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The subject of developed theme is xenotransplantation as future alternative to allotransplantation. because of its an experimental nature and being unrecognized as therapy method, xenotransplantation causes ethical and legal problems, which must be solved before the clinical trials start. Variuos sources of information and reports of councils on bioethics are used to examine the ethical issues raised by xenotransplantation. The other part of work is based on overlooking and analysis of World health organization, council of Europe and European Union laws concerning xenotransplantation, which help to reveal the legal problems. The situation in Lithuania is presented.

Islet transplantation has the potential to be an effective treatment for patients with type 1 diabetes. However, a shortage of human donor islets and the need for continuous immunosuppressive therapy currently limit this therapy to patients with brittle type 1 diabetes. Neonatal pigs may provide an unlimited source of islets for transplantation; however, the barrier of islet xenograft rejection must still be overcome. Understanding the mechanism of neonatal porcine islet (NPI) rejection will help to develop targeted therapies to prevent rejection. This thesis studied the early immune cells and molecules involved in NPI xenograft rejection, compared the role of NK cells in two models of islet xenotransplantation and investigated the role of T cell co-stimulatory and adhesion pathways in NPI xenograft rejection. Targeting these aspects of the immune response to NPI xenografts with short-term therapies may play a role in improving NPI xenograft acceptance and induce long-term xenograft survival.

Islet transplantation is an attractive method to achieve blood glucose homeostasis. However, β-cell function declines over time. Therefore, it is necessary to explore strategies to enhance the β-cell mass and function. Also, because there is a severe shortage of human cadaver tissue, alternative sources of insulin secreting tissue need to be examined. Neonatal porcine islet (NPI) tissue has emerged as an attractive alternative source of β-cells. The aim of this thesis was to optimize the culturing conditions of NPIs pre-transplantation so that the available tissue can be used as efficiently and economically as possible. The results from this study indicate that the treatment of NPI cultures with z-VAD-FMK, a pan caspase inhibitor and general protease inhibitor significantly enhances β-cell survival. Additionally, the optimum length of culturing NPIs pre-transplantation appears to be 3-5 days. Since widespread cell death stimulates immunogenic response, this treatment also has the potential benefit of reducing immunosuppression needs in the recipient. / Experimental Surgery

