Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders of the gut, which are characterized by an uncontrolled inflammatory response to lumenal content. The main forms of IBD are Crohn’s disease and ulcerative colitis. Only palliative care is available to the several million patients afflicted with IBD worldwide, and the short-term goal of medical treatments is to bring symptoms under control to prevent complications. The mechanisms of IBD pathogenesis are not fully understood, and the development of more effective, ideally curative treatments for IBD and other inflammatory diseases depends upon a better understanding of the regulation of the inflammatory response. Several studies have demonstrated a crucial role for proteases, notably trypsin-like and elastolytic activties, in the maintenance of a chronic inflammatory response of the gastrointestinal tract and associated pain. Therefore, the control of the protease/anti-protease balance appears to be crucial to the development of IBD. The potential therapeutic benefits of protease inhibitors are highlighted by the association of over-expression of the elastase inhibitor, ELAFIN, with a strong protective effect against colitis. Since several years, I have carried out research programs on the pathophysiology of a genodermatosis, Netherton syndrome (NS), where defective control of protease activity leads to severe dysregulation of inflammation and immunity. We have dissected the respective contribution of three hyperactive serine proteases to the NS phenotype, and we identified elastase 2 (ELA2) as a new epidermal protease whose expression has not been reported in any epithelium previously. The present project proposes to investigate the regulation of protease activity in chronically inflamed bowel diseases, based on my expertise on the functional characterization of proteases, and on the strong rationale recently presented for a role of proteases in IBD. Our preliminary data indicate that, in both human and mice, ELA2 is expressed in the monolayer intestinal epithelium under physiological and ulcerative conditions. Moreover, the elastolytic activity is increased in monolayer intestinal epithelial cells during colitis. Consequently, further characterization of ELA2 hyperactivity will focus on its capability to induce pro-inflammatory mediators. To provide an in vivo demonstration of the involvement of ELA in colitis, an ELA2 conditional knockout mouse model will be developed. In addition, the protein targets of ELA2 activity will be identified by using an innovative proteomic approach (ICAT, Isotope-Coded Affinity Tag). This study should bring forth new insights into bowel homeostasis, and reveal new therapeutic targets for IBD treatment. The project will be extended to the identification of hyperactive proteases released within the intestinal tissue of IBD patients. Proteolytic activities will be detected by zymographic analysis of gel-fractionated protein extracts, and then assigned to specific proteases using mass spectrometry. The characterization of the hyperactive proteases hyperactive in the inflamed gut, their substrates, and the consequences of their increased activity will advance our knowledge of the biological cascades initiated by protease dysregulation in IBD. We expect our analysis of protease hyperactivity to identify control points in the mechanisms of disease that could be addressed by the development of specific inhibitors, as the basis of novel therapeutic approaches for IBD.

Heart failure (HF) is a clinical syndrome in which pathological stress or injury is associated with a failure of cardiac performance to meet the metabolic demands of the body. Age is a major risk factor for HF, at least in part because it prolongs exposure to hypertension, diabetes and other cardiovascular risks. However, intrinsic cardiac aging, the slowly progressive structural changes and functional declines with age also makes the heart more susceptible to stress and contributes to increased cardiovascular mortality in the elderly. HF is the end consequence of a “long-term” cardiac remodelling. In the early phase, ventricular remodelling is a physiological compensatory response mainly characterized by cardiomyocyte hypertrophy. Late remodelling, characterized by cardiomyocyte loss and fibrotic response, leads to progressive HF. One of the major pitfalls in treating HF is a lack of appropriate explanation on the mechanisms responsible for myocardial decompensation. Thus, every step toward this goal will represent a new therapeutic potential to avoid decompensation and increase patient survival. Reactive oxygen species (ROS) appear to play a prominent role in triggering and maintaining ventricular damage, thus accelerating the progression of HF. Indeed, it has been shown that enhanced oxidative stress is involved in myocyte death in the aging heart. In addition, progression of cardiac damages in animals is attenuated by the administration of antioxidants. Therefore, it has been proposed that oxidative stress plays an important role in cardiac remodelling, by precipitating the progression from hypertrophy to failure. The mitochondrial monoamine-degrading enzyme monoamine oxidase (MAO)-A has recently been identified as a major source of ROS in the heart. Besides its classical role in the degradation of monoamines (5-HT, catecholamines), cardiac MAO-A is also involved in receptor-independent effects of 5-HT, by generating hydrogen peroxide (H2O2). In rat cardiomyocytes, depending on the concentration of substrate, ROS generated by MAO-A can induce cardiomyocyte apoptosis or necrosis. In addition, we showed that cardiac MAO-A was strongly upregulated in human dilated cardiopathy (unpublished data) and in the aging heart. These observations prompted us to analyse the consequences of MAO-A upregulation in cardiomyocytes. For this purpose, we generated two independent lines of transgenic mice with cardiac-specific overexpression of MAO-A. We found that transgenic mice died prematurely from HF due to enhanced oxidative stress and myocyte drop-out (unpublished data). These results prompted us to hypothesize that MAO-A may be a death and senescence-associated factor in the heart. In this proposal, our goal is to better understand the mechanisms of action of MAO-A metabolites (ROS and aldehydes) in the transition from hypertrophy to failure and in oxidative damage associated with aging. This project will have complementary in vitro and in vivo approaches. In vitro experiments will be performed on rat neonatal cardiomyocytes infected with MAO-A adenovirus to evaluate: 1) the participation of biogenic aldehydes generated by MAO-A in cardiomyocyte death; 2) the apparition of senescence markers following MAO-A overexpression; 3) the potential of MAO-A inhibitors in preventing cardiomyocyte death induced by pro-apoptotic agents. For our in vivo studies, we will evaluate the role of aldehyde in the development of HF induced by aortic banding or by MAO-A overexpression in mice, by treating mice with different carbonyl scavengers. The evolution toward HF will be followed, together with the apparition of senescence markers. The role of MAO-A in the transition from hypertrophy to failure will be evaluated by using selective MAO-A inhibitors in these same models. This project should help us understand the importance of oxidative stress and aldehydes in the development of HF associated with MAO-A, and give new therapeutics directions

Memory impairment in predemential Alzheimer's disease has usually been related to medial temporal lobe dysfunction. However, atrophy of this region also induces remote cortical reorganization, resulting in both deleterious and compensatory effects with respect to memory performance. The interplay between deleterious and compensatory mechanisms remains improperly understood, partly because these effects have been shown using different neuroimaging methods (basal state (SPECT, PET), resting state (fMRI) or activation studies (PET, fMRI)). The general objective of this research project is to gain a better understanding of the mechanisms underlying cortical reorganization in relation to memory impairment in predemential AD. We identified a series of issues (section 1.2) to propose a research project based on five different studies (section 1.3). We expect our findings will provide new insights in the memory impairment of predemential AD, its functional reorganisation and ultimately, in how to monitor and modulate these reorganization mechanisms (section 1.4). The five studies described in the following project follow a broad logical order, although each is independent and is used to tackle specific scientific questions. Basically, Study 1 aims at showing positive and negative reorganisation mechanisms using PET at basal state, Study 2 aims at assessing the effect of the reorganisation of these mechanisms on memory and Study 3 will complement these findings using cerebral structural connectivity. Study 4 will be focused on bridging methodological issues between basal state PET and resting state fMRI, building mainly on study 1. We will be using CMRGlu in FDG-PET at rest for this study. Study 5 is somehow the result of all previous studies as we will incorporate in this one the methodological issues that will have been addressed in previous studies, notably Study 1 and 4. We will investigate in this study the issue of the baseline condition during plasticity imaging and during activation tasks. This project is lead by a young neurologist, Dr. Jérémie Pariente ('MCU-PH' Toulouse University Hospital and Inserm U 825). The present project involves three other young scientists, Dr. Emmanuel Barbeau (CR2, psychologist), CNRS UMR 5549, Dr. Florence Remy (MCU, physicist), CNRS UMR 5549 and Xavier Franceries (MCU, physicist), Inserm UMR 825 all in Toulouse, France. They come from two different labs that support their project, want to build a new expert group on AD and have significant and complementary expertise on the different fields required to complete this project. The synergy between the different participants to this project is good and balanced between clinical and methodological expertise. It relies on true (and demonstrated through publications) expertise on the different fields required to complete the project satisfactorily: neurology, neuropsychology, memory assessment, neuroimaging techniques, plasticity, methodological developments, research project management.

