chimeric recipients did not cause rejection but rather established stable chimerism in all tissue-resident populations including dermal T cells with the phenotype of Tregs (CD25+FoxP3+); findings which suggest tissue-specific, and regulatory, mechanisms may play important roles. These data support the hypothesis that mixed chimerism is sufficient for whole-skin tolerance of VCAs, but further work is required to demonstrate the necessity of stable, rather than transient, chimerism and to confirm the necessity of other systemic or tissue-specific factors for prevention of epidermal rejection.

Les amputations d’un membre supérieur sont non seulement dévastatrices pour le bien-être physique, psychologique et social des patients, mais elles comportent également des répercussions financières importantes pour l’individu et le système de santé. Les allotransplantations de tissus composites vascularisés ont été proposées en tant que solution permettant de rétablir la forme et la fonction au détriment d’une immunosuppression à vie et d’un taux élevé de rejet chronique. Les prothèses myoélectriques combinent l’expertise chirurgicale avec les avancées technologiques pour réhabiliter les fonctions motrices d’un moignon amputé, mais elles demeurent limitées par un taux élevé d’abandon et des coûts importants. Dans les systèmes de santé avec des ressources limitées, les dirigeants ont la tâche complexe de partager équitablement l’allocation de ressources entre plusieurs maladies et interventions. Dans le domaine de l’économie de la santé, les analyses de type coût-bénéfice ont été développées pour répondre à ces questions. Les mesures d’utilité doivent incorporer l’impact que le traitement suscite sur l’espérance de vie et la qualité de vie. Ces utilités sont ensuite rapportées en fonction du coût, ce qui permet aux dirigeants de la santé de déterminer dans quels traitements il serait préférable d’investir les ressources. Dans cette thèse, nous proposons un modèle pour étudier les coûts-utilité des allotransplantations de la main et des prothèses myoélectriques. Pour commencer, une étude pilote a été effectuée sur les amputations du pouce traitées avec des lambeaux libres de l’orteil, ce qui nous a permis de confirmer la faisabilité des questionnaires d’utilité conçus. Par la suite, les utilités ont été mesurées dans une population d’amputés du membre supérieur, de patients réimplantés proximalement et de contrôles en santé. Les résultats démontrent que 1) les patients réimplantés rapportent la meilleure utilité avec les prothèses myoélectriques, 2) les amputés unilatéraux préfèrent significativement les prothèses myoélectriques également, et 3) aucune différence n’a été recelée entre les deux traitements chez les amputés bilatéraux. Au final, une analyse des coûts-bénéfices a été effectuée dans le contexte du système de santé canadien, démontrant que le traitement des patients amputés unilatéralement avec des prothèses myoélectriques permettrait de sauver davantage de coûts, alors que l’écart en épargnes monétaires se rétrécit pour les amputés bilatéraux traités avec une allotransplantation ou une prothèse. Avec les résultats rapportés dans cette thèse, nous pouvons proposer une mise à jour des indications de traitements pour les patients avec une amputation du membre supérieur. Basé sur l’analyse de type coût-utilité, nous concluons que les amputés unilatéraux sont de meilleurs candidats pour des prothèses myoélectriques, alors que les deux traitements sont encore adéquats pour les amputations bilatérales. / Amputations of the upper extremity are not only devastating for the patient’s physical, psychological and social well-being, but they also yield significant financial repercussions to the individual and the healthcare system. Vascularized composite allotransplantations of the upper extremity were proposed as a solution to restore form and function, albeit to the detriment of lifelong immunosuppression and high rates of chronic rejection. New-generation myoelectric prostheses combine surgical prowess with technological refinements to rehabilitate motor functions of the amputated stump, but remain plagued by high rates of abandonment and significant costs. In healthcare systems wherein resources are limited, financial regulators have the difficult task of proposing an equitable divide of resource allocations between a multitude of diseases and interventions. In the field of health economics, cost-benefit analyses were developed to assist in this decision-making process. Utility outcome measures need to encompass the impact that a treatment elicits on life expectancy and quality of life. Comparison of utilities of different interventions