Malarial hemozoin is a Nalp3 inflammasome activating danger signal.

BACKGROUND: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents. METHODOLOGY/PRINCIPAL FINDINGS: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged. SIGNIFICANCE/CONCLUSIONS: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.

The role of macrophage migration inhibitory factor in mouse islet transplantation.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells. METHODS: This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice. RESULTS: Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups. CONCLUSION: These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.

Intravitreal ranibizumab for AMD : the individual rhythm of OCT guided retreatment intervals

Purpose: We intended to determine whether the need for retreatmentwith intravitreal ranibizumab follows an individual rhythm in patientswith exudative AMD. Setting: Prospective mono-centre cohort study.Methods: Prospective study. 48 patients with exudative AMD. 3 loadingdoses of ranibizumab, followed by a 12 months PRN regimen guided byearly exudative signs on SD-OCT. An intensified follow-up allowed todetect recurrences early.Results:Mean VA improved by 6.4 letters at month 3 and by 13.1 letters atmonth 12, with a mean of 8.0 injections (range 0-12) during the maintenancephase. The intra-individual variance of the intervals was relativelysmall and ranged within 20% of themean interval in 91% of patients.Thefirst interval was within 1 week of the mean interval in 84% of patients.The retreatment criteria were stable in 89% of patients.Conclusion: The relative stability of the intra-individual intervalsmay allowsimplifying the care for AMDpatients.Theremay be a predictive role for thefirst interval after the loading phase. The functional results of this PRNregimenwith early retreatmentwere excellent. Financial disclosure:None.

Isolamento de Aeromonas hydrophila em girinos de rã-touro na metamorfose

O objetivo deste trabalho foi isolar o agente causador da doença-da-perna-vermelha em girinos de Rana catesbeiana em fase de transformação. Coletaram-se 20 girinos, na fase G4, apresentando prostração, anorexia, pele ressecada, pernas posteriores hemorrágicas e natação errática. Amostras do coração, fígado e partes da perna foram inoculadas em meio de cultura ágar triptona de soja e ágar sangue, a 25°C, por 48 horas. Aeromonas hydrophila foi a principal causa do surto de mortalidade. A fim de avaliar o efeito dessa bactéria nos girinos, realizou-se inoculação por via oral e intraperitoneal de 10(6) unidades formadoras de colônia por mililitro, e verificou-se o aparecimento de petéquias hemorrágicas na boca e nos órgãos internos.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

Th2 activities induced during virgin T cell priming in the absence of IL-4, IL-13, and B cells.

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-gamma as they begin to proliferate. Parallel respective induction of B cells to produce gamma1 or gamma2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and gamma1 and epsilon switch transcript production were underway 48-72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The gamma1 and epsilon switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.

Substance P-induced skin inflammation is not modulated by a single dose of sitagliptin in human volunteers.

Substance P (SP), an undecapeptide belonging to the tachykinin family, is released during the activation of sensory nerves, and causes vasodilation, edema and pain through activation of tissular Neurokinin 1 receptors. SP proinflammatory effects are terminated by angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP), while the aminopeptidase dipeptidylpeptidase IV (DPPIV) can also play a role. The aim of this randomized, crossover, double-blind study was to assess the cutaneous vasoreactivity (flare and wheal reaction, burning pain sensation) to intradermal injection of ascending doses of SP in six volunteers receiving a single therapeutic dose of the DPPIV inhibitor sitagliptin or a matching placebo. Cutaneous SP challenges produced the expected, dose-dependent flare and wheal response, while eliciting mild to moderate local pain sensation with little dose dependency. However, no differences were shown in the responses observed under sitagliptin compared with placebo, while the study would have been sufficiently powered to detect a clinically relevant increase in sensitivity to SP. The results of this pilot study are in line with proteolytic cleavage of SP by ACE and NEP compensating the blockade of DPPIV to prevent an augmentation of its proinflammatory action.

Therapeutic applications of viscous and injectable poly(ortho esters).

Poly(ortho esters) (POE) are hydrophobic and bioerodible polymers that have been investigated for pharmaceutical use since the early 1970s. Among the four described generations of POE, the third (POE III) and fourth (POE IV) are promising viscous and injectable materials which have been investigated in numerous biomedical applications. POE III has been extensively studied for ophthalmic drug delivery, it presents an excellent biocompatibility and is currently being investigated as a vehicle for sustained drug delivery to treat diseases of the posterior segment of the eye. POE IV is distinguishable by a highly reproducible and controlled synthesis, a higher hydrophobicity, and an excellent biocompatibility. It is currently under development for a variety of applications, such as ocular delivery, periodontal disease treatment and applications in veterinary medicine. This review will also focus on new perspectives for this promising family of polymers, such as guided tissue regeneration, treatment of osteoarthritis, as well as peptide and protein delivery.

