acase@tulane.edu / Astrocytes are the most numerous nonneuronal cell-type within the brain. These glial cells support activities essential for neuronal function, regulating extracellular concentrations of ions and neurotransmitters, providing metabolic substrates, and maintaining the blood-brain barrier. In addition, astrocytes respond to central nervous system (CNS) disease by becoming reactive, a nonspecific but highly characteristic response that involves various molecular and morphological changes. Quantifiable changes in astrocyte activation and remodeling have been documented in chronic stress, neurodegenerative disease, mood disorders, and CNS infections. However, whether astrocytes remodel in the same way and whether all insults produce the same effect is largely unknown. Here, studies examine astrocyte morphology and immune activation in nonhuman primates across a wide range of CNS diseases including bacterial and viral infection, and self-injurious behaviors. Paraffin-embedded tissue was selected from the Tulane National Primate Research Center archives, and immunofluorescence with TLR2 and GFAP was used to examine immune activation of astrocytes in cortical tissue. In addition, reconstruction of astrocyte morphology using Neurolucida software revealed specific remodeling patterns for each CNS disease state. While immune activation through Toll-like receptor and GFAP expression generally increased in pathological states, specific astrocyte remodeling patterns were observed relative to pathology indicating that astrocyte response to CNS disease is dependent upon cortical location and pathological state. / 1 / Kim Lee

Humans are exceptional in their willingness to and frequency with which they help one another. However, nonhuman primates also exhibit prosocial behavior. Recently, a number of laboratory studies examining prosociality among primates have yielded conflicting results. These contradictory findings may be due to a reliance on human interaction, tokens, or interactions in the direct context of food, a highly valued resource for animals. The current study examined prosocial behavior among capuchin monkeys (Cebus apella) in a tool task designed to address these issues by examining whether capuchins would transfer a necessary tool to a partner in different payoff conditions. Some capuchins’ behavior indicated that they understood the task, passing the tool when a partner and food were present. Notably, tool transfer in both tasks was overwhelmingly active rather than passive, which is unusual in the context of food; indicating active prosocial behavior is present amongst primates other than cooperative breeders.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18124号 / 医博第3844号 / 新制||医||1001(附属図書館) / 30982 / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 武嗣, 教授 大森 治紀, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

La persistance du VIH-1 est associée au maintien de l’inflammation chronique chez les patients infectés, malgré la mise en place de combinaison de traitements antirétroviraux. L’inflammation chronique est associée à un risque augmenté de développer des comorbidités, non associées au SIDA. Les polynucléaires neutrophiles (PNN) sont des cellules myéloïdes qui ont été impliqués dans de multiples maladies inflammatoires chroniques. Néanmoins, leur rôle dans l’infection par le VIH-1 est moins bien connue. Afin de pallier ce manque de connaissances, nous avons évalué l’hétérogénéité des PNNs dans le modèle macaque cynomolgus infecté par le SIVmac251. L’analyse phénotypique par cytométrie de masse a révélé la circulation de PNNs immatures en phase chronique de l’infection. En caractérisant l’hétérogénéité des PNNs au cours de l’infection par le SIV, nous avons observé une augmentation des fréquences des neutrophiles immatures et activés dans le sang dès la primo-infection. En phase chronique, les PNNs immatures et activés étaient toujours significativement augmentés dans le sang et la moelle osseuse. Au cours de l’infection, les PNNs avaient une fonction immunostimulatrice envers la prolifération et la sécrétion cytokinique des lymphocytaire T. L’initiation d’un traitement antirétroviral précoce a permis de restaurer le phénotype des PNNs. Les PNNs sont des cellules à fort potentiel pro-inflammatoire abondantes qui devraient être ainsi considérés comme de nouveaux effecteurs de l’inflammation chronique associée au VIH-1. / Even under combinational antiretroviral treatments (cART), HIV-1 persistence is associated with chronic inflammation in infected patients, leading to an increased risk of non-AIDS-related comorbidities. Polymorphonuclear neutrophils (PMN), have been less studied in HIV infection whereas they were associated with chronic inflammation diseases. To evaluate PMN heterogeneity in SIVmac251 nonhuman primate infection model, we first performed multiparameter single-cell phenotyping by mass cytometry giving a global vision of the immune system. This analysis demonstrated circulation of immature PMN with impaired during chronic infection. Then, we characterized neutrophils heterogeneity in the course of SIV infection. In primary infection, there was an increased frequency of CD10- immature and CD62L-low primed PMNs in peripheral blood. In chronic phase, CD10- immature PMNs were significantly higher in bone marrow and blood, maintaining a primed profile. During SIV infection, PMNs demonstrated variable immunomodulatory function against T cells proliferation and cytokine production. Early cART allowed to restore PMN phenotype. In this study, we provide unprecedented insight into PMN heterogeneity in the course of SIV infection. Since PMN represent 40-70% of circulating leukocytes and primed PMN are more potent to release pro-inflammatory cytokines and to transmigrate, they should be considered as a new player in HIV-1 chronic inflammation.