as a function of cost further indicates which route healthcare regulators should partake. In this thesis, we propose a model to study cost-utilities of hand allotransplantation and myoelectric prostheses. To begin, a pilot study was performed on thumb amputations treated with free toe flaps, which allowed to confirm the feasibility of the utility questionnaires that we developed. Afterwards, utilities and quality adjusted life years were measured in a population of upper extremity amputees, proximally replanted patients and healthy controls. Findings demonstrated that 1) replanted patients reported the highest utility outcomes for myoelectric prostheses, 2) unilateral amputees significantly preferred myoelectric prostheses as well, and 3) no significant preference between both interventions was obtained in patients with bilateral amputations. Finally, a cost-benefit analysis was performed in the context of the Canadian healthcare system, demonstrating that significant savings can be achieved with treatment of unilateral amputations with myoelectric prostheses, whereas the gap in cost savings between both treatment groups becomes less significant in bilateral amputees. With the findings reported in this thesis, we can propose an update of the indications for treatment in patients with upper extremity amputations. From the perspective of cost-utility analyses, we conclude that unilateral amputees are better candidates for myoelectric prostheses, and that both treatments can still be offered in cases of bilateral amputations.

Les interrogations éthiques sur la greffe du visage semblent incontournables. Quels sont les risques tolérables, non pas de manière irresponsable, mais, en conscience, dans l'intérêt du patient ? Dans quelles mesures les questions éthiques ont-elles été abordées par les équipes chirurgicales avant la réalisation des greffes ? L'éthique peut-elle légitimer cette intervention ? Une étude de la littérature scientifique et de la littérature grise des équipes ayant réalisé une greffe de la face a été menée pour déterminer les questions éthiques soulevées par cette greffe. Elles touchent à la fois au donneur, à sa famille et au receveur. A partir de ces items, une enquête par questionnaire a été conduite auprès de ces mêmes équipes chirurgicales pour évaluer leur prise en charge. Pour ouvrir le débat, associer la collectivité à des fins pédagogiques et tracer la route des nouvelles acceptations, 1000 chirurgiens français ont aussi été interrogés par un questionnaire. Les résultats ont permis d'explorer le problème du consentement de la famille du donneur, la place des proches dans le respect de la mémoire et de la volonté du défunt. Explorer aussi le respect du cadavre, le secret médical, le respect de l'anonymat du donneur, le rôle des médias, et enfin la notion de consentement d'un patient vulnérable et celle de la balance bénéfice / risque pour un traitement dont les bénéfices sont encore incertains. Dans le cadre du visage, ces questions prennent une dimension humaine essentielle... Pour en témoigner les premiers travaux ont porté sur le symbolisme du visage, la défiguration, les enjeux historiques, juridiques, institutionnels et religieux. / The ethical questions about the face transplant seem unavoidable. What risks can be tolerated, not irresponsibly, but in conscience, in the interest of the patient ? To what extent the ethical issues have been addressed by surgical teams prior to the completion of transplants ? Can ethics justify this intervention? A study of the scientific literature and general press of every team having performed the face transplant was conducted to determine the ethical issues raised by this transplantation. They affect both the donor and his family and the recipient. From these items, a questionnaire survey was conducted with the same surgical team to assess the management of these ethical issues. To open the debate, involving the community for educational and trace the way of the new acceptances, 1000 French surgeons were also interviewed by a questionnaire. The results were used to explore the issue of consent of the donor's family, the place of family in respect of memory and the will of the deceased. Explorer also respect the body, medical confidentiality, respect for donor anonymity, the role of media in the first major surgery, and finally the concept of consent of a vulnerable patient and that of the risk / benefit ratio for treatment whose benefits are still uncertain. As part of the face such questions are essential human dimension ... To testify early work focused on the symbolism of the face, disfigurement, historical, legal, institutional, and religious challenges.

Xenotransplantation (XTP; animal-to-human transplantation) is a controversial technology of contemporary scientific, medical, ethical and social debate in Australia and internationally. The complexities of XTP encompass immunology, immunosuppression, physiology, technology (genetic engineering and cloning), microbiology, and animal/human relations. As a result of these controversies, the National Health and Medical Research Council (NHMRC), Australia, formed the Xenotransplantation Working Party (XWP) in 2001. The XWP was designed to advise the NHMRC on XTP, if and how it should proceed in Australia, and to provide draft regulatory guidelines. During the period 2001-2004, the XWP produced three publicly available documents one of which, ‘Animal-to-Human Transplantation Research: A Guide for the Community’ (2003), was specifically designed to introduce the general public to the major issues and background of XTP. This thesis examines XTP in Australia as guided and influenced by this community document. Explicitly, drawing upon actor (actant)- network theory, I will reveal the Australian XTP network and explore, describe and explain XTP problematisations and network negotiations by the enrolled actants on two key concepts and obligatory passage points - animals and risk. These actants include those providing regulatory advice (members of the XWP and the associated Animal Issues Subcommittee), those developing and/or critiquing XTP (official science and scientists), and those targeted by the technology (people on dialysis, with Type-1 diabetes, Huntington’s disease, Parkinson’s disease, pre-or post-human-tohuman transplantation, and their partner/spouse). The stories are gathered through focus groups, semi-structured interviews and document analysis. They reveal ambiguous and sometimes contradictory stories about animals and risk, which influence and impact the problematisations of XTP and its networks. Therefore, XTP mobilises tension; facilitating both support and apprehension of the XTP network and its construction by both the sciences and the publics.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study concerning the lifelong dynamic of undifferentiated cell populations present in the organism expanded the therapeutics frontiers aiming to healing several kinds of wounds and diseases. Different cell populations have been used for this purpose, as those derived from pre-implantation embryos, called embryonic stem cells (ESC); those derived from pluripotent somatic cells niches, responsible for organic regeneration throughout life, named adult stem cells (ASC); and cells derived from the bone marrow mononuclear fraction (BMNC), which has ASC and hematopoietic lineage cells at different stages of differentiation. Important barriers have been overcome aiming to elucidate benefits and risks inherent to cell therapy. However, the knowledge derives from an evolution process fraught with doubts, and there are many things to be unveiled for safe exploration of different kinds of cell therapy, mainly when allogeneic cells are used. Toxoplasmosis is an opportunistic zoonosis caused by the protozoan Toxoplasma gondii, which has a worldwide distribution and affects about a third of the human population. In the acute infection, T. gondii tachyzoites spread up and multiply themselves into host cells. Immune response leads to evasion of the protozoan in tissue cysts of (semi-latent) bradyzoites, mantaining the chronic infection of the host. Immunosuppression, as used in conditioning for allogeneic cells transplantation, is a risk factor for toxoplasmosis reactivation in chronically infected hosts. The studies presented in this thesis were performed aiming to contribute with: a. methods used for molecular detection of allogeneic cells after systemic transplantation by endovenous injection, allowing to mapping the organic distribution and the fate of these cells in recipient body; and b. understanding about interrelationships among conditioning (pharmacological immunosuppression) for transplantation, allogeneic cell therapy with BMNC and toxoplasmosis reactivation in chronically infected hosts. The study described in the Chapter 1 shows a nested-PCR test with increased sensitivity and assurance for molecular diagnosis of presence or absence of male mice cells in body fluids and tissues of female recipients after intravenous transplantation. This method, based on detection of specific DNA fragment from the Y chromosome, was effective for cells from two distinct populations: BMNC and in vitro cultured cells derived from adipose tissue. Therefore, this method can be suitable for other cell types. The study presented in the Chapter 2 shows the effects caused by an immunosuppressive pharmacological regimen with high doses of busulfan and cyclophosphamide (PCT), followed by allogeneic BMNC transplantation in a model of chronic toxoplasmosis in mice. This study was divided in three subsequent steps: I. experimental infection with type II cystogenic strain of T. gondii (ME-49) and characterization of acute and chronic toxoplasmosis; II. evaluation of the pharmacological immunosuppression effects in female Balb/c; and III. evaluation of the effects resulting from transplantation of male mice BMNC to recipient females. The pharmacological immunosuppression caused worsening of the general clinical condition of the animals and high mortality rates. This effect occurred remarkably earlier in animals chronically infected with T. gondii, showing the high risk of the combination between persistent toxoplasmosis and pharmacological immunosuppression in conditioning for allogeneic transplantation. This study showed the safety of the allogeneic BMNC transplantation in female Balb/c independently of the infection by T. gondii, provided that they have not been subjected to conditioning regimen before transplantation. In the Chapter 3, a pharmacological immunosuppressive regimen, with reduced doses of busulfan and cyclophosphamide (PCTrd), was tested as an alternative to minimize conditioning regimen inherent risks in mice chronically infected with T. gondii and submitted to allogeneic transplantation of BMNC. This study was also divided in three sequential steps: I. oral infection of female Balb/c with tissue cysts of T. gondii (cystogenic VEG strain) and monitoring over a 120 days period (chronic toxoplasmosis); II. evaluation of conditioning regimen effects using reduced doses of busulfan and cyclophosphamide (PCTrd); III. endovenous transplantation of allogeneic BMNC from male donors to the female Balb/c. The results showed the safety of the PCTrd for use in female Balb/c chronically infected with T. gondii. Reduced doses of busulfan and cyclophosphamide (PCTrd) resulted in lower incidence and intensity of clinical signs and absence of mortality, in contrast to results that were observed with high doses (PCT) recommended for myeloablation. Transplantation of allogeneic bone marrow-derived mononuclear cells from male donors was safe in the short term for female Balb/c mice submitted to PCTrd regardless of persistent infection by T. gondii. / O estudo da dinâmica das populações celulares indiferenciadas, presentes no organismo ao longo da vida, ampliou a fronteira de alternativas para o tratamento de vários tipos de lesões e de doenças. Diversas populações celulares têm sido usadas para isso, como as derivadas de embriões em fase de pré-implantação, denominadas células-tronco embrionárias (CTE); as derivadas de nichos de células somáticas pluripotentes, responsáveis pela regeneração dos tecidos orgânicos ao longo da vida, chamadas de células-tronco adultas (CTA); e as células da fração mononuclear da medula óssea (CFMO), que conta com CTA e células da linhagem hematopoiética em variados estágios de diferenciação. Importantes obstáculos têm sido superados no que se refere ao conhecimento de benefícios e de riscos inerentes à terapia celular. Contudo, a construção do conhecimento é permeada de dúvidas e há muito a ser desvendado para que se possam explorar com segurança as potencialidades das terapias celulares, especialmente usando células alogênicas. A toxoplasmose é uma zoonose oportunista causada pelo protozoário Toxoplasma gondii, que apresenta distribuição mundial e acomete cerca de um terço da população humana. Na fase aguda da infecção, taquizoítos de T. gondii disseminam-se e multiplicam-se nas células hospedeiras. A resposta imune induz a evasão do protozoário em cistos teciduais de bradizoítos (forma de semilatência), mantendo o hospedeiro cronicamente infectado. A imunossupressão, como a utilizada no pré-condicionamento para transplante de células alogênicas, é um fator de risco de reativação da toxoplasmose em portadores crônicos. Na presente tese, são apresentados estudos realizados com objetivo de contribuir com: a. métodos de detecção molecular de células alogênicas após o transplante sistêmico pela via endovenosa, permitindo o mapeamento da distribuição e do destino destas células no organismo receptor; e b. compreensão das inter-relações envolvendo pré-condicionamento (imunossupressão farmacológica) para o transplante, terapia celular alogênica com CFMO e reativação da toxoplasmose em portadores crônicos. No Capítulo 1 demonstra-se uma nested-PCR que aumenta a sensibilidade e a segurança do diagnóstico molecular de presença ou ausência de células de camundongos machos em fluidos e tecidos de receptoras fêmeas após transplante endovenoso. O método, que envolve a detecção de fragmento de DNA específico do cromossomo Y, foi eficaz para células de duas populações distintas: CFMO e células cultivadas in vitro derivadas do tecido adiposo. Portanto, o método pode ser útil para outros tipos celulares. O estudo apresentado no Capítulo 2 demonstra os efeitos de um protocolo de imunossupressão farmacológica com altas doses de busulfan e ciclofosfamida (PCT), seguido do transplante alogênico de CFMO em um modelo de toxoplasmose crônica em camundongos. Esse estudo foi dividido em três etapas subsequentes: I. infecção experimental com cepa cistogênica do tipo II de T. gondii (ME-49) e caracterização da toxoplasmose aguda e crônica; II. avaliação dos efeitos da imunossupressão farmacológica nas fêmeas Balb/c; e III. avaliação dos efeitos do transplante de CFMO de camundongos machos para as fêmeas receptoras. A imunossupressão farmacológica provocou agravamento do quadro clínico geral dos animais e altas taxas de mortalidade. Esse efeito foi notavelmente precoce nos animais cronicamente infectados por T. gondii, demonstrando o alto risco da combinação entre toxoplasmose persistente e imunossupressão farmacológica na preparação para o transplante alogênico. Demonstrou-se a segurança do transplante alogênico de CFMO em fêmeas Balb/c infectadas ou livres de infecção por T. gondii, desde que não tenham sido submetidas ao protocolo de pré-condicionamento para o transplante. No Capítulo 3, um regime de imunossupressão farmacológica com doses reduzidas de busulfan e ciclofosfamida (PCTdr) foi testado como alternativa para minimizar os riscos inerentes ao pré-condicionamento em camundongos cronicamente infectados com T. gondii e submetidos ao transplante alogênico de CFMO. Esse estudo também foi dividido em três etapas sequenciais: I. infecção oral de fêmeas Balb/c com cistos teciduais de T. gondii (cepa VEG, cistogênica) e monitoramento durante um período de 120 dias (toxoplasmose crônica); II. avaliação dos efeitos do regime de pré-condicionamento, com doses reduzidas de busulfan e ciclofosfamida (PCTdr); III. transplante alogênico endovenoso de CFMO de doadores machos para as fêmeas Balb/c. Os resultados demonstraram a segurança do PCTdr para uso em Balb/c fêmeas cronicamente infectadas com T. gondii. Doses reduzidas de busulfan e ciclofosfamida (PCTdr) resultaram em menor incidência e intensidade de sinais clínicos e ausência de mortalidade, em oposição ao ocorrido com as altas doses (PCT) recomendadas para mieloablação. O transplante alogênico de células mononucleares da medula óssea de doadores machos foi seguro no curto prazo em fêmeas Balb/c submetidas ao PCTdr, independentemente de infecção persistente pelo T. gondii.

Since its beginning, graft rejection remains the key problem of solid organ transplantation. This reaction of the recipient's immune system against mismatched antigens of the transplanted organ causes graft damage and consequently loss of its function. Rejection involves cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Among the genetic factors which may have a prognostic value in rejection risk evaluation are the Human Leukocyte Antigens (HLA) genotype, the Killer Immunoglobuline-like Receptor (KIR) gene repertoir, cytokine and other gene polymorphisms. These factors could be screened for before transplantation to find the best possible combination of genetic characteristics of the donor and recipient and to reveal patients with "risky" genotypes, who may need more intensive immunosuppression and more careful post-transplant follow-up. Molecular factors, such as HLA and non-HLA antibodies, soluble CD30 molecule (sCD30), Hepatocyte Growth Factor (HGF) and other cytokines, measured before and/or after transplantation in the recipient's blood may be helpful for rejection risk estimation and may also be used as post-transplant rejection onset markers. In our study, we focused on some of the above mentioned factors. We found that ethnicity plays a significant role in the...