Fibrose rétropéritonéale: cause rare d'insuffisance rénale postrénale, a ne pas méconnaître [Retroperitoneal fibrosis: a rare cause of postrenal kidney failure, no to be missed].

We report the observation of a fifty years old man, admitted in the emergency room for bilateral lumbar pain and hyperkaliemic metabolic acidosis, and postrenal kidney failure induced by bilateral hydronephrosis. Radiographic exploration and histologic studies of biopsy confirmed an idiopathic retroperitoneal fibrosis that clinically and biologicaly responded to three seances of hemodialysis, and insertion in each uretere of one double J stent, and long term corticotherapy. The retroperitoneal fibrosis is a little common inflammatory disease, characterized by the development of a fibrous mass around the retroperitoneal structures. His diagnostic means evolved. On the other hand, his treatment was the object of no checked controlled and randomized trial. This article proposes an updating of the knowledge on this subject.

Postnatal intracerebroventricular administrations of NGF alter spatial memory in adulthood

The present work assessed the effects of intracerebroventricular injections (2x5 mg/2.5 ml) of recombined human nerve growth factor (rhNGF) at postnatal days 2 and 3 upon the development of spatial learning capacities in rats. The treated rats were trained at the age of 22 days to escape onto an invisible platform at a fixed position in space in a Morris navigation task. For half of the subjects, the training position was also cued, a procedure aimed at facilitating escape and reducing attention to the distant spatial cues. At the age of 2 months all the rats were retrained in the same task. Treatment effects were found in both immature and adult rats. The injection of NGF induced a slight alteration of the immature rats' performance. In contrast, a marked impairment of spatial abilities was shown in the 2-month-old rats. The most consistent effects were a significant increase in the escape latency and a decrease bias towards the training platform area during probe trials. The reduction of spatial memory was particularly marked if the subjects had been trained in a cued condition. Taken together, these experiments reveal that an acute pharmacological treatment that leads to transient modifications during early development might induce a behavioural change long after treatment. Thus, the development and the maintenance of an accurate spatial representation are tightly related to the development of brain structures that could be altered by precocious NGF administrations.

Childhood and malaria vaccines combined in biodegradable microspheres produce immunity with synergistic interactions.

Biodegradable microspheres may represent a potential tool for the delivery of combination vaccines. We demonstrate strong immunogenicity of five co-encapsulated antigens after a single subcutaneous inoculation in guinea pigs. Tetanus- and diphtheria-specific antibodies were not significantly affected by the presence of either antigen or by the presence of pertussis or Haemophilus influenzae type b (Hib) antigens. Microsphere formulations gave better protection against diphtheria toxin than did two injections of a licensed tetravalent vaccine. Finally, a synthetic malaria peptide antigen (PfCS) also encapsulated in PLGA microspheres increased diphtheria and tetanus-specific immunity and improved protection against diphtheria. These findings demonstrate the potential of microspheres as an alternative and promising strategy for combination vaccines with a further aptitude in reducing the number of inoculations required to gain functional immunity.

Treatment of osteitis pubis with on single iv injection of Ibandronate: report of two cases

Objectives: Osteitis pubis is a noninfectious painfulinflammatory disorder of the symphysis pubis. Etiologicfactors are numerous, the most common are: osseousextension of adductor enthesis due to sport overuse,irritation after urological and abdominal procedures, andsystemic inflammatory disorders in particular spondylarthropathies.Many cases are idiopathic. The symptomsconsist of regional chronic mechanical and sometimenocturnal pain. Diagnosis is usually confirmed by eitherbone scintigraphy or by MRI. There are no standardtreatments but conservative approaches including rest andNSAIDS are generally recommended. In 2001, a goodclinical and radiological response of three refractory caseswith 3-6 monthly perfusions of pamidronate was reported(1). Ibandronate is a much more powerful and long-lastingbisphosphonate than pamidronate, and has not yet beenreported in literature to our knowledge in this indication.Materials/Methods: We present two cases of idiopathicorigin: one woman (63 years old) and one man (36 yearsold).The symptoms were present >3 months in the firstpatient and 1 year in the second. The diagnosis wasconfirmed by MRI which showed bone edema on bothsizes of symphysis and in the second case bony erosionsadjacent to the joint were seen. Both cases failed to respondto conservative measures. Both patients received one singledirect iv Injection of 3 mg of Ibandronate.Results: The injections resulted in a rapid (within a fewdays) resolution of pain that lasted more than 6 months inboth patients. No side effects were observed. In the firstcase, an isotope bone scan performed 4 months after theinjection showed no residual uptake. The second patienthad a repeated MRI after 6 months. It demonstrated anattenuation of bone edema compared to the first MRI.Conclusions: IV Ibandronate may constitute a safe andeffective treatment option for patients with refractoryosteitis pubis.Reference: 1: Maksymowych WP, Aaron SL, Russell AS, JRheumatol 28:2754, 2001.Disclosure of Interest: None declared.

What impact do systemically administrated bisphosphonates have on oral implant therapy? A systematic review.

OBJECTIVES: The aim of this systematic review is to evaluate, analysing the dental literature, whether: * Patients on intravenous (IV) or oral bisphosphonates (BPs) can receive oral implant therapy and what could be the risk of developing bisphosphonate-related osteonecrosis of the jaw (BRONJ)? * Osseointegrated implants could be affected by BP therapy. MATERIAL AND METHODS: A Medline search was conducted and all publications fulfilling the inclusion and exclusion criteria from 1966 until December 2008 were included in the review. Moreover, the Cochrane Data Base of Systematic Reviews, and the Cochrane Central Register of Controlled Trials and EMBASE (from 1980 to December 2008) were searched for English-language articles published between 1966 and 2008. Literature search was completed by a hand research accessing the references cited in all identified publications. RESULTS: The literature search rendered only one prospective and three retrospective studies. The prospective controlled non-randomized clinical study followed patients with and without BP medication up to 36 months after implant therapy. The patients in the experimental group had been on oral BPs before implant therapy for periods ranging between 1 and 4 years. None of the patients developed BRONJ and implant outcome was not affected by the BP medication. The three selected retrospective studies (two case-controls and one case series) yielded very similar results. All have followed patients on oral BPs after implant therapy, with follow-up ranging between 2 and 4 years. BRONJ was never reported and implant survival rates ranged between 95% and 100%. The literature search on BRONJ including guidelines and recommendations found 59 papers, from which six were retrieved. Among the guidelines, there is a consensus on contraindicating implants in cancer patients under IV-BPs and not contraindicating dental implants in patients under oral-BPs for osteoporosis. CONCLUSIONS: From the analysis of the one prospective and the three retrospective series (217 patients), the placement of an implant may be considered a safe procedure in patients taking oral BPs for <5 years with regard to the occurrence of BRONJ since in these studies no BRONJ has been reported. Moreover, the intake of oral-BPs did not influence short-term (1-4 years) implant survival rates.

Viral- and myelin-specific cellular immune response in patients treated with natalizumab: a cross-sectional and a longitudinal prospective study

Introduction: Natalizumab, a monoclonal antibody binding to the alpha4 integrins, is efficient in preventing relapses and progression of disability in multiple sclerosis (MS) patients. However, a total of seven MS patients treated with natalizumab suffered from progressive multifocal leukoencephalopathy (PML), on a total of 53?000 patients (data of March 6, 2009) treated with this drug. PML is a disease affecting immunosuppressed people, which is caused by the polyomavirus JC (JCV). This virus produces a lytic infection of the oligodendrocytes. Yet, natalizumab cannot be considered as a classical immunosuppressant, such as suggested by the fact that no increased incidence of other opportunistic infections was reported with this drug. It has been postulated that, by closing the blood-brain, natalizumab might prevent JCV-specific CD8_ T cells to reach the CNS and perform immune surveillance. Alternatively, it has been suggested that this drug acts by releasing JCV from the bone marrow, one of its site of latency. In this study, we address the question whether there is an increased activity of JCV in the blood of natalizumab-treated MS patients. Material and Methods: In this prospective longitudinal study, we are following a cohort of 24 MS patients receiving monthly injections of natalizumab. Blood and urine are drawn every one to three months, up to 12 months. As a control group, we follow 16 MS patients treated with IFN-beta. For this control group, there are two time-points: before and 1094 months after treatment onset. We are analysing the viral (JCV-, EBV- and CMV-) as well as the myelin- (MOG-, MOBP-) specific cellular immune responses using proliferation and ELISPOT (IFNgamma) assays. For JCV, we study the response against VP1, the major capsid protein. For JCV VP1, MOG and MOBP, we use 15-mer peptides overlapping by 10 amino acids, thus eliciting CD4_ as well as CD8_ T cell response. These peptides encompasse the whole sequence of the proteins. For EBV and CMV, we use pools of immunodominant 8- to 10-mer peptides eliciting CD8_ T cells. At the same time-points, using RTPCR, we determine the presence of JCV DNA coding for the VP1 protein in the PBMC, plasma, and urine. Results: At the time of writing this abstract, 16 patients have reached the 9-month (T9), and 11 the T12 time-point. We expect that by the ISNV meeting in June 2009, 18 and 14 patients will be at T9 and T12, respectively. Virological and immunological results will be presented. 9th International Symposium on NeuroVirology 2_6 June 2009 39 J Neurovirol Downloaded from informahealthcare.com by Cantonale et Universitaire on 06/25/10 For personal use only. Conclusions: This ongoing longitudinal prospective study should tell us whether there is an enhanced JCV activity in the peripheral blood of patients on natalizumab. This work is supported by the FNS (PP00B-106716), the Swiss MS Society and a research grant from Biogen Dompe.