No description available.

La transplantation d'organes est souvent la meilleure approche thérapeutique pour l'insuffisance organique au stade terminal. Le rejet de greffe est le principal obstacle pour une transplantation réussie, car cette dernière est le plus fréquemment réalisée entre individus génétiquement distincts. Les tissus ou organes transplantés sont généralement reconnus par le système immunitaire comme corps étrangers et sont rapidement détruits. Une série d'approches a été réalisée en clinique pour augmenter l'acceptation de la transplantation d’organes. Les immunosuppresseurs ont un rôle clé dans le combat contre le rejet de greffe. Actuellement, les résultats à court terme de la transplantation d'organes ont été considérablement améliorés avec l'émergence des inhibiteurs de la calcineurine (ICN), mais les résultats à long terme sont encore insatisfaisants. L'une des principales raisons est que les médicaments immunosuppresseurs actuels manquent de spécificité. Ces agents, en particulier ICN, sont considérés comme les facteurs de risque pour la perte tardive du greffon et les décès avec un greffon fonctionnel en raison de la toxicité des médicaments, de la sur-immunosuppression et d'autres effets secondaires. Ainsi, il y a un besoin urgent de rechercher de nouvelles thérapies idéales. Les lymphocytes T jouent un rôle central dans l'initiation du rejet des allogreffes. La pleine activation des cellules T nécessite au moins deux signaux combinés, mais distincts. En plus du signal généré par l'interaction entre le récepteur des cellules T (TCR) et les complexes CMH-peptides, le second signal, appelé signal de costimulation qui est délivré par l'engagement du récepteur de costimulation avec son ligand, est essentiel. L'engagement du TCR en l'absence de signaux de costimulation peut donner lieu à l'anergie des cellules T, un état d'absence de réponse immunitaire. Les molécules costimulatrices acquièrent ainsi l'attention en tant que cibles thérapeutiques potentielles. Du fait que ces molécules soient largement limitées à des cellules T et/ou des cellules présentatrices d'antigène, cibler la voie de signalisation de costimulation offre la possibilité de moduler le système immunitaire d'une manière plus sélective par rapport à des agents immunosuppresseurs conventionnels. À ce jour, de nombreuses molécules costimulatrices ont été identifiées et certaines ont été testées en tant que cibles thérapeutiques dans des modèles de transplantation d'organes. Les axes CD28–CD80/86 et CD40–CD40L sont importants et les deux voies de signalisation de costimulation les mieux connues. Bélatacept est un variant de l'antigène 4 des lymphocytes T cytotoxiques-immunoglobuline G (CTLA4-Ig) qui bloque la voie de signalisation CD28–CD80/86. C'est le seul bloqueur de la costimulation à être approuvé pour utilisation clinique en transplantation d'organes. Par rapport à la thérapie basée sur les ICN pour les receveurs de transplantation rénale, les thérapies à base de bélatacept montrent un taux similaire de survie, une fonction supérieure du greffon et l'amélioration du profil de risque cardiovasculaire. Cependant, bélatacept est également associée à des taux plus élevés de rejet aigu et de syndrome lymphoprolifératif post-greffe (SLPG). Dans notre étude, l'efficacité d'ASKP1240, un nouvel AcM anti-CD40 complètement humain qui bloque la voie de costimulation CD40–CD40L, a été évaluée sur la prévention du rejet d'allogreffe avec le modèle de transplantation rénale chez le singe cynomolgus. Quand ASKP1240 a été administré seul, il a réduit l'incidence du rejet aigu et a prolongé la survie de l'allogreffe rénale dépendamment de la dose administrée. L'acceptation de l'allogreffe rénale a été encore améliorée chez des singes qui ont reçus des traitements d'ASKP1240 combiné avec le tacrolimus (dose sub-thérapeutique) ou le mycophénolate mofétil (MMF). Le rejet aigu d'allogreffe a été totalement éliminé chez ces animaux et la médiane de survie du greffon