Abstract :

Background: Airways progenitors may be involved in embryogenesis and lung repair. The characterization of these important populations may enable development of new therapeutics to treat acute or chronic lung disease. In this study, we aimed to establish the presence of bronchioloalveolar progenitors in ovine lungs and to characterize their potential to differentiate into specialized cells. Results: Lung cells were studied using immunohistochemistry on frozen sections of the lung. Immunocytochemistry and flow cytometry were conducted on ex-vivo derived pulmonary cells. The bronchioloalveolar progenitors were identified by their co-expression of CCSP, SP-C and CD34. A minor population of CD34(pos)/SP-C-pos/CCSPpos cells (0.33% +/- 0.31) was present ex vivo in cell suspensions from dissociated lungs. Using CD34 magnetic positive-cell sorting, undifferentiated SP-C-pos/CCSPpos cells were purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrices, SP-C-pos/CCSPpos cells differentiated into either club cells (formerly named Clara cells) or alveolar epithelial type-II cells. Furthermore, these ex vivo and in vitro derived bronchioloalveolar progenitors expressed NANOG, OCT4 and BMI1, specifically described in progenitors or stem cells, and during lung development. Conclusions: We report for the first time in a large animal the existence of bronchioloalveolar progenitors with dual differentiation potential and the expression of specialized genes. These newly described cell population in sheep could be implicated in regeneration of the lung following lesions or in development of diseases such as cancers.

Nov-08  2013     

DOI : 10.1186/1746-6148-9-224                   

Pubmed : 24206786

Abstract :

BtpA/Btp1/TcpB is a virulence factor produced by Brucella species that possesses a Toll interleukin-1 receptor (TIR) domain. Once delivered into the host cell, BtpA interacts with MyD88 to interfere with TLR signalling and modulates microtubule dynamics. Here the crystal structure of the BtpA TIR domain at 3.15 angstrom is presented. The structure shows a dimeric arrangement of a canonical TIR domain, similar to the Paracoccus denitrificans Tir protein but secured by a unique long N-terminal alpha-tail that packs against the TIR: TIR dimer. Structure-based mutations and multi-angle light scattering experiments characterized the BtpA dimer conformation in solution. The structure of BtpA will help with studies to understand the mechanisms involved in its interactions with MyD88 and with microtubules. Structured digital abstract: BtpA and BtpA bind by x-ray crystallography (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.                  

NOV  01    2013         

DOI: 10.1016/j.febslet.2013.09.007      

Pubmed : 24076024

Abstract :

We describe the design of original nanocarriers that allows for ultrahigh chromophore loading while maintaining the photo-activity of each individual molecule. They consist in shells of charged biocompatible polymers grafted on gold nanospheres. The self-organization of extended polymer chains results from repulsive charges and steric interactions that are optimized by tuning the surface curvature of nanoparticles. This type of nano-scaffolds can be used as light-activated theranostic agents for fluorescence imaging and photodynamic therapy. We demonstrate that, labeled with a fluorescent photosensitizer, it can localize therapeutic molecules before triggering the cell death of B16-F10 melanoma with an efficiency that is similar to the efficiency of the polymer conjugate alone, and with the advantage of extremely high local loading of photosensitizers (object concentration in the picomolar range). (C) 2013 Elsevier Ltd. All rights reserved.

NOV 2013     OK

DOI : 10.1016/j.biomaterials.2013.07.032             

Pubmed : 23915950

Abstract :

 TGF- signaling induces epithelial to mesenchymal transition (EMT) and plays an important role in hepatocellular carcinoma (HCC) development. Clinical observations indicate that hepatitis C virus (HCV) chronic infection, which is a major cause of HCC, induces TGF- signaling perturbations. Here, we investigate the mechanisms by which HCV nonstructural proteins interfere with TGF- signaling, in human hepatoma cell lines expressing HCV subgenomic replicon. A transcriptomic study showed that TGF- stimulation of these cells resulted in a protumoral gene expression profile and in up-regulation of EMT-related genes compared to control interferon-treated cells not expressing HCV proteins. We found that the viral protease NS3-4A interacted with SMURF2, a negative regulator of TGF- signaling. In cells expressing HCV subgenomic replicon or NS3-4A, TGF- stimulation induced an increased expression of SMAD-dependent genes compared to control cells. This enhanced signaling was suppressed by SMURF2 overexpression and mimicked by SMURF2 silencing. In addition, NS3-4A expression resulted in an increased and prolonged TGF--induced phosphorylation of SMAD2/3 that was abrogated by SMURF2 overexpression. Neither NS3-4A protease activity nor SMURF2 ubiquitin-ligase activity was required to affect TGF- signaling. Therefore, by targeting SMURF2, NS3-4A appears to block the negative regulation of TGF- signaling, increasing the responsiveness of cells to TGF-. Verga-Gerard, A., Porcherot, M., Meyniel-Schicklin, L., Andre, P., Lotteau, V., and Perrin-Cocon, L. Hepatitis C virus/human interactome identifies SMURF2 and the viral protease as critical elements for the control of TGF- signaling.  

