Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation.

Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs , , , , ). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

Optimising approaches to active immunotherapy of cancer

The design of therapeutic cancer vaccines is aimed at inducing high numbers and potent T cells that are able to target and eradicate malignant cells. This calls for close collaboration between cells of the innate immune system, in particular dendritic cells (DCs), and cells of the adaptive immune system, notably CD4+ helper T cells and CD8+ cytotoxic T cells. Therapeutic vaccines are aided by adjuvants, which can be, for example, Toll¬like Receptor agonists or agents promoting the cytosolic delivery of antigens, among others. Vaccination with long synthetic peptides (LSPs) is a promising strategy, as the requirement for their intracellular processing will mainly target LSPs to professional antigen presenting cells (APCs), hence avoiding the immune tolerance elicited by the presentation of antigens by non-professional APCs. The unique property of antigen cross-processing and cross-presentation activity by DCs plays an important role in eliciting antitumour immunity given that antigens from engulfed dead tumour cells require this distinct biological process to be processed and presented to CD8+T cells in the context of MHC class I molecules. DCs expressing the XCR1 chemokine receptor are characterised by their superior capability of antigen cross- presentation and priming of highly cytotoxic T lymphocyte (CTL) responses. Recently, XCR1 was found to be also expressed in tissue-residents DCs in humans, with a simitar transcriptional profile to that of cross- presenting murine DCs. This shed light into the value of harnessing this subtype of XCR1+ cross-presenting DCs for therapeutic vaccination of cancer. In this study, we explored ways of adjuvanting and optimising LSP therapeutic vaccinations by the use, in Part I, of the XCLl chemokine that selectively binds to the XCR1 receptor, as a mean to target antigen to the cross-presenting XCR1+ DCs; and in Part II, by the inclusion of Q.S21 in the LSP vaccine formulation, a saponin with adjuvant activity, as well as the ability to promote cytosolic delivery of LSP antigens due to its intrinsic cell membrane insertion activity. In Part I, we designed and produced XCLl-(OVA LSP)-Fc fusion proteins, and showed that their binding to XCR1+ DCs mediate their chemoattraction. In addition, therapeutic vaccinations adjuvanted with XCLl-(OVA LSP)-Fc fusion proteins significantly enhanced the OVA-specific CD8+ T cell response, and led to complete tumour regression in the EL4-OVA model, and significant control of tumour growth in the B16.0VA tumour model. With the aim to optimise the co-delivery of LSP antigen and XCLl to skin-draining lymph nodes we also tested immunisations using nanoparticle (NP)-conjugated OVA LSP in the presence or absence of XCLl chemokine. The NP-mediated delivery of LSP potentiated the CTL response seen in the blood of vaccinated mice, and NP-OVA LSP vaccine in the presence of XCLl led to higher blood frequencies of OVA-specific memory-precursor effector cells. Nevertheless, in these settings, the addition XCLl to NP-OVA LSP vaccine formulation did not increase its antitumour therapeutic effect. In the Part II, we assessed in HLA-A2/DR1 mice the immunogenicity of the Melan-AA27L LSP or the Melan-A26. 35 AA27l short synthetic peptide (SSP) used in conjunction with the saponin adjuvant QS21, aiming to identify a potent adjuvant formulation that elicits a quantitatively and qualitatively strong immune response to tumour antigens. We showed a high CTL immune response elicited by the use of Melan-A LSP or SSP with QS21, which both exerted similar killing capacity upon in vivo transfer of target cells expressing the Melan-A peptide in the context of HLA-A2 molecules. However, the response generated by the LSP immunisation comprised higher percentages of CD8+T cells of the central memory phenotype (CD44hl CD62L+ and CCR7+ CD62L+) than those of SSP immunisation, and most importantly, the strong LSP+QS21 response was strictly CD4+T cell-dependent, as shown upon CD4 T cell depletion. Altogether, these results suggest that both XCLl and QS21 may enhance the ability of LSP to prime CD8 specific T cell responses, and promote a long-term memory response. Therefore, these observations may have important implications for the design of protein or LSP-based cancer vaccines for specific immunotherapy of cancer -- Les vacans thérapeutiques contre le cancer visent à induire une forte et durable réponse immunitaire contre des cellules cancéreuses résiduelles. Cette réponse requiert la collaboration entre le système immunitaire inné, en particulier les cellules dendrites (DCs), et le système immunitaire adaptatif, en l'occurrence les lymphocytes TCD4 hdper et CD8 cytotoxiques. La mise au point d'adjuvants et de molécules mimant un agent pathogène tels les ligands TLRs ou d'autres agents facilitant l'internalisation d'antigènes, est essentielle pour casser la tolérance du système immunitaire contre les cellules cancéreuses afin de générer une réponse effectrice et mémoire contre la tumeur. L'utilisation de longs peptides synthétiques (LSPs) est une approche prometteuse du fait que leur présentation en tant qu'antigénes requiert leur internalisation et leur transformation par les cellules dendrites (DCs, qui sont les mieux à même d'éviter la tolérance immunitaire. Récemment une sous-population de DCs exprimant le récepteur XCR1 a été décrite comme ayant une capacité supérieure dans la cross-présentation d'antigènes, d'où un intérêt à développer des vaccins ciblant les DCs exprimant le XCR1. Durant ma thèse de doctorat, j'ai exploré différentes approches pour optimiser les vaccins avec LSPs. La première partie visait à cibler les XCR1-DCs à l'aide de la chemokine XCL1 spécifique du récepteur XCR1, soit sou s la forme de protéine de fusion XCL1-OVA LSP-Fc, soit associée à des nanoparticules. La deuxième partie a consisté à tester l'association des LSPs avec I adjuvant QS21 dérivant d'une saponine dans le but d'optimiser l'internalisation cytosolique des longs peptides. Les protéines de fusion XCLl-OVA-Fc développées dans la première partie de mon travail, ont démontré leur capacité de liaison spécifique sur les XCRl-DCs associée à leur capacité de chemo-attractio. Lorsque inclues dans une mmunisation de souris porteuse de tumeurs établies, ces protéines de fusion XCL1-0VA LSP-Fc et XCLl-Fc plus OVA LSP ont induites une forte réponse CDS OVA spécifique permettant la complète régression des tumeurs de modèle EL4- 0VA et un retard de croissance significatif de tumeurs de type B16-0VA. Dans le but d'optimiser le drainage des LSPs vers es noyaux lymphatiques, nous avons également testé les LSPs fixés de manière covalente à des nanoparticules co- injectees ou non avec la chemokine XCL1. Cette formulation a également permis une forte réponse CD8 accompagnée d'un effet thérapeutique significatif, mais l'addition de la chemokine XCL1 n'a pas ajouté d'effet anti-tumeur supplémentaire. Dans la deuxième partie de ma thèse, j'ai comparé l'immunogénicité de l'antigène humain Melan A soit sous la forme d un LSP incluant un épitope CD4 et CD8 ou sous la forme d'un peptide ne contenant que l'épitope CD8 (SSP) Les peptides ont été formulés avec l'adjuvant QS21 et testés dans un modèle de souris transgéniques pour les MHC let II humains, respectivement le HLA-A2 et DR1. Les deux peptides LSP et SSP ont généré une forte réponse CD8 similaire assoc.ee a une capacité cytotoxique équivalente lors du transfert in vivo de cellules cibles présentant le peptide SSP' Cependant les souris immunisées avec le Melan A LSP présentaient un pourcentage plus élevé de CD8 ayant un Phénotype «centra, memory» (CD44h' CD62L+ and CCR7+ CD62L+) que les souris immunisées avec le SSP, même dix mois après I'immunisation. Par ailleurs, la réponse CD8 au Melan A LSP était strictement dépendante des lymphocytes CD4, contrairement à l'immunisation par le Melan A SSP qui n'était pas affectée. Dans l'ensemble ces résultats suggèrent que la chemokine XCL1 et l'adjuvant QS21 améliorent la réponse CD8 à un long peptide synthétique, favorisant ainsi le développement d'une réponse anti-tumeur mémoire durable. Ces observations pourraient être utiles au développement de nouveau vaccins thérapeutiques contre les tumeurs.

Antagonistic peptide technology for functional dissection of CLE peptides revisited.

In the Arabidopsis thaliana genome, over 1000 putative genes encoding small, presumably secreted, signalling peptides can be recognized. However, a major obstacle in identifying the function of genes encoding small signalling peptides is the limited number of available loss-of-function mutants. To overcome this, a promising new tool, antagonistic peptide technology, was recently developed. Here, this antagonistic peptide technology was tested on selected CLE peptides and the related IDA peptide and its usefulness in the context of studies of peptide function discussed. Based on the analyses, it was concluded that the antagonistic peptide approach is not the ultimate means to overcome redundancy or lack of loss-of-function lines. However, information collected using antagonistic peptide approaches (in the broad sense) can be very useful, but these approaches do not work in all cases and require a deep insight on the interaction between the ligand and its receptor to be successful. This, as well as peptide ligand structure considerations, should be taken into account before ordering a wide range of synthetic peptide variants and/or generating transgenic plants.

The development of decision limits for the GH-2000 detection methodology using additional insulin-like growth factor-I and amino-terminal pro-peptide of type III collagen assays.

The GH-2000 and GH-2004 projects have developed a method for detecting GH misuse based on measuring insulin-like growth factor-I (IGF-I) and the amino-terminal pro-peptide of type III collagen (P-III-NP). The objectives were to analyze more samples from elite athletes to improve the reliability of the decision limit estimates, to evaluate whether the existing decision limits needed revision, and to validate further non-radioisotopic assays for these markers. The study included 998 male and 931 female elite athletes. Blood samples were collected according to World Anti-Doping Agency (WADA) guidelines at various sporting events including the 2011 International Association of Athletics Federations (IAAF) World Athletics Championships in Daegu, South Korea. IGF-I was measured by the Immunotech A15729 IGF-I IRMA, the Immunodiagnostic Systems iSYS IGF-I assay and a recently developed mass spectrometry (LC-MS/MS) method. P-III-NP was measured by the Cisbio RIA-gnost P-III-P, Orion UniQ? PIIINP RIA and Siemens ADVIA Centaur P-III-NP assays. The GH-2000 score decision limits were developed using existing statistical techniques. Decision limits were determined using a specificity of 99.99% and an allowance for uncertainty because of the finite sample size. The revised Immunotech IGF-I - Orion P-III-NP assay combination decision limit did not change significantly following the addition of the new samples. The new decision limits are applied to currently available non-radioisotopic assays to measure IGF-I and P-III-NP in elite athletes, which should allow wider flexibility to implement the GH-2000 marker test for GH misuse while providing some resilience against manufacturer withdrawal or change of assays. Copyright © 2015 John Wiley & Sons, Ltd.

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimer's disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.

The predictive value of preoperative B-type natriuretic peptide in children undergoing cardiac surgery

Introduction: B-type natriuretic peptide (BNP) is a biomarker of myocardial stress. In children, the value of preoperative BNP on postoperative outcome is unclear. The aim of this study was to determine the predictive value of preoperative NT-proBNP on postoperative outcome in children after congenital heart surgery. Results: Ninety-seven patients were included in the study with a median age of 3.3 years [0.7-5.2]. Preoperative median NT-proBNP was 412 pg/ml [164-1309]. NT-proBNP was above the P95 reference value for age in 56 patients (58%). Preoperative NT-proBNP was significantly higher in patients who had mechanical ventilation duration of more than 2 days (1156 pg/ml [281-1951] vs. 267 pg/ml [136-790], p=0.003) and who stayed more than 6 days in the pediatric intensive care unit (727 pg/ml [203-1951] vs. 256 pg/ml [136-790], p=0.007). However, preoperative NT-proBNP was not significantly higher in patients with an increased inotropic score, a prolonged cardiopulmonary bypass time or an increased surgical risk category. Conclusions: An elevated preoperative NT-proBNP reflects hemodynamic status and cardiac dysfunction, and therefore is a valuable adjunct in predicting a complicated postoperative course. ___________________________________ Introduction: Le peptide natriurétique type B (BNP) est un marqueur reflétant le stress myocardique. Dans la population pédiatrique, la signification des valeurs préopératoire de BNP, en particulier sur l'évolution postopératoire, n'est pas clairement établie. Le but de l'étude est de déterminer la valeur prédictive de la partie NT sérique du BNP (NT-proBNP) sur l'évolution post opératoire d'enfants porteur d'une cardiopathie congénitale et ayant eu une chirurgie cardiaque. Résultats: Nonante-sept enfants ont été inclus dans l'étude, avec un âge médian de 3.3 ans [0.7-5.2]. La valeur médiane du NT-proBNP préopératoire était de 412 pg/ml [164-1309]. Le NT-proBNP préopératoire était supérieur au P95 des valeurs de référence pour l'âge chez 56 patients (58%). Le NT-proBNP préopératoire était significativement plus élevé chez les patients ayant eu plus de deux jours de ventilation mécanique dans la période postopératoire (1156 pg/ml [281-1951] vs. 267 pg/ml [136-790], p=0.003) et ayant été hospitalisés plus de 6 jours dans l'unité de soins intensifs pédiatrique (727 pg/ml [203-1951] vs. 256 pg/ml [136-790], p=0.007). Par contre, le NT-proBNP préopératoire n'était pas significativement plus élevé chez les patients ayant eu un score d'inotrope élevé pendant leur hospitalisation aux soins intensifs, un temps de circulation extracorporelle prolongé ou ayant subi une chirurgie avec un risque chirurgical élevé. Conclusions: Un NT-proBNP sérique élevé en préopératoire reflète l'importance du stress myocardique induit par l'hémodynamique et la dysfonction myocardique, il est un marqueur qui permet d'améliorer l'identification des patients à risque d'avoir une évolution post opératoire compliquée.

Synthetic long peptide booster immunization in rhesus macaques primed with replication-competent NYVAC-C-KC induces a balanced CD4/CD8 T-cell and antibody response against the conserved regions of HIV-1.

The Thai trial (RV144) indicates that a prime-boost vaccine combination that induces both T-cell and antibody responses may be desirable for an effective HIV vaccine. We have previously shown that immunization with synthetic long peptides (SLP), covering the conserved parts of SIV, induced strong CD4 T-cell and antibody responses, but only modest CD8 T-cell responses. To generate a more balanced CD4/CD8 T-cell and antibody response, this study evaluated a pox-vector prime/SLP boost strategy in rhesus macaques. Priming with a replication-competent NYVAC, encoding HIV-1 clade C gag, pol and nef, induced modest IFNγ T-cell immune responses, predominantly directed against HIV-1 Gag. Booster immunization with SLP, covering the conserved parts of HIV-1 Gag, Pol and Env, resulted in a more than 10-fold increase in IFNγ ELISpot responses in four of six animals, which were predominantly HIV-1 Pol-specific. The animals showed a balanced polyfunctional CD4 and CD8 T-cell response and high Ab titres.

Development of the ability to inhibit a prepotent response: influence of working memory and processing speed.

This study aimed to examine developmental trends in response inhibition during childhood and to control for possible developmental influence of other basic cognitive processes (such as working memory and processing speed). In addition, we explored the relationships between response inhibition, working memory, and processing speed, as they are thought to be integral to cognitive control. Therefore, we assessed these three cognitive abilities in 159 children aged from 5 to 12. Results showed an improvement in response inhibition ability from 5 to 10 years of age. This improvement remained significant after controlling for the influence of working memory and processing speed. Furthermore, the developmental relationships showed an early differentiation between response inhibition, working memory, and processing speed. Thus, these processes were independent and need to be treated as such in further studies.

MHC/Peptide-Specific Interaction of the Humoral Immune System: A New Category of Antibodies.

Abs bind to unprocessed Ags, whereas cytotoxic CD8(+) T cells recognize peptides derived from endogenously processed Ags presented in the context of class I MHC complexes. We screened, by ELISA, human sera for Abs reacting specifically with the influenza matrix protein (IMP)-derived peptide58-66 displayed by HLA-A*0201 complexes. Among 653 healthy volunteers, blood donors, and women on delivery, high-titered HLA-A*0201/IMP58-66 complex-specific IgG Abs were detected in 11 females with a history of pregnancies and in 1 male, all HLA-A*0201(-). These Abs had the same specificity as HLA-A*0201/IMP58-66-specific cytotoxic T cells and bound neither to HLA-A*0201 nor the peptide alone. No such Abs were detected in HLA-A*0201(+) volunteers. These Abs were not cross-reactive to other self-MHC class I alleles displaying IMP58-66, but bound to MHC class I complexes of an HLA nonidentical offspring. HLA-A*0201/IMP58-66 Abs were also detected in the cord blood of newborns, indicating that HLA-A*0201/IMP58-66 Abs are produced in HLA-A*0201(-) mothers and enter the fetal blood system. That Abs can bind to peptides derived from endogenous Ags presented by MHC complexes opens new perspectives on interactions between the cellular and humoral immune system.

Respiratory health effects of fifteen years of improved collective protection in a wheat-processing worker population.

INTRODUCTION: Occupational exposure to grain dust causes respiratory symptoms and pathologies. To decrease these effects, major changes have occurred in the grain processing industry in the last twenty years. However, there are no data on the effects of these changes on workers' respiratory health. OBJECTIVES: The aim of this study was to evaluate the respiratory health of grain workers and farmers involved in different steps of the processing industry of wheat, the most frequently used cereal in Europe, fifteen years after major improvements in collective protective equipment due to mechanisation. MATERIALS AND METHOD: Information on estimated personal exposure to wheat dust was collected from 87 workers exposed to wheat dust and from 62 controls. Lung function (FEV1, FVC, and PEF), exhaled nitrogen monoxide (FENO) and respiratory symptoms were assessed after the period of highest exposure to wheat during the year. Linear regression models were used to explore the associations between exposure indices and respiratory effects. RESULTS: Acute symptoms - cough, sneezing, runny nose, scratchy throat - were significantly more frequent in exposed workers than in controls. Increased mean exposure level, increased cumulative exposure and chronic exposure to more than 6 mg.m (-3) of inhaled wheat dust were significantly associated with decreased spirometric parameters, including FEV1 and PEF (40 ml and 123 ml.s (-1) ), FEV1 and FVC (0.4 ml and 0.5 ml per 100 h.mg.m (-3) ), FEV1 and FVC (20 ml and 20 ml per 100 h at >6 mg.m (-3) ). However, no increase in FENO was associated with increased exposure indices. CONCLUSIONS: The lung functions of wheat-related workers are still affected by their cumulative exposure to wheat dust, despite improvements in the use of collective protective equipment.

Alzheimer's disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression.

BACKGROUND: Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing. RESULTS: The human APP, βCTF and Aβ42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy ((1)H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks. CONCLUSIONS: Altogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, βCTF and Aβ42/Aβ40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.