Tolerogenic dendritic cells and their role in induction of immune tolerance

Les cellules dendritiques sont des cellules du système immunitaire qui permettent d'instruire les lymphocytes T, autres cellules de ce système, pour mettre en place une réponse immunitaire adaptée afin de combattre et vaincre une infection. Ces cellules dendritiques vont reconnaître des motifs spécifiquement exprimés par des pathogènes par l'intermédiaire de récepteurs exprimés à leur surface. En détectant ces molécules, elles vont s'activer et subir diverses modifications pour pouvoir activer les lymphocytes T. Elles vont alors interagir avec les lymphocytes Τ et transférer les informations nécessaires pour que ces cellules s'activent à leur tour et produisent différentes protéines de façon à éliminer le pathogène. En fonction du type de pathogène, les informations transférées entre les cellules dendritiques et les lymphocytes seront différentes de manière à produire la réponse immunitaire la mieux adaptée pour supprimer l'élément infectieux. Dans le corps, les cellules dendritiques circulent continuellement afin de détecter les éléments étrangers. Quand elles reconnaissent une protéine étrangère, elles la phagocytent, c'est-à-dire qu'elles la mangent afin de pouvoir la présenter aux lymphocytes T. Mais quand elles phagocytent un élément étranger, elles peuvent également prendre des éléments du soi, comme par exemple quand elles phagocytent une cellule infectée par un virus. Les cellules dendritiques doivent alors être capables de différentier les molécules du soi et du non-soi de façon à ne pas induire une réponse en présentant un antigène du soi aux lymphocytes T. D'autant plus que lors de leur développement, les lymphocytes Τ qui sont capables de reconnaître le soi sont éliminés mais ce système n'est pas parfait et donc certains lymphocytes Τ auto-reactifs peuvent se trouver dans le corps. Il existe ainsi d'autres mécanismes en périphérie du site de développement pour inhiber ces lymphocytes Τ auto-reactifs. Ce sont les mécanismes de tolérance. Quand les lymphocytes Τ induisent une réponse aux antigènes du soi, cela résulte à des maladies auto-immunes. Dans mon projet de recherche, nous avons travaillé avec des lignées de cellules dendritiques, c'est-à-dire des cellules dendritiques semblables à celles que l'on peut trouver in vivo mais qui sont immortalisées, elles peuvent donc être cultiver et manipuler in vitro. Nous avons génétiquement modifiées ces lignées cellulaires pour qu'elles expriment des molécules immunosuppressives afin d'étudier comment induire une tolérance immunitaire, c'est-à-dire si l'expression de ces molécules permet d'éviter de générer une réponse immunitaire. Pour cela, nous avons utilisé des modèles murins de tumeurs et de maladies auto-immunes. Nous avons démontré que ces lignées de cellules dendritiques peuvent être un grand outil de recherche pour étudier les bénéfices de différentes molécules immuno-modulatrices afin d'induire une tolérance immunitaire à différents antigènes. - Les cellules dendritiques sont responsables de l'induction des réponses immunitaires adaptatives. Suite à une infection microbienne, les cellules dendritiques s'activent, elles induisent l'expression de molécules de costimulation à leur surface, sécrètent des cytokines et induisent la différentiation des cellules Τ effectrices et mémoires. De plus, les cellules dendritiques ont un rôle important dans l'induction et la maintenance de la tolérance immunitaire au niveau du thymus et en périphérie, en induisant l'anergie, la délétion ou la conversion des cellules Τ naïves en cellules régulatrices. Dans notre groupe, une nouvelle lignée de cellules dendritiques appelée MuTu a été crée par la culture de cellules dendritiques tumorales isolées à partir d'une rate d'une souris transgénique, dans laquelle l'expression de l'oncogène SV40 et du GFP sont sous le contrôle du promoteur CD1 le, et sont ainsi spécifiquement exprimés dans les cellules dendritiques. Ces nouvelles lignées appartiennent au sous-type des cellules dendritiques conventionnelles exprimant CD8a. Elles ont conservé leur capacité d'augmenter l'expression des marqueurs de