OCT       2013     

DOI : 10.1096/fj.13-229187          

Pubmed : 23781096

Abstract :

Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent Pmet17-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced Pmet17-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

OCT   2013     

DOI : 10.1038/msb.2013.53             

Pubmed : 24104478 p53

Abstract :

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

SEP        2013     

DOI : 10.3791/50610            

Pubmed : 24084155

Abstract :

An unexplained increase in the incidence of parapneumonic empyema (PPE) in pneumonia cases has been reported in recent years. The present study investigated the genetic and biological specifications of new isolates of torque teno mini virus (TTMV) detected in pleural effusion samples from children hospitalised for severe pneumonia with PPE. A pathogen discovery protocol was applied in undiagnosed pleural effusion samples and led to the identification of three new isolates of TTMV (TTMV-LY). Isolated TTMV-LY genomes were transfected into A549 and human embryonic kidney 293T cells and viral replication was assessed by quantitative real-time PCR and full-length genome amplification. A549 cells were further infected with released TTMV-LY virions and the induced-innate immune response was measured by multiplex immunoassays. Genetic analyses of the three TTMV-LY genomes revealed a classic genomic organisation but a weak identity (<64%) with known sequences. We demonstrated the in vitro replication of TTMV-LY in alveolar epithelial cells and the effective release of infectious viral particles. We also showed a selective production of inflammatory mediators in response to TTMV infection. This study reports the description of replicative TTMV-LY isolated from parapneumonic effusions of children hospitalised with PPE, suggesting a potential role of the virus in the pathogenesis of pneumonia.          

AUG      2013         

DOI : 10.1183/09031936.00107212               

Pubmed : 23060626

Abstract :

Objective. Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomalrecessive form of SLE. Methods. We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. Results. We identified a homozygous missense mutation in PRKCD, encoding protein kinase (PKC), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKC (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKC delta, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. Conclusion. Our findings indicate that PKC is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKC deficiency represents a novel genetic defect of apoptosis leading to SLE.       

AUG      2013          

DOI : 10.1002/art.38008              

Pubmed : 23666743

Abstract :

 Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-beta 1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-beta 1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-beta 1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte-agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 siRNA combination is effective in reinitializing correct production and assembly of the cartilage-characteristic matrix in agarose hydrogel, without production of type I collagen. Because agarose is known to favor long-term expression of the chondrocyte phenotype and agarose-based hydrogels are approved for clinical trials, this strategy appears very promising to repair hyaline cartilage.         

AUG      2013          

DOI : 10.1089/ten.tec.2012.0396          

Pubmed : 23311625

Abstract :

Background: Most quantitative measures of phenotypic traits represent macroscopic contributions of large numbers of cells. Yet, cells of a tissue do not behave similarly, and molecular studies on several organisms have shown that regulations can be highly stochastic, sometimes generating diversified cellular phenotypes within tissues. Phenotypic noise, defined here as trait variability among isogenic cells of the same type and sharing a common environment, has therefore received a lot of attention. Given the potential fitness advantage provided by phenotypic noise in fluctuating environments, the possibility that it is directly subjected to evolutionary selection is being considered. For selection to act, phenotypic noise must differ between contemporary genotypes. Whether this is the case or not remains, however, unclear because phenotypic noise has very rarely been quantified in natural populations. Results: Using automated image analysis, we describe here the phenotypic diversity of S. cerevisiae morphology at single-cell resolution. We profiled hundreds of quantitative traits in more than 1,000 cells of 37 natural strains, which represent various geographical and ecological origins of the species. We observed abundant trait variation between strains, with no correlation with their ecological origin or population history. Phenotypic noise strongly depended on the strain background. Noise variation was largely trait-specific (specific strains showing elevated noise for subset of traits) but also global (a few strains displaying elevated noise for many unrelated traits). Conclusions: Our results demonstrate that phenotypic noise does differ quantitatively between natural populations. This supports the possibility that, if noise is adaptive, microevolution may tune it in the wild. This tuning may happen on specific traits or by varying the degree of global phenotypic buffering.