One of the major challenges in transplantation medicine is to control the very strong immune-responses to foreign antigens responsible for graft-rejection. Whereas immunosuppressive drugs efficiently inhibit acute graft rejection, a non-diminishing proportion of patients suffers from chronic rejection which ultimately leads to functional loss of the graft. Moreover, these drugs have severe side-effects (most notably opportunistic infections, cancer, and nephrotoxicity), in part due to the general immunosuppression they cause. Induction of specific immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with toxic immunosuppressive drugs, and is therefore the 'Holy Grail' of transplantation medicine. Tolerance is not only important in transplantation, but also in physiological conditions. During their development, T and B lymphocytes somatically mutate the genes encoding their receptors for antigen. This process being stochastic in nature, many of these developing cells express antigen-receptors specific for autoantigens and will therefore need to be controlled by tolerance-inducing mechanisms. A major mechanism relies on the activity of regulatory T cells. These cells play a crucial role in maintenance of tolerance to self and their absence leads to a lethal autoimmune syndrome. They also play an important role in the control of immunity to infections, to tumors, and to the fetus. We have hypothesized that regulatory T cells could induce tolerance to transplanted tissues and organs. Indeed, injection of regulatory T cells together with a bone-marrow graft in mice, fully and definitively prevented rejection. However, when we tried to apply this strategy to skin or heart allografts, we failed to prevent rejection. Data from the literature suggested that survival of regulatory T cells required continued stimulation with antigen. Since only limited numbers of antigen-presenting cells are present in transplanted tissues, we argued that this might limit the survival of the regulatory T cells we injected. Transplantation of bone-marrow, which contains many antigen-presenting cells and their precursors, would allow for a better survival of regulatory T cells. We have been able to confirm this hypothesis, and argued that co-transplantation of bone-marrow and a skin or heart allograft might allow for prevention of rejection. We have tested this hypothesis by grafting mice with bone-marrow, injecting them with regulatory T cells, and then grafting them allogeneic skin or hearts. Our recently published results showed that this protocol allows for prevention allograft-rejection. Very importantly, in contrast to immunosuppressive drugs, regulatory T cells not only prevented acute but also chronic rejection. Regulatory T cell-based therapy for prevention of allograft-rejection is therefore better than drug-based therapies. In contrast to immunosuppressive drugs, regulatory T cells are specific for antigen and their therapeutic use should be free of the side effects of drugs. On the other hand, regulatory T cells are known to control of immunity to infections and to tumors. It will therefore now be important to verify that their therapeutic use does not render mice more susceptible to infections, to de novo tumorigenesis, and to reactivation of dormant tumors. In our hands, one single injection of regulatory T cells was sufficient to prevent rejection of allografts in mice for up to 250 days. However, will this very good durability be sufficient in Man? Key to this question are the cellular mechanisms involved in the tolerant state. We will therefore analyze these mechanisms. Most importantly, we will assess if injected regulatory T cells need to persist to assure tolerance or if they can transfer their tolerogenic capacity to other T cells, thereby assuring durability of tolerance. Other important issues will need to be addressed before regulatory T cell-based therapy for prevention of allograft-rejection can be transposed to the clinic. The preconditioning regimen we used may not be totally applicable in clinical settings and we will need to evaluate if other regimens are compatible with our protocol. The conditions used for the in vitro culture of injected regulatory T cells, required in our protocol, will need to be adapted. We also feel that it is important to assess if the induced tolerant state resists to very strong immunogenic stimuli associated with a variety of infections. Therapeutic use of regulatory T cells, central players in control of immunological homeostasis, may turn out to be the ideal (long sought-after) means for induction of tolerance to allografts. Phase I clinical trials with regulatory T cells are currently in progress, and a more solid understanding of their in vivo characteristics should in the near future allow for their use in cell-based therapies that will permanently prevent allograft rejection, without undesired side-effects.