costimulation à leur surface ainsi que le production de cytokines en réponse à des ligands des récepteurs Toll, ainsi que leur capacité à présenter des antigènes associés aux molécules du complexe majeur d'histocompatibilité (CMH) de classe I ou II pour activer la prolifération et la différentiation des lymphocytes T. En utilisant un système de transduction de lentivirus de seconde génération, ces nouvelles lignées de cellules dendritiques ont été génétiquement modifiées pour sur-exprimer des molécules immunosuppressives (IL-10, TGFP latent, TGFp actif, Activin A, Arginase 1, IDO, B7DC et CTLA4). Ces lignées permettent d'étudier de manière reproductible le rôle de ces molécules potentiellement tolérogènes sur les réponses immunitaires in vitro et in vivo. Ces lignées potentiellement tolérogènes ont été testées, tout d'abord, in vitro, pour leur capacité à inhiber l'activation des cellules dendritiques, à bloquer la prolifération des cellules Τ ou à modifier leur polarisation. Nos résultats démontrent qu'en réponse à une stimulation, la sur-expression des molécules costimulatrices et la sécrétion de molécules pro- inflammatoires est réduite quand les cellules dendritiques sur-expriment l'IL-10. La sur¬expression de TGFp sous sa forme active induit le développement de cellules régulatrices CD4+ CD25+ Foxp3+ et bloque la réponse CD8 cytotoxique tandis que la sur-expression de CTLA4 à la surface des cellules dendritiques inhibe une réponse Thl et induit des lymphocytes Τ anergiques. Ces lignées ont également été utilisées pour étudier l'induction de tolérance in vivo. Tout d'abord, nous avons étudié l'induction de tolérance dans un modèle de développement de tumeurs. En effet, quand les lignées tumorales sont transférées dans les lignées de souris C57BL/6, elles sont reconnues comme du non-soi du à l'expression de l'oncogène SV40 et du GFP et sont éliminées. Ce mécanisme d'élimination a été étudié en utilisant une lignée de cellules dendritiques modifiée pour exprimer la luciférase et qui a permis de suivre le développement des tumeurs par de l'imagerie in vivo dans des animaux vivants. Ces lignées de cellules dendritiques MuTu sont éliminées dans la souris C57BL/6 par les lymphocytes CD8 et l'action cytotoxique de la perforine. Après plusieurs injections, les cellules dendritiques sur-exprimant CTLA4 ou l'actif TGFp peuvent casser cette réponse immunitaire inhérente aux antigènes de la lignée et induire le développement de la tumeur dans la souris C57BL/6. Le développement tumoral a pu être suivi en mesurant la bioluminescence émise par des cellules dendritiques modifiées pour exprimer à la fois l'actif TGFp et la luciférase. Ces tumeurs ont pu se développer grâce à la mise en place d'un microenvironnement suppressif pour échapper à l'immunité en recrutant des cellules myéloïde suppressives, des lymphocytes CD4 régulateurs et en induisant l'expression d'une molécule inhibitrice PD-1 à la surface des lymphocytes CD8 infiltrant la tumeur. Dans un deuxième temps, ces lignées tolérogènes ont également été testées dans un modèle murin de maladies auto-immunes, appelé l'encéphalomyélite auto-immune expérimental (EAE), qui est un modèle pour la sclérose en plaques. L'EAE a été induite dans la souris par le transfert de cellules de ganglions prélevées d'une souris donneuse préalablement immunisée avec une protéine du système nerveux central, la glycoprotéine myéline oligodendrocyte (MOG) émulsifiée dans de l'adjuvant complet de Freund. La vaccination des souris donneuses et receveuses avec les cellules sur-exprimant l'actif TGFP préalablement chargées avec la protéine MOG bloque l'induction de l'EAE. Nous sommes actuellement en train de définir les mécanismes qui permettent de protéger la souris du développement de la maladie auto-immune. Dans cette étude, nous avons ainsi démontré la possibilité d'induire la tolérance in vivo et in vitro à différents antigènes en utilisant nos nouvelles lignées de cellules dendritiques et en les modifiant pour exprimer des molécules immunosuppressives. En conséquence, ces nouvelles lignées de cellules dendritiques représentent un outil pour explorer les bénéfices de différentes molécules