Juil-03   2013          

DOI : 10.1186/1752-0509-7-54                  

Pubmed : 23822767

Abstract :

Background: Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Results: Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 angstrom resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. Conclusion: The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins. Crystallographic interfaces revealed several interactions that might be involved in FIV replication. Further studies are needed to better understand their biological relevance in the function of FIV Gag during viral replication.              

Juin-24  2013     

DOI : 10.1186/1742-4690-10-64              

Pubmed : 23800358

Abstract :

How morphogen gradients are shaped is a major question in developmental biology, but remains poorly understood. Hedgehog (Hh) is a locally secreted ligand that reaches cells at a distance and acts as a morphogen to pattern the Drosophila wing and the vertebrate neural tube. The proper patterning of both structures relies on the precise control over the slope of Hh activity gradient. A number of hypotheses have been proposed to explain Hh movement and hence graded activity of Hh. A crux to all these models is that the covalent binding of cholesterol to Hh N-terminus is essential to achieve the correct slope of the activity gradient. Still, the behavior of cholesterol-free Hh (Hh-N) remains controversial: cholesterol has been shown to either increase or restrict Hh range depending on the experimental setting. Here, in fly embryos and wing imaginal discs, we show that cholesterol-free Hh diffuses at a long-range. This unrestricted diffusion of cholesterol-free Hh leads to an absence of gradient while Hh signaling strength remains uncompromised. These data support a model where cholesterol addition restricts Hh diffusion and can transform a leveled signaling activity into a gradient. In addition, our data indicate that the receptor Patched is not able to sequester cholesterol-free Hh. We propose that a morphogen gradient does not necessarily stem from the active transfer of a poorly diffusing molecule, but can be achieved by the restriction of a highly diffusible ligand. (C) 2013. Published by The Company of Biologists Ltd.

JUN -15         2013     

DOI : 10.1242/bio.20134952        

Pubmed : 23789110

Abstract :

Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naive pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naive pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naive and primed pluripotency, which represents a key step toward the generation of bona fide naive PSC lines in rabbits. (C) 2013. Published by The Company of Biologists Ltd. 

JUN -15         2013     

DOI : 10.1242/bio.20134242       

Pubmed : 23789112

Abstract :

Human metapneumovirus (HMPV) is a paramyxovirus that causes acute respiratory-tract infections in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We engineered HMPV viral-like particles (HMPV-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two HMPV viruses of either lineage A or B. These VLPs functionally display F and G HMPV surface glycoproteins. When injected in mice, HMPV-VLPs induce strong humoral immune response against both homologous and heterologous strains. Moreover, the induced neutralizing antibodies prevented mortality upon subsequent infection of the lungs with both homologous and heterologous viruses. Upon challenge, viral titers in the lungs of immunized animals were significantly reduced as compared to those of control animals. In conclusion, a HMPV-VLP vaccine that induces cross-protective immunity in mice is a promising approach to prevent HMPV infections. (C) 2013 Elsevier Ltd. All rights reserved.   

Juin-07  2013     

DOI : 10.1016/j.vaccine.2013.03.051      

Pubmed : 23583815

Abstract :

Patrolling Ly6C(-) monocytes are blood-circulating cells that play a role in inflammation and in the defense against pathogens. Here, we show that similar to natural killer (NK) cells, patrolling monocytes express high levels of S1PR5, a G-coupled receptor for sphingosine-1 phosphate. We found that S1pr5(-/-) mice lack peripheral Ly6C(-) monocytes but have a normal number of these cells in the bone marrow (BM). Various lines of evidence exclude a direct contribution of S1PR5 in the survival of Ly6C(-) monocytes at the periphery. Rather, our data support a role for S1PR5 in the egress of Ly6C(-) monocytes from the BM. In particular, we observed a reduced frequency of patrolling monocytes in BM sinusoids of S1PR5 KO mice. Unexpectedly, S1P was not a chemoattractant for patrolling monocytes and had no significant effect on their viability in vitro. Moreover, the disruption of S1P gradients in vivo did not alter Ly6C(-) monocyte trafficking and viability. These data suggest that S1PR5 regulates the trafficking of monocytes via a mechanism independent of S1P gradients.