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-22Bitstream added on 2014-06-13T19:45:12Z : No. of bitstreams: 1 bezerra_mb_dr_jabo.pdf: 930909 bytes, checksum: f1899f875197e48c17afaf824f63cfca (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Objetivou-se testar diferentes protocolos de cultivo in vitro e in vivo de folículos ovarianos pré-antrais de fetos bovinos. Para tanto, um total de 41 ovários de fetos bovinos foram obtidos em matadouro, transportados e utilizados para o cultivo in vitro (n=20) e para o xenotransplante (n=21). Após processados no laboratório em fragmentos entre 0,5 e 1 mm3 foram encaminhados para os cultivos O cultivo in vitro baseou-se em protocolo bem sucedido de cultivo de FOPA em caprinos e testou diferentes fontes de macromoléculas e a utilização do azul de tripan na viabilidade dos tecidos cultivados a uma atmosfera controlada de 5% CO2 em ar, a 38,5°C e nutridos com dMEM (300 mOsm/L, pH 7,2) suplementado com antibióticos, ITS, piruvato de sódio, glutamina, hipoxantina, dAMPc, bFSH e IGF-I. A depender do tratamento, foi adicionado BSA (0,1%) SFB (10%) ou PVA (1%). O cultivo in vivo de FOPA foi executado por xenotransplante sob a cápsula renal num total de 65 camundongas imunodeficientes. Desenvolveu-se uma técnica de biopsia e verificou-se o efeito do tempo de transplante (30, 60 e 30 e 60 dias após o transplante) sobre a percentagem e a viabilidade de FOPA bem como a possível presença de folículos antrais. Num segundo momento, 32 receptoras receberam estímulo hormonal de 10 UI de eCG (n=18) e 10 UI r-hFSH (n=14). Os resultados mostraram que o cultivo com PVA apresentou FOPA normais em percentagem semelhante aos cultivos com BSA e PVA. Quanto ao cultivo por xenotransplante, observou-se o crescimento sucessivo de FOPA até estádios antrais ao longo do tempo de transplante (> 30 dias). O resultado das estimulações exógenas apresentou folículos antrais com oócitos que apresentaram o cumulus expandido em 2/5 (40%) dos oócitos selecionados para MIV de cada um dos tratamentos propostos. Concluindo, FOPA oriundos de fetos bovinos podem ser cultivados por pelo menos 8 dias em PVA... / This study aimed to evaluate different protocols of in vitro and in vivo preantral follicles (PFs) culture from bovine fetus. Thus, a total of 41 fetal bovine ovaries, from a slaughterhouse, were collected and transported at laboratory for in vitro culture (n=20) and for xenotransplantation (n=21), after processing into small cortical pieces measuring between 0,5 and 1mm3 the slices were cultured. The in vitro culture was based on well successful protocol of preantral follicles in caprine and tested different sources of macromolecules and trypan blue viability of cultured tissues cultured at controlled atmosphere (5%CO2 in air, 39°C). The culture medium used was dMEM (300 mOsm/L, pH 7, 2) supplemented with antibiotics, ITS, sodium pyruvate, glutamine, hypoxanthine, dAMPc, bFSH, and IGF-I. Depending of treatment, was added BSA (0,1%), FCS (10%) or PVA (1%). In vivo culture of preantral follicles was carried out by xenotransplantation under renal capsule of immunodeficient females mice (total of 65) that were submitted to a biopsy technique previously developed for tissue collection and to verify the effectiveness of time of transplantation (30, 60 and 30 and 60 days post surgery) under percentage and viability as well as putative growth of PFs to antral follicles. At second stage, 32 recipient mice were submitted to hormonal stimuli with 10 IU of eCG (n=18) and 10 IU of r-hFSH (n=14). The results showed that PVA culture presented normally PF in similar distribution when compared with BSA and PVA culture. Regarding xenotransplantation, successive growth of PF until antral stages was observed belong time of transplantation. Exogenous stimulation presented oocytes with expanded cumulus in 2/5 (40%) of selected oocytes for IVM from each treatment. Conclusively PFs from fetal bovine ovaries can be cultured at PVA at least 8 days and grows until antral stages after xenotransplantation procedures... (Complete abstract click electronic access below)

L'intégrase (IN) est une enzyme essentielle du cycle réplicatif des rétrovirus, qui catalyse l'intégration de l'ADN viral rétrotranscrit dans le génome de la cellule cible. Des données structurales, précédemment obtenues par notre équipe, ont conduit à la conception rationnelle de molécules susceptibles de bloquer l'IN sous une forme oligomérique inactive. Des tests d'intégration concertée in vitro ont permis d'étudier leur effet sur l'activité enzymatique de l'IN.Le porc est porteur d'un Gammaretrovirus endogène appelé Porcine Endogenous RetroVirus (PERV), susceptible d'être transmis à l'homme lors d'une xénotransplantation. L'étude structurale de l'IN du virus recombinant PERV-A/C a pour but de mieux comprendre son fonctionnement et de guider le développement rationnel d'inhibiteurs limitant le risque de xénozoonose. J'ai modélisé l'intasome de l'IN de PERV-A/C en complexe avec le raltégravir, un médicament utilisé comme traitement anti-VIH. J'ai ensuite mis au point des protocoles de purification permettant d'étudier le domaine carboxy-terminal (Carboxy Terminal Domain ou CTD) de l'IN de PERV-A/C isolé et en complexe avec le CTD du cofacteur cellulaire humain Brd2. L'enveloppe SAXS de ce complexe a été déterminée. En parallèle, une étude par histone array, réalisée avec le CTD seul de l'IN de PERV-A/C, a révélé un profil de spécificité pour des queues d'histones H2B et H3 portant des modifications post-traductionnelles / Integrase (IN) is an essential enzyme in retroviral replicative cycle, which catalyzes the integration of the viral DNA into the target cell genome. Structural data previously obtained by our team led to rational design of molecules likely to block IN in an inactive oligomeric form. In vitro concerted integration assays made it possible to study their effect on IN enzymatic activity. Pig has an endogenous gammaretrovirus called Porcine Endogenous RetroVirus (PERV) which can be transmitted to humans during xenotransplantation. The aim of the structural study of the recombinant virus PERV-A/C IN is to better understand its mechanism and thus guide the rational design of inhibitors limiting xenozoonosis risk. I modeled the PERV-A/C IN intasome in complex with raltegravir, a drug used for HIV treatment. Then I developed purification protocols to study the isolated and complexed PERV-A/C IN Carboxy-Terminal Domain (CTD) with the human cellular cofactor Brd2. The SAXS envelope of the complex was determined. In parallel, a histone array study, performed with the PERV-A/C IN CTD alone, revealed a specificity profile for modified H2B and H3 histone tails