ayant des propriétés immuno-modulatrices pour manipuler le système immunitaire vers un phénotype tolérogène. - Dendritic cells (DC) are widely recognized as potent inducers of the adaptive immune responses. Importantly, after microbial infections, DC become activated, induce co- stimulation, secrete cytokines and induce effector and memory Τ cells. DC furthermore play an important role in inducing and maintaining central and peripheral tolerance by inducing anergy, deletion or commitment of antigen-specific naïve Τ cells into regulatory Τ cells. In our group, stable MuTu DC lines were generated by culture of splenic DC tumors from transgenic mice expressing the SV40 large Τ oncogene and the GFP under DC-specific CDllc promoter. These transformed DC belong to the CD8a+ conventional DC subtype and have fully conserved their capacity to upregulate co-stimulatory markers and produce cytokines after activation with Toll Like Receptors-ligands, and to present Major Histocompatibility class-I or MHCII-restricted antigens to activate Τ cell expansion and differentiation. Using a second- generation lentiviral transduction system, these newly developed MuTu DC lines were genetically modified to overexpress immunosuppressive molecules (IL-10, latent TGFp, active TGFp, Activin A, Arginase 1, IDO, B7DC and CTLA4). This allows to reproducibly investigate the role of these potentially tolerogenic molecules on in vitro and in vivo immune responses. These potentially tolerogenic DC were tested in vitro for their ability to inhibit DC activation, to prevent Τ cell proliferation and to modify Τ cell polarization. Our results show that the upregulation of costimulatory molecules and the secretion of pro-inflammatory cytokines were reduced upon stimulation of DC overexpressing IL-10. The overexpression of active TGFP induced the development of CD4+ CD25+ Foxp3+ regulatory Τ cells and inhibited the cytotoxic CD8 Τ cell response as shown by using the OT-II Τ cell system whereas the surface expression of CTLA-4 on DC prevented the Thl response and prompted an anergic antigen-specific Τ cell response. These MuTu DC lines were also used in vivo in order to study the induction of tolerance. First we addressed the induction of tolerance in a model of tumorogenesis. The adoptively transferred tumor cell lines were cleared in C57BL/6 mice due to the foreign expression of SV40 LargeT and GFP. The mechanism of clearance of MuTu DC line into C57BL/6 mice was investigated by using luciferase-expressing DC line. These DC line allowed to follow, by in vivo imaging, the tumor development in living animals and determined that MuTu DC lines were eliminated in a perforin-mediated CD8 Τ cell dependent and CD4 Τ cell independent response. After multiple injections, DC overexpressing CTLA4 or active TGFp could break the immune response to these inherent antigens and induced DC tumorogenesis in wild type mice. The tumor outgrowth in C57BL/6 mice was nicely observed by double-transduced DC lines to express both luciferase and active TGFp. actTGFp-DC tumor was shown to recruit myeloid-derived suppressor cells, induce CD4+ CD25+ Foxp3+ regulatory Τ cells and induce the expression of the inhibitory receptor PD-1 on tumor- infiltrating CD8+ Τ cells in order to escape tumor immunity. Tolerogenic DC lines were also tested for the induction of tolerance in a murine model of autoimmune disease, the experimental autoimmune encephalitis (EAE) model for human multiple sclerosis. EAE was induced in C57BL/6 mice by the adoptive transfer of lymph node cells isolated from donor mice previously immunized by a protein specific to the central nervous system, the myelin oligodendrocyte glycoprotein (MOG) emulsified in the complete freund adjuvant. The vaccination of donor and recipient mice with MOG-pulsed actTGFP-DC line prevented EAE induction. We are still investigating how the active TGFP protect mice from EAE development. We generated tolerogenic DC lines inducing tolerance in vitro and in vivo. Thereby these MuTu DC lines represent a great tool to explore the benefits of various immuno-modulatory molecules to manipulate the immune system toward a tolerogenic phenotype.