Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries, and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. It has been speculated that HEV replicates in sites other than the liver. Since HEV is presumably fecal-orally transmitted it is unclear how the virus reaches the liver and extrahepatic replication could be a possible explanation. The recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication in a swine model. We experimentally infected specific-pathogen-free (SPF) pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were each inoculated intravenously with swine HEV, nineteen pigs (group 2) with the US-2 strain of human HEV, and seventeen pigs (group 3) as uninoculated controls. To identify the potential extrahepatic sites of HEV replication using the swine model, two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days post inoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive strand HEV RNA in each tissue collected during necropsy at different DPIs. A negative strand-specific RT-PCR was standardized and used to detect the replicative, negative-strand of HEV RNA from tissues that tested positive for the positive strand RNA. As expected, positive strand HEV RNA was detected in almost every type of tissue at some time point during viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine and human HEV inoculated pigs. However, replicative, negative strand of HEV RNA was detected primarily in the small intestine, lymph nodes, colon, and liver. Our results demonstrate for the first time that HEV replicates in tissues other than the liver and that the gastrointestinal tract is also the target of virus infection. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV. / Master of Science

Les conversions enzymatiques des antigènes du système des groupes sanguins ABO et de l'antigène majeur de xénotransplantation α3Gal représentent des alternatives thérapeutiques afin d'améliorer l'approvisionnement des hôpitaux en sang de groupe O et la transplantation d'organes. La famille GH110 des glycoside hydrolases comprend des enzymes actives sur les antigène B et α3Gal à pH neutre et fonctionnant avec un mécanisme catalytique par inversion. La sous‐famille GH110_A regroupe des membres actifs exclusivement sur l'antigène B, tandis que la sous‐famille GH110_B regroupe des membres actifs sur les antigènes B et α3Gal. Le travail présenté dans ce manuscrit décrit les études structurales et fonctionnelles de deux α‐galactosidases, BtGal110A (GH110_A) et BtGal110B (GH110_B), de la famille GH110 provenant de la bactérie commensale Bacteroides thetaiotaomicron et représentatives des chacune des ces deux sous‐familes. La spécificité de substrat et la grande efficacité catalytique de ces enzymes à pH neutre ont été confirmées grâce à des oligosaccharides mimant l'extrémité des antigènes B et α3Gal. BtGal110A et BtGal110B se replient en hélice β droite et se distinguent d'autres protéines au repliement similaire par la présence de deux domaines additionnels qui adoptent un repliement en demi tonneau β et participent à l'architecture du site actif. La machinerie catalytique de BtGal110A et BtGal110B est constituée de trois acides aspartiques, semblable à celle des enzymes des familles GH28 et GH49, qui partagent la même repliement en hélice β. / Enzymatic conversions of the ABO blood group antigens and of the major xenotransplantation α3Gal antigen represent therapeutic alternatives intended to improve supply of hospitals with requested blood type O and organs. The family GH110 of glycoside hydrolases includes enzymes active towards B and α3Gal antigen within neutral pH and employs an inverting catalytic mechanism. The GH110_A subfamily gathers enzymes with exquisite substrate specificity towards the B antigen, while the GH110_B subfamily gathers enzymes active towards the B and the α3Gal antigens. The present manuscript describes the functional and structural studies of two α‐galactosidases BtGal110A (GH110_A) and BtGal110B (GH110_B) from the commensal bacteria Bacteroides thetaiotaomicron, two representative members of each of these two subfamilies. The substrate specificity and high catalytic efficiency of these enzymes at neutral pH were confirmed using oligosaccharides corresponding to the terminal carbohydrate sequences of the B and α3Gal antigens. BtGal110A and BtGal110B contain a right‐handed β‐helix, but he presence of two additional domains tfolded as half β‐barrels, which participate to the architecture of the active site distinguishes BtGal110A and BtGal110B fro other related glycoside hydrolases. The catalytic machinery consists of three aspartic acids, reminiscent of that of enzymes from the GH28 and GH49 families, which share a similar β‐helix fold. Our observations support the membership of family GH110 to the clan‐N of glycoside hydrolases.

Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (similar to 3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 +/- 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 mu m), and purity was on average 63 +/- 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates=175 +/- 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized conditions. These immature islets undergo plasticity in culture and form fully functional multicellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissue-derived organoids termed "pancreatites," as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.

Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.

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Islet transplantation is a more physiological way to treat type 1 diabetes. However, shortage of donor tissue and chronic administration of immune suppressive drugs has limited the widespread application of this therapy for all patients with type 1 diabetes, particularly children suffering from this disease. Xenogeneic islet transplantation particularly neonatal porcine islets (NPI) holds promise for clinical transplantation because of the potentially unlimited supply of islets. New evidence suggests that monoclonal antibodies (mAbs) specific for immune cell surface molecules could be employed in the prevention of islet graft rejection as well as induction of immunological tolerance to the transplanted grafts without the need for continuous administration of harmful immune suppressive drugs. It was shown by our group that short-term administrations of a combination of anti-LFA-1 and anti-CD154 mAbs which targets both adhesion and costimulatory pathways of T cell activation, is highly effective in preventing NPI xenograft rejection. In this thesis, we determined whether short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs could induce tolerance to NPI xenografts. Our data show that this combination of mAbs can induce dominant, species and tissue specific tolerance to NPI xenografts which is mediated by regulatory T cells in non-autoimmune prone B6 mice. We also found that T cell subsets such as CD4+ and CD8+ T cells as well as antigen presenting cells (APC) play an important role in the induction and maintenance of tolerance to NPI xenografts. In addition we found that PD-1/PDL interaction is important for induction and maintenance of tolerance to NPI xenografts. Finally, we found that this combined mAb therapy was effective in preventing NPI xenografts rejection in autoimmune prone NOD mice when it was combined with anti-CD4 mAb. It is may hope that the research presented in this thesis will provide insight into the nature of the immune responses to xenogeneic islet transplantation in humans and aid in the development of effective, tolerance inducing therapies, so that patients with T1DM will once again know a life free from their disease. / Experimental Surgery

Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.

Resumo: 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... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: 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) / Orientador: Maria Rita Pacheco / Coorientador: Wilter Ricardo Russiano Vicente / Banca: José Ricardo de Figueiredo / Banca: José Antonio Visintin / Banca: Joaquim Mansano Garcia / Banca: Paulo Henrique Franceschini / Doutor

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18896号 / 医博第4007号 / 新制||医||1009(附属図書館) / 31847 / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂井 義治, 教授 伊達 洋至, 教授 前川 平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM