Search

The identification of patients at risk of developing a severe form of acute pancreatitis is a major issue. The goal of this study was to identify parameters at admission associated with severe pancreatitis to develop a predictive severity score.We conducted a retrospective study at Caen University Hospital between January 2014 and December 2017, including 504 patients hospitalized for acute pancreatitis, of whom 74 had a severe form. We developed a predictive score named Admission Severe Acute Pancreatitis (ASAP) score based on parameters associated with a severe form in multivariate analysis. We validated our score in an independent validation cohort of 80 patients.Hypothermia, low oxygen saturation or albumin levels, and high creatinine levels were significantly associated with severe pancreatitis. The ASAP score showed notable predictive accuracy (area under receiver operating characteristic, 0.82), which was significantly higher than Sequential Organ Failure Assessment, persistent Systemic Inflammatory Response Syndrome, and Balthazar. Using the -2.1742 threshold, the ASAP score had a sensitivity and specificity of 74% and a negative predictive value of 95%. These predictive performances for ASAP score were confirmed in the validation cohort.The ASAP score demonstrates remarkable predictive accuracy in distinguishing severe forms of acute pancreatitis.

There is intensive research using gold nanoparticles for biomedical purposes, which have many advantages such as ease of synthesis and high reactivity. Their possible small size (&lt, 10 nm) can lead to the crossing of biological membranes and then to problematic dissemination and storage in organs that must be controlled and evaluated. In this work, a simple isocratic HPLC method was developed and validated to quantify the gold coming from nanoparticles in different biological samples. After a first carbonization step at 900 °C, the nanoparticles were oxidized by dibroma under acidic conditions, leading to tetrachloroaurate ions that could form ion pairs when adding rhodamine B. Finally, ion pairs were extracted and rhodamine B was evaluated to quantify the corresponding gold concentration by reversed-phase HPLC with visible detection. The method was validated for different organs (liver, spleen, lungs, kidneys, or brain) and fluids (plasma and urine) from rats and mice. Lastly, the developed method was used to evaluate the content of gold in organs and fluids after intravenous (IV) injection of nanoparticles.

International audience; Health risk assessments of chemical substances consider aggregate and/or cumulative exposures in order to characterise population health risks realistically.Should environmental exposure to chemical substances via indoor settled dust be taken into account and if so, how?Exposure to the chemicals in settled dust can occur through ingestion, inhalation, and dermal contact. Ingestion appears to be the most important route of exposure, and the only one that can be estimated from current knowledge. The recommended measurement technique is sampling by vacuuming a given surface, then sieving the dust at 250 μm (maximum size of the particles adhering to the hands) to document the mass and surface concentrations. Oral bioaccessibility would provide a better exposure estimate, but its methods have not yet been validated for most compounds. Guideline values for indoor dust are relevant if exposure via dust is non-negligible for a fraction of the population. They could be developed by simply allocating a fraction of the toxicological reference value to dust.; Les évaluations des risques sanitaires des substances chimiques considèrent de plus en plus fréquemment les expositions agrégées et/ou cumulées afin de caractériser de manière la plus réaliste les risques sanitaires pour la population.L’exposition environnementale aux substances chimiques via la poussière sédimentée sur les surfaces intérieures doit-elle être prise en compte et de quelle manière ?La population est exposée aux substances chimiques présentes dans la poussière sédimentée par ingestion, inhalation et contact cutané. L’ingestion semble la voie d’exposition la plus importante, et la seule à pouvoir être estimée au vu des connaissances actuelles. Pour la mesure, il est recommandé d’utiliser la technique de prélèvement par aspiration sur une surface déterminée, suivie d’un tamisage à 250 μm (taille maximum des particules adhérant aux mains) afin de documenter les concentrations massique et surfacique. La mesure de la bioaccessibilité orale permettrait de mieux estimer l’exposition mais les méthodes ne sont pas validées à ce jour pour la majorité des composés. Les valeurs guides pour les poussières intérieures (VGPI) sont pertinentes si l’exposition via la poussière est non négligeable pour une fraction de la population. Elles pourraient être élaborées simplement en allouant à la poussière une fraction de la valeur toxicologique de référence.

International audience; No abstract available

Thiols are very important molecules in the biomedical field involved for example in redox homeostasis. Their detection and quantification remain difficult due to their poor stability (oxidation) linked to their strong reactivity towards other thiols (by the formation of S-S bonds) or other interfering molecules in the medium. Cellulose membranes with immobilized gold nanoparticles (AuNP) were developed to capture and quantify thiols in simple and complex matrices. This device was first optimized and characterized in terms of nanostructuration and thiol adsorption. N-Acetylcysteine (NAC) and reduced glutathione (GSH), chosen as model molecules, were filtered through the device demonstrating a maximal adsorption capacity of 270 and 60 nmol respectively. In a second step, the adsorbed species were subjected to ligand exchange using a more reactive thiol, dithiothreitol. The results showed release rates of approximately 90% for NAC and GSH. Finally, the amount of endogenous GSH in rat plasma was determined without any pretreatment. For the first time to our knowledge, a nanostructured device for the capture, selective and sensitive quantification of thiols is proposed. This device is easy to handle and overcomes matrix effects. Moreover, the very large concentration factor induced by this technology will be a valuable asset to decrease the quantification limits of analytical methods.

International audience; The design of layer-by-layer (LbL) polyelectrolyte films including nanoparticles is a growing field of innovation in a wide range of biomedical applications. Gold nanoparticles (AuNPs) are very attractive for further biomolecule coupling to induce a pharmacological effect. Nanostructured LbL films coupled with such metallic species show properties that depend on the conditions of construction, i.e. the polymer nature and dissolution buffer. Tripartite LbL films (polycation, AuNP, and polyanion) were evaluated using two different polycationic polymers (poly(allylamine hydrochloride) (PAH), poly(ethylene imine) (PEI)) and various medium conditions (salts, i.e. phosphate, Tris or Tris-NaCl buffers, and concentration). AuNP incorporation and film stability were analysed by visible spectrophotometry, capillary zone electrophoresis, a quartz crystal microbalance, and high-performance liquid chromatography. The ideal compromise between AuNP loading and film stability was obtained using PAH prepared in Tris-NaCl buffer (0.01-0.15 M). This condition allowed the formation of a LbL film that was more stable than the film with PEI and provided an AuNP quantity that was 4.8 times greater than that of the PAH-PBS-built film. In conclusion, this work presents an analytical strategy for the characterization of nanostructured multilayer films and optimization of LbL films enriched with AuNPs to design biomedical device coatings.

ObjectiveTo study blood anti-Aβ antibodies in the context of spontaneous inflammatory or hemorrhagic CAA manifestations, which are similar to complications occurring after monoclonal anti-Aβ antibody immunotherapies.MethodsIn this case-control study, serum anti-Aβ antibody isotype, concentration, avidity, and reactivity toward soluble or fibrillary Aβ1-40 and Aβ1-42 isoforms were assessed using an ELISA-based multiplex analysis. Anti-Aβ serologic patterns were defined in CAA and CAA subgroups using multivariable logistic regression analyses.ResultsFourty-one healthy aged controls and 64 CAA patients were recruited: 46 with hemorrhagic features (CAA-he) and 18 with CAA-related inflammation (CAA-ri). As compared to controls, the most striking features of CAA-related serological profiles were the following: i) both CAA-he and CAA-ri patients displayed lower binding diversity of anti-soluble Aβ1-40 IgM; ii) CAA-he patients displayed higher anti-soluble Aβ1-40 / fibrillary Aβ1-42 IgG4 concentrations ratio and higher anti-soluble Aβ1-42 IgG4 and IgA avidity; iii) CAA-ri patients displayed higher binding diversity of anti-soluble Aβ1-40 IgG3 and higher anti-fibrillary/soluble Aβ1-42 IgG4 dilution curve steepness ratio.ConclusionThis proof-of-concept study revealed anti-Aβ antibody variations in CAA patients, some of which were associated to CAA clinical phenotypes, unveiling pathophysiological insights regarding CAA-hemorrhagic and inflammatory related events.

Objective Recent studies have underlined the effect of systemic inflammation on the pathophysiology of Alzheimer's disease (AD). Neutrophils are key components of early innate immunity and contribute to uncontrolled systemic inflammation if not tightly regulated. The aim of our study was to fully characterize human circulating neutrophils at different disease stages in AD. Methods We analyzed neutrophil phenotypes and functions in 42 patients with AD (16 with mild cognitive impairment and 26 with dementia), and compared them to 22 age-matched healthy subjects. This study was performed directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. Results Blood samples from AD patients with dementia revealed neutrophil hyperactivation associated with increased reactive oxygen species production and increased levels of intravascular neutrophil extravascular traps. The homeostasis of circulating neutrophils in these patients also changed: The ratio between the harmful hyperreactive CXCR4high /CD62Llow senescent and the CD16bright /CD62Ldim immunosuppressive neutrophil subsets rose in the later stage of the disease. These abnormalities were greater in fast-decliner than in slow-decliner patients. Interpretation Our results indicate that the inflammatory properties of circulating neutrophils shift as the percentage of aged neutrophils expands in patients with AD-changes that may play an instrumental role in establishing systemic chronic inflammation. Most important, our data strongly suggest that the neutrophil phenotype may be associated with the rate of cognitive decline and may thus constitute an innovative and prognostic blood biomarker in patients with AD. Ann Neurol 2018;83:387-405.

ObjectiveTo better understand the functional state of circulating neutrophils in patients with ischemic stroke (IS) for planning future clinical trials.MethodsWe analyzed by flow cytometry activation state of circulating neutrophils and the distribution of neutrophil peripheral subsets in 41 patients with acute IS less than 6 hours before admission and compared them with 22 age-matched healthy controls.ResultsOur results demonstrated continuous basal hyperactivation of circulating neutrophils during acute IS, characterized by lower l-selectin expression and higher CD11b expression at the cell surface, increased ROS production by neutrophils, and greater circulating levels of neutrophil elastase. Neutrophil hyperactivation was associated with deregulation of the equilibrium between apoptotic and necrotic. Patients also had higher percentages than controls of the overactive senescent (CXCR4bright/CD62Ldim) neutrophil subset and increased percentage of neutrophils with a reverse transendothelial migration (CD54highCXCR1low) phenotype. Importantly, neutrophil alterations were associated with the clinical severity of the stroke, evaluated by its NIH Stroke Scale score.ConclusionAltogether, our results indicate that during acute IS, the inflammatory properties of circulating neutrophils rise, associated with the expansion of harmful neutrophil subsets. These changes in neutrophil homeostasis, associated with disease severity, may play an instrumental role by contributing to systemic inflammation and to the blood-brain barrier breakdown. Our findings highlight new potential therapeutic approaches of stroke by rebalancing the ratio of senescent to immunosuppressive neutrophils or decreasing reverse neutrophil transmigration or both.

OS; L’Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail endosse les conclusions et recommandations de l’expertise collective relative à la caractérisation de l’exposition de la population générale aux substances chimiques présentes dans la poussière déposée sur les surfaces intérieures. Ces travaux d’expertise confirment la pertinence de considérer l’ingestion de poussières sédimentées en environnements intérieurs dans l’évaluation de l’exposition globale de la population aux substances chimiques non volatiles et semi-volatiles. Concernant le prélèvement de la poussière, l’Anses recommande l’harmonisation des méthodes entre laboratoires en utilisant l’aspiration pour le prélèvement de la poussière suivie d’un tamisage à 250 µm pour la taille des particules qui vont être analysées. Cette recommandation ne s’applique pas au cas particulier du plomb, pour lequel il existe une norme spécifique qui préconise le prélèvement par lingette. L’Agence encourage par ailleurs la conduite d’études en France afin de disposer d’estimations robustes et représentatives, dans le contexte français, de paramètres particulièrement impactants dans les calculs de doses d’exposition via l’ingestion de poussières. Il s’agit en particulier de l’empoussièrement en intérieur, correspondant à la quantité de poussière par unité de surface, et des quantités de poussière ingérées par jour et par tranche d’âges. Enfin, concernant l’élaboration de valeurs guides pour les poussières intérieures (VGPI) relatives à des substances pour lesquelles l’ingestion de poussière peut être non négligeable pour une partie de la population, l’Agence va poursuivre des travaux d’expertise et se propose de les associer à sa mission pérenne sur l’élaboration de valeurs guides de qualité d’air intérieur (VGAI). A court terme, une réflexion sur la méthode d’élaboration sera donc lancée pour ensuite l’appliquer à des substances d’intérêt. Compte tenu d’expertises antéreures et de la littérature scientifique passée en revue, le plomb et les phtalates apparaissent être des substances prioritaires à investiguer dans ce cadre.

International audience; Gold nanoparticle (AuNP) interaction with the blood compartment as a function of their charge and the binding energy of their surface ligand was explored. Citrate, polyallylamine and cysteamine stabilized AuNP along with dihydrolipoic acid and polyethylene glycol capped AuNP were synthesized and fully characterized. Their interactions with model proteins (human albumin and human fibrinogen) were studied. Complexes formed between AuNP and protein revealed several behaviors ranging from corona formation to aggregation. Protein fluorescence quenching as a function of temperature and AuNP concentration allowed the determination of the thermodynamic parameters describing these interactions. The hemolysis induced by AuNP was also probed: an increasing or a decreasing of hemolysis ratio induced by AuNP was observed as of function of protein corona formation. Taken together, our results drew up a composite sketch of an ideal surface ligand for blood compatible AuNP. This capping agent should be strongly bound to the gold core by one or more thiol groups and it must confer a negative charge to the particles.

There is to date no effective way of preventing or curing neurodegenerative diseases such as Alzheimer disease or transmissible spongiform encephalopathies. The idea of treating those conditions by immunological approaches has progressively emerged over the last ten years. Encouraging results have been reported in Alzheimer disease and in peripheral forms of mouse prion diseases following passive injection of Abs or active immunization against the peptides or proteins presumably at the origin of those disorders. Still, major difficulties persist due to some characteristics of those conditions such as slow evolution, brain location, uncertainties regarding precise pathogenic pathways, and, above all, the fact that the target Ag is self, meaning that it is poorly immunogenic and potentially harmful if tolerance was transgressed. To analyze some of those difficulties, we are developing adoptive cell transfer approaches. In this study, lymphocytes sensitized against the prion protein in nontolerant Prnp−/− mice were transferred into histocompatible wild-type recipients which were partly or totally devoid of their own lymphocytes. Under such conditions, we found that the engrafted T lymphocytes resisted peripheral tolerance, remained reactive for several months against epitopes of the prion protein, and significantly attenuated the progression of prions in secondary lymphoid organs with subsequent delay in the evolution of the neurological disease. Interestingly, those protective T lymphocytes secreted lymphokines and migrated more readily into the host CNS but did not appear to be engaged in cooperation with host B cells for Ab production.

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The aim of this study was to determine whether the chemokine stromal cell–derived factor 1 (SDF-1) induces the growth, migration, and invasion of human hepatoma cells. We show that SDF-1 G protein–coupled receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), and SDF-1 mRNA are expressed in human hepatoma Huh7 cells, which secrete and bind SDF-1. This binding depends on CXCR4 and glycosaminoglycans. SDF-1 associates with CXCR4, and syndecan-4 (SDC-4), a heparan sulfate proteoglycan at the plasma membrane of Huh7 cells, induces the growth of Huh7 cells by promoting their entry into the cell cycle, and inhibits the tumor necrosis factor-α–mediated apoptosis of the cells. SDF-1 also reorganizes Huh7 cytoskeleton and induces tyrosine phosphorylation of focal adhesion kinase. Finally, SDF-1 activates matrix metalloproteinase-9, resulting in increased migration and invasion of Huh7 cells. These biological effects of SDF-1 were strongly inhibited by the CXCR4 antagonist AMD3100, by a glycosaminoglycan, heparin, as well as by β-d-xyloside treatment of the cells, or by c-jun NH2-terminal kinase/stress-activated protein kinase inhibitor. Therefore, the CXCR4, glycosaminoglycans, and the mitogen-activated protein kinase signaling pathways are involved in these events. The fact that reducing SDC-4 expression by RNA interference decreased SDF-1–induced Huh7 hepatoma cell migration and invasion strongly indicates that SDC-4 may be an auxiliary receptor for SDF-1. Finally, the fact that CXCR4 is expressed in hepatocellular carcinoma cells from liver biopsies indicates that the in vitro results reported here could be extended to in vivo conditions. (Mol Cancer Res 2007;5(1):21–33)

Recent studies highlight the implication of innate and adaptive immunity in the pathophysiology of Alzheimer's disease, and foster immunotherapy as a promising strategy for its treatment. Vaccines targeting amyloid-β peptide provided encouraging results in mouse models, but severe side effects attributed to T cell responses in the first clinical trial AN1792 underlined the need for better understanding adaptive immunity in Alzheimer's disease. We previously showed that regulatory T cells critically control amyloid-β-specific CD4(+) T cell responses in both physiological and pathological settings. Here, we analysed the impact of regulatory T cells on spontaneous disease progression in a murine model of Alzheimer's disease. Early transient depletion of regulatory T cells accelerated the onset of cognitive deficits in APPPS1 mice, without altering amyloid-β deposition. Earlier cognitive impairment correlated with reduced recruitment of microglia towards amyloid deposits and altered disease-related gene expression profile. Conversely, amplification of regulatory T cells through peripheral low-dose IL-2 treatment increased numbers of plaque-associated microglia, and restored cognitive functions in APPPS1 mice. These data suggest that regulatory T cells play a beneficial role in the pathophysiology of Alzheimer's disease, by slowing disease progression and modulating microglial response to amyloid-β deposition. Our study highlights the therapeutic potential of repurposed IL-2 for innovative immunotherapy based on modulation of regulatory T cells in Alzheimer's disease.

We recently demonstrated that stromal cell-derived factor-1 (SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4 expressed by human primary lymphocytes and macrophages, and HeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signaling molecule. Here, we demonstrate that SDF-1 strongly accelerates the shedding of syndecan-4 ectodomains and to a lesser extent that of syndecan-1 from HeLa cells. The fact that this acceleration was not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4 mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence. Pre-treating the cells with heparitinases I, III, or with the protein kinase C (PKC) inhibitor, bisindolylmaleimide, significantly inhibited this accelerated shedding, which suggests the involvement of both cell-surface heparan sulfate and PKC transduction pathway. In contrast, Map Kinase or NF-kappaB pathway inhibitors had no effect. Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9) mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9 silencing by RNA interference strongly decreases the syndecan-1 and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1 also accelerates in a CXCR4-independent manner, the shedding of syndecan-1 and -4 from human primary macrophages, which is significantly inhibited by anti-MMP-9 antibodies. This strongly indicates the role of MMP-9 in these events occurring in both a tumoral cell line and in human primary macrophages. Because MMP-9 plays a crucial role in extracellular matrix degradation during cancer cell metastasis and invasion, and shed ectodomains of syndecans may likely be involved in tumor cell proliferation, these data further indicate the multiplicity of the roles played by SDF-1 on tumor cell biology.

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.

We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase--and protein kinase C-dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.

International audience; Prion diseases are caused by the transconformation of the host cellular prion protein PrPc into an infectious neurotoxic isoform called PrPSc. While vaccine-induced PrP-specific CD4(+)T cells and antibodies partially protect scrapie-infected mice from disease, the potential autoreactivity of CD8(+) cytotoxic T lymphocytes (CTLs) received little attention. Beneficial or pathogenic influence of PrPc-specific CTL was evaluated by stimulating a CD8(+) T-cell-only response against PrP in scrapie-infected C57BL/6 mice. To circumvent immune tolerance to PrP, five PrP-derived nonamer peptides identified using prediction algorithms were anchored-optimized to improve binding affinity for H-2D(b) and immunogenicity (NP-peptides). All of the NP-peptide; elicited a significant number of IFN gamma secreting CD8(+) T cells that better recognized the NP-peptides than the natives; three of them induced T cells that were lytic in vivo for NP-peptide-loaded target cells. Peptides 168 and 192 were naturally processed and presented by the 1C11 neuronal cell line. Mini:genes encoding immunogenic NP-peptides inserted into adenovirus (rAds) vectors enhanced the specific CD8(+)T-cell responses. Immunization with rAd encoding 168NP before scrapie inoculation significantly prolonged the survival of infected mice. This effect was attributable to a significant lengthening of the symptomatic phase and was associated with enhanced CD3(+) T cell recruitment to the CNS. However, immunization with Ad168NP in scrapie-incubating mice induced IFN gamma-secreting CD8(+) T cells that were not cytolytic in vivo and did not influence disease progression nor infiltrated the brain. In conclusion, the data suggest that vaccine-induced PrP-specific CD8(+) T cells interact with prions into the CNS during the clinical phase of the disease. (C) 2012 Elsevier Inc. All rights reserved.

Article Number: e4917; International audience; In prion diseases, PrP(c), a widely expressed protein, is transformed into a pathogenic form called PrP(Sc), which is in itself infectious. Antibodies directed against PrP(c) have been shown to inhibit PrP(c) to PrP(Sc) conversion in vitro and protect in vivo from disease. Other effectors with potential to eliminate PrPSc-producing cells are cytotoxic T cells directed against PrP-derived peptides but their ability to protect or to induce deleterious autoimmune reactions is not known. The natural tolerance to PrP(c) makes difficult to raise efficient adaptive responses. To break tolerance, adenovirus (Ad) encoding human PrP (hPrP) or control Ad were administered to wild-type mice by direct injection or by transfer of Ad-transduced dendritic cells (DCs). Control Ad-transduced DCs from Tg650 mice overexpressing hPrP were also used for immunization. DC-mediated but not direct administration of AdhPrP elicited antibodies that bound to murine native PrP(c). Frequencies of PrP-specific IFN gamma-secreting T cells were low and in vivo lytic activity only targeted cells strongly expressing hPrP. Immunohistochemical analysis revealed that CD3(+) T cell infiltration was similar in the brain of vaccinated and unvaccinated 139A-infected mice suggesting the absence of autoimmune reactions. Early splenic PrP(Sc) replication was strongly inhibited ten weeks post infection and mean survival time prolonged from 209 days in untreated 139A-infected mice to 246 days in mice vaccinated with DCs expressing the hPrP. The efficacy appeared to be associated with antibody but not with cytotoxic cell-mediated PrP-specific responses.

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.

Several hurdles must be overcome in order to achieve efficient and safe immunotherapy against conformational neurodegenerative diseases. In prion diseases, the main difficulty is that the prion protein is tolerated as a self protein, which prevents powerful immune responses. Passive antibody therapy is effective only during early, asymptomatic disease, well before diagnosis is made. If efficient immunotherapy of prion diseases is to be achieved, it is crucial to understand precisely how immune tolerance against the prion protein can be overcome and which effector pathways may delay disease progression. To this end, we generated a transgenic mouse that expresses the ß-chain of a T cell receptor recognizing a PrP epitope presented by the class II major histocompatibility complex. The fact that the constraint is applied to only one TCR chain allows adaptation of the other chain according to the presence or absence of tolerogenic PrP. We first show that transgene-bearing T cells, pairing with rearranged α-chains conferring anti-PrP specificity, are systematically eliminated during ontogeny in PrP+ mice, suggesting that precursors with good functional avidity are rare in a normal individual. Second, we show that transgene-bearing T cells with anti-PrP specificity are not suppressed when transferred into PrP+ recipients and proliferate more extensively in a prion-infected host. Finally, such T cells provide protection through a cell-mediated pathway involving IL-4 production. These findings support the idea that cell-mediated immunity in neurodegenerative conditions may not be necessarily detrimental and may even contribute, when properly controlled, to the resolution of pathological processes., Author Summary It is generally accepted that prion-specific antibodies can protect against mouse scrapie infection. However, passive antibody therapy is limited to the lymphoinvasion stage of the disease. Active immunization has been attempted but the results have been disappointing. There is therefore a need for developing analytical models that will allow a fine dissection of the immune mechanisms at play in prion diseases and help distinguish between protective effects mediated by B cells and antibodies, and the effect of T cells. The aim of our study was to thoroughly examine T cell tolerance to the prion protein and to evaluate whether a pure specific population of T cells adoptively transferred to a normal host could proliferate and confer protection against scrapie. We designed a transgenic mouse in which the majority of T lymphocytes recognize the prion protein. Our key findings are that prion-specific T cells remain functional when transferred to normal recipients, even more so when the host is infected with scrapie, and confer partial protection against the disease by slowing down prion replication, in complete absence of anti-prion antibodies. Anti-prion T cells may therefore be considered as a therapeutic tool in the future.

Introduction Alors que le systeme immunitaire adaptatif tolere naturellement les antigenes du soi, plusieurs etudes recentes suggerent qu’il est capable de reagir au peptide amyloide Aβ, aussi bien dans des modeles animaux transgeniques qu’au cours de la maladie d’Alzheimer humaine. Ainsi, des methodes d’immunisation classiques induisent des reponses ayant un effet sur le plan pathologique et eventuellement clinique. La mise au point de vaccins therapeutiques s’avere cependant difficile car les reponses cellulaires T peuvent se compliquer de meningoencephalites graves. Par ailleurs, si les anticorps anti-Aβ seuls semblent efficaces contre l’accumulation des plaques seniles, leur effet therapeutique clinique est moins certain. Dans l’ensemble, il apparait indispensable de mieux connaitre les relations entre le systeme immunitaire et le peptide Aβ pour concevoir des approches immunotherapeutiques efficaces et sures. Un interet particulier a ete porte aux cellules T regulatrices (Treg), responsables des principaux mecanismes de la tolerance immunitaire peripherique. Materiel & methodes Des souris C57BL/6 sauvages ou doubles transgeniques APPPS1 (APP K670N/M671L et PS-1 L166P) ont ete traitees ou non a l’âge de 8 ou 16 semaines par un anticorps anti-CD25 (PC61) pour depleter de facon transitoire les Treg. L’effet de cette depletion sur l’activation des reponses T CD4+ anti-Aβ a ete analyse apres vaccination par Aβ42 en presence d’adjuvant complet de Freund. En parallele, l’impact de la depletion des Treg sur l’evolution naturelle de la maladie a ete etudie par analyses histologiques et comportementales sur un groupe de souris APPPS1 traitees ou non par PC61 mais non-vaccinees. Resultats Apres suppression des Treg les reponses proliferatives T CD4+ anti-Aβ post-vaccinales sont plus augmentees chez les souris transgeniques APPPS1 que chez les souris sauvages. D’autre part, nos resultats preliminaires suggerent que la tolerance due aux cellules Treg peut favoriser la progression des signes pathologiques. Discussion Les cellules Treg inhibent les reponses T CD4+ anti-Aβ se developpant spontanement au cours de la maladie, et pourraient ainsi contribuer a la physiopathologie de la maladie d’Alzheimer. La depletion des cellules T regulatrices represente donc un outil therapeutique potentiel, sous reserve que les reponses immunitaires chez l’homme soient semblables a celles du modele murin APPPS1. Des travaux recents vont dans ce sens, montrant que les reponses immunitaires adaptatives contribueraient au controle des depots amyloides.

International audience; Background : Active immunization against Aβ was reported to have a therapeutic effect in murine models of Alzheimer’s disease. Clinical Aβ vaccination trial AN1792 was interrupted due to the development in 6 % of the patients of meningoencephalitis likely involving pro-inflammatory CD4+ T cells. However, the potential implication of auto-aggressive anti-Aβ CD8+ T cells has been poorly investigated.Methods : Potential MHC-I-restricted Aβ-derived epitopes were first analyzed for their capacity to recruit functional CD8+ T cell responses in mouse models. Their impact on migration of CD8+ T cells into the brain parenchyma and potential induction of meningoencephalitis and/or neuronal damage was investigated upon vaccination in the APPPS1 mouse model of AD.Results :We identified one nonamer peptide, Aβ33-41, which was naturally processed and presented in association with H-2-Db molecule on neurons and CD11b+ microglia. Upon optimization of anchor residues for enhanced binding to H-2-Db, immunization with the modified Aβ33-41NP peptide elicited Aβ-specific IFNγ-secreting CD8+ T cells, which are cytotoxic towards Aβ-expressing targets. Whereas T cell infiltration in the brain of APPPS1 mice is dominated by CD3+CD8− T cells and increases with disease evolution between 4 and 7 months of age, a predominance of CD3+CD8+ over CD3+CD8− cells was observed in 6- to 7-month-old APPPS1 but not in WT animals, only after vaccination with Aβ33-41NP. The number of CD11b+ mononuclear phagocytes, which significantly increases with age in the brain of APPPS1 mice, was reduced following immunization with Aβ33-41NP. Despite peripheral activation of Aβ-specific CD8+ cytotoxic effectors and enhanced infiltration of CD8+ T cells in the brain of Aβ33-41NP-immunized APPPS1 mice, no clinical signs of severe autoimmune neuroinflammation were observed.Conclusions : Altogether, these results suggest that Aβ-specific CD8+ T cells are not major contributors to meningoencephalitis in response to Aβ vaccination.