JUN       2013     

DOI : 10.1002/eji.201343312        

Pubmed : 23519784

Abstract :

Epidemiological studies indicate a role of genetic and environmental factors in Parkinsons disease involving alterations of the neuronal -synuclein (-syn) protein. In particular, a relationship between Parkinsons disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in -syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express -syn endogenously, and overexpression of -syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous -syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous -syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the -syn adenovirustransduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased -syn in the SK-MEL-2 adenovirustransduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase -syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinsons disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinsons disease. 

JUN       2013     

DOI : 10.1093/toxsci/kft076      

 Pubmed :  23535362

Abstract :

A functional study of mutants of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) was conducted with the support of a recently proposed HIV-1 intasome model. Firstly, we investigated the predicted position of the C-terminal domain (CTD) and the flexibility of the alpha-6 helix by mutating the residue Ile-203. This had no impact on the 3 '-processing reaction but reduced the strand transfer reaction and the formation of tetramers. Secondly, the residues Ile-141 of the catalytic loop and Glu-246 of the CTD are predicted to bind the Td-3 base of the viral DNA maintaining it in a "flipped out" position and stabilizing the catalytic core domain (CCD)-CTD interface. Our data showed that the Ile-141/Td-3 interaction was important for the strand transfer activity and the oligomerization of IN. Interestingly, mutating the Glu-246 residue by an alanine enhanced half- and full-site integrations, suggesting that this residue may not be optimized for integration. (C) 2013 Elsevier Inc. All rights reserved.

MAY 10 2013     

DOI : 10.1016/j.virol.2013.02.001  

 Pubmed : 23473371          

Abstract :

The inflammasome is an innate immune signaling platform leading to caspase-1 activation, maturation of pro-inflammatory cytokines and cell death. Recognition of DNA within the host cytosol induces the formation of a large complex composed of the AIM2 receptor, the ASC adaptor and the caspase-1 effector. Francisella tularensis, the agent of tularemia, replicates within the host cytosol. The macrophage cytosolic surveillance system detects Francisella through the AIM2 inflammasome. Upon Francisella novicida infection, we observed a faster kinetics of AIM2 speck formation in ASC(KO) and Casp1(KO) as compared to WT macrophages. This observation was validated by a biochemical approach thus demonstrating for the first time the existence of a negative feedback loop controlled by ASC/caspase-1 that regulates AIM2 complex formation/stability. This regulatory mechanism acted before pyroptosis and required caspase-1 catalytic activity. Our data suggest that sublytic caspase-1 activity could delay the formation of stable AIM2 speck, an inflammasome complex associated with cell death.

APR 2013     

DOI : 10.3389/fcimb.2013.00014          

Pubmed : 23630667

Abstract :

Invariant natural killer T (iNKT) cells expressing a CD1d-restricted invariant alpha beta TCR have key regulatory roles in autoimmunity, pathogen immunity, and tumor surveillance, but their function in the control of allergic skin diseases remains poorly documented. Using a model of contact hypersensitivity (CHS) to the hapten DNFB, we show here that iNKT cell deficiency results in enhanced skin inflammation due to augmented hapten-specific IFN-gamma-producing CD8(+) effectors in skin draining lymph nodes (dLNs) and their massive recruitment into the allergen-exposed skin. Adoptive transfer and antibody depletion experiments as well as in vitro studies revealed that iNKT cells (1) reduce the severity of CHS, even in presensitized mice, (2) require hapten presentation by CD1d(+) dendritic cells (DCs) to dampen skin inflammation, and (3) produce IL-4 and IL-13 after CD1d-dependent in vitro stimulation by hapten-loaded DCs only in the presence of IFN-gamma released from activated CD8(+) effector T cells. In corollary, mice double deficient in IL-4 and IL-13 exhibit an exacerbated CHS. Finally, iNKT-suppressive function is independent of Foxp3(+) regulatory T cells (Tregs). These data highlight that, besides Foxp3(+) Tregs, iNKT cells are potent downregulators of CD8(+) T cell mediated CHS, and underscore that both cell types are important for the regulation of allergic skin inflammation. Journal of Investigative Dermatology (2013) 133, 980-987; doi:10.1038/jid.2012.404; published online 29 November 2012         

APR       2013     

DOI : 10.1038/jid.2012.404           

Pubmed : 23190881

Abstract :

Microglial cells constitute the first line of defense of the central nervous system (CNS) against microbial invasion. Pathogens are detected thanks to an array of innate immune receptors termed pattern recognition receptors (PRRs). PRRs have