rénal de ces groupes était significativement plus longue que ceux des groupes avec un traitement monothérapie. ASKP1240 a été bien toléré pour un traitement allant jusqu'à 180 jours. Il n'y avait pas d'effets secondaires évidents, y compris les complications thromboémboliques liées au médicament. L'administration d'ASKP1240 n'a pas induite de libération de cytokines. Ensuite, nous avons étudié les effets d'ASP2409 sur le rejet de l'allogreffe rénale et la survie chez des singes cynomolgus. ASP2409 est une nouvelle CD86-sélective variante de CTLA4-Ig, qui possède une affinité de liaison au CD86 14 fois plus élevée que le bélatacept in vitro et une amélioration de la puissance immunosuppressive. Une haute dose d'ASP2409 en monothérapie a montré des résultats supérieurs dans la réduction de rejet aigu et la prolongation de la survie de l'allogreffe rénale en comparaison avec une faible dose d'ASP2409 en monothérapie. Une faible dose d'ASP2409 en combinaison avec tacrolimus (dose sub-thérapeutique) inhibe complètement le rejet aigu d'allogreffe et prolonge significativement la survie de l'allogreffe rénale par rapport à une monothérapie avec une faible dose d'ASP2409 ou une dose sub-thérapeutique de tacrolimus. La médiane de survie de l'allogreffe des animaux recevant un traitement à base d'une dose élevée d'ASP2409, bélatacept, ou une dose thérapeutique de tacrolimus étaient identiques (> 91 jours). Les traitements à base d'une dose élevée d'ASP2409 présentaient de meilleurs résultats histopathologiques que le traitement à base de bélatacept. En outre, la fréquence des cellules FoxP3+ dans les allogreffes rénales a été observée plus haute dans les traitements à base d'ASP2409 et de bélatacept comparés aux traitements à base de tacrolimus. L'étude a également montré que ASP2409 est sans danger pour les animaux pour les doses qui ont été testées. Nous n'avons pas trouvé de graves effets secondaires liés à ASP2409 au cours des 91 jours d'étude. Collectivement, ces résultats suggèrent que la modulation sélective de la costimulation des cellules T avec des bloqueurs de la costimulation est une stratégie réalisable pour la prévention et le traitement du rejet d'allogreffe. ASKP1240 et ASP2409 sont tous deux des agents immunosuppresseurs prometteurs pour les régimes d'évitement ou de réduction des inhibiteurs de la calcineurine. / Organ transplantation is often the best therapeutic approach for end-stage organ failure. Graft rejection is the major obstacle to successful transplantation because transplantation is most frequently carried out between genetically distinct individuals. Transplanted tissues or organs are usually recognized by the immune system as foreign and are rapidly destructed without immune interventions. A series of approaches have thus been applied in clinic to inhibit the allogenic immune responses and in turn increase organ transplant acceptance. Immunosuppressive drugs are the key players in the "war" against immune cell-mediated rejection of allogenic transplants. Currently, the use of calcineurin inhibitors (CNIs) has dramatically decreased the risk of acute transplant rejection and improved the short-term outcomes of organ transplantation, but the long-term outcomes are still unsatisfied. One of the main reasons causing unsatisfied long-term outcomes is that current immunosuppressive drugs do not specifically target immune cells that cause transplant rejection. These immunosuppressive agents, especially CNIs, are the risk factors for late graft loss and death with functioning graft (DWFG) due to drug toxicity, over-immunosuppression, and other side-effects. Thus there is an urgent need for seeking novel therapies that can selectively eliminate the alloreactive immune responses while preserving the integrity of the remainder of the host immune system. It has been known that T cells play a central role in initiating allograft rejection. Full activation of T cells requires at least two collaborative but distinct signals. The first signal is generated by the interaction between T cell receptor (TCR) and MHC-peptide complex. The second signal, termed costimulatory signal, is delivered through the engagement of costimulatory receptors by their ligands. Importantly, TCR engagement in the absence of costimulatory signals can result in T cell anergy, a state of T cell unresponsiveness. Costimulatory molecules thus gain attention as potential therapeutic targets. Because these molecules are primary expressed on T cells and/or antigen-presenting cells, targeting costimulatory signaling pathway offers the oppertumities to modulate immune system in a more selective way relative to conventional immunosuppressive agents. To date, numerous costimulatory molecules have been identified and some have been tested as therapeutic targets in organ transplant models. CD28–CD80/86 and CD40–CD40L axis are two important and the most well known costimulatory signaling pathways. Belatacept, a variant of cytotoxic T lymphocyte antigen 4-immunoglobulin G (CTLA4-Ig) that blocks CD28–CD80/86 signaling pathway, is the only costimulation blocker to be approved for clinical use in organ transplantation. Compared to CNI-based regimen for kidney transplant recipients, belatacept-based regimens show similar patient and graft survival rate, superior graft function, and improved cardiovascular risk profile. However, belatacept is also associated with higher rates of acute rejection and posttransplant lymphoproliferative disorder (PTLD). Disruption of CD40–CD40L interaction with anti-CD40L mAbs has also been demonstrated to be a reliable approach for preventing acute rejection and for prolonging allograft survival. Unfortunately, unexpected thromboembolic complications in preclinical studies and clinical trials discontinued the development of anti-CD40L mAbs. The main objective of this thesis is to identify the optimal T cell costimulation blockers that can show improved safety and non-inferior efficacy in promoting allograft acceptance relative to current costimulatory blocking agents. Anti-CD40 mAbs are an alternative approach to block CD40–CD40L costimulatory pathway. CD40 is not involved in the stability of platelet thrombi and anti-CD40 mAbs are expected to not cause thromboembolic events. ASKP1240 is a novel fully human anti-CD40 mAb. In this study, the efficacy of ASKP1240 in the prevention of renal allograft rejection was evaluated in cynomolgus monkey kidney transplantation model. When ASKP1240 was administered alone, it dose-dependently reduced the incidence of acute rejection and prolonged renal allograft survival. Renal allograft acceptance was further improved in the monkey which received regiments using ASKP1240 combined with conventional immunosuppressive drugs tacrolimus (sub-therapeutic dose) or mycophenolate mofetil (MMF). Acute allograft rejection was totally eliminated in these animals and the kidney allograft median survival times (MST) of these groups were significantly longer than those of monotherapy groups. ASKP1240 administration did not induce cytokine release. This agent was well tolerated in monkey kidney transplant recipients during the 180-day treatment period. There were no obvious side effects including drug-related thromboembolic complications. Next, we tested the hypothesis that a CD86-selective variant of CTLA4-Ig might show non-inferior efficacy on the prevention of allograft rejection and prolongation of graft survival in comparison with belatacept. CD86 is the dominating ligand between the two natural ligands for CD28 in alloimmune response. Improvements in CD86 binding affinity of CTLA4-Igs confer increased immunosuppressive potency. A CD86-selective CTLA4-Ig variant may reach therapeutic levels of CD86 occupancy while occupies substantially less CD80 ligand than non-CD86-selective CTLA4-Igs. Preservation of CD80 signaling may be favoring to retain protective immunity and to improve safety of CTLA4-Ig therapy. This thesis investigated the effects of a novel CD86-selective CTLA4-Ig variant, ASP2409, on renal allograft rejection and survival in cynomolgus monkeys. ASP2409 possesses 14-fold higher in vitro CD86 binding affinity than belatacept and improved immunosuppressive potency. Compared to no-treatment control, low-dose (0.3 mg/kg) ASP2409 monotherapy significantly prolonged renal allograft survival (MST = 5 and 26 days, respectively) but did not decrease the incidence of acute rejection (8/8). The rate of acute renal allograft rejection was reduced in the group of high-dose ASP2409 monotherapy (2/7) and, correspondingly, a much longer MST (>91 days) was shown in this group. Combination therapy of low-dose ASP2409 and tacrolimus (sub-therapeutic dose) completely eliminated acute allograft rejection and notably prolonged renal allograft survival (MST >91 days). The MSTs of renal allografts in the animals receiving high-dose ASP2409-, belatacept-, and therapeutic dose tacrolimus-based immunosuppressive regimens were same (>91 days). High-dose ASP2409-based regimens exhibited better histopathological results than belatacept-based regimen. Furthermore, higher frequencies of FoxP3+ Tregs in renal allografts were observed in ASP2409- and belatacept-based regimens compared to tacrolimus-based regimen. ASP2409 was also demonstrated to be safe for animals with the doses to be tested. There were no serious side effects related to ASP2409 to be found in term of 91-day study. This study demonstrates that ASKP1240 is as effective as anti-CD40L mAbs on inhibition of acute rejection and prolongation of renal allograft survival, and do not cause thromboembolic complications. Previous studies indicated that CNIs could diminish the effects of anti-CD40L mAbs. In our study, ASKP1240 in combination with tacrolimus or MMF shows improved efficiency. Furthermore, the effects of ASP2409 on promoting renal allograft acceptance are not inferior to belatacept. These results collectively suggest that ASKP1240 and ASP2409 both are the promising immunosuppressive agents for solid organ transplantation.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18186号 / 医科博第51号 / 新制||医科||4(附属図書館) / 31044 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 松岡 雅雄, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’électroporation (EP) in vivo est utilisée comme stratégie d’amélioration de la réponse immune induite par les vaccins ADN. Cependant son effet sur les acteurs du système immunitaire inné reste méconnu. Dans l’objectif de mettre en évidence le comportement cellulaire sur le site de la vaccination, nous avons développé des approches d’imagerie par fluorescence in vivo chez le macaque. Nos résultats montrent que l’EP locale, augmente non seulement la quantité et la distribution de l’antigène vaccinal, mais induit également la mobilisation et la migration des cellules de Langerhans. De plus, l’EP cause un recrutement de leucocytes dans la peau et le tissu sous-cutané et favorise la production de cytokines pro-inflammatoires dans la peau. Ces évènements précoces, qui résultent de l’utilisation de l’EP en tant que système de délivrance des vaccins ADN, mettent en évidence le potentiel de l’EP en tant qu’adjuvant vaccinal. / In vivo electroporation (EP) is used as a strategy to improve the immune response induced by DNA vaccines. However, its local effect on the innate immune cells has not been fully described. We developed in vivo fluorescence imaging approaches to highlight the cell behavior in the site of vaccination in macaques. Our results show that the local EP not only increases the amount and the distribution of the vaccine antigen, but also induces the mobilization and migration of Langerhans cells. Furthermore, EP causes the recruitment of leukocytes into the skin and subcutaneous tissue and promotes the production of pro-inflammatory cytokines. These early events that result from the use of the EP as a delivery system for DNA vaccines, highlight its potential as a vaccine adjuvant.

No description available.