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Clinical islet transplantation is today an established alternative treatment for a selected group of type 1 diabetes patients. The predominant technique for transplantation is infusion of islets in the liver via the portal vein. Obstacles to advancing islet transplantation include limited engraftment resulting from an immediate blood-mediated inflammatory reaction (IBMIR), a life-long need for immunosuppression and the shortage of organs available. In this thesis, innate and adaptive immunity were explored in allogeneic and xenogeneic settings, with the long-term goal of preventing islet graft destruction. Methods for studying immune responses to islets in blood and engrafted islets in liver tissue (intragraft gene expression) were developed and refined. The innate response to human islets and exocrine tissue in ABO-compatible blood was characterized up to 48 h using a novel whole-blood model. Physiological changes in the blood during incubations were explored and adjusted to allow prolonged experiments. Increased production of chemokines targeting CXCR1/2, CCR2 and CXCR3 was observed, accompanied by massive intra-islet neutrophil infiltration. Notably, endocrine and exocrine tissue triggered a similarly strong innate immune response. Two studies of adult porcine islet transplantation to non-human primates (NHPs) were performed. Expression of immune response genes induced in liver tissue of non-immunosuppressed NHPs (≤72 h) was evaluated after porcine islet transplantation. Up-regulation of CXCR3 mRNA, together with IP-10, Mig, MIP-1α, RANTES, MCP-1 and cytotoxic effector molecule transcripts, was associated with T-cell and macrophage infiltration at 48-72 h. Long-term survival (>100 days) of adult porcine islets in a NHP model was later demonstrated using T-cell-based immunosuppression, including co-stimulatory blockade (anti-CD154 mAb). Graft failure was associated with increased levels of circulating, indirectly activated T cells, non-Gal pig-specific IgG and gene transcripts of inflammatory cytokines. Microarray analysis of the response to inflammatory cytokines in cultured porcine islets identified genes involved in cell death, immune responses and oxidative stress; this gene pattern coincided with physiological changes (decrease in insulin and ATP content). In summary, allogeneic whole-blood experiments and xenogeneic in vivo studies underscored the importance of preventing early inflammation and cell-recruitment to avoid islet graft loss in islet transplantation. Long-term survival of porcine islets in NHPs was shown to be feasible using T-cell-directed immunosuppression, including anti-CD154 mAb.

The need for an unlimited source of islets and a safer method of immunosuppression has limited the widespread application of islet transplantation. To remedy the shortage of donor tissue, xenotransplantation of neonatal porcine islets (NPI) has been proposed. In this study we sought to determine if combining co-transplantation of NPI with Sertoli cells (SC) with a short-term monoclonal antibody (mAb) therapy would prevent NPI xenograft rejection. We hypothesize that this combination of treatments will lead to long-term NPI xenograft survival. Our result show a significant increase in the proportion of mice achieving long-term graft survival compared to untreated mice transplanted with NPI alone, as 7/7 mice treated with anti-LFA-1 mAb (p=0.001), 7/8 mice treated with anti-CD154 mAb (p=0.003), and 4/9 mice treated with anti-CD45RB mAb (p=0.020) achieved and maintained normoglycemia long-term. Therefore, we conclude that the combination of mAb therapy with SC is highly efficacious in preventing NPI xenograft rejection. / Experimental Surgery

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23602号 / 医博第4789号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 川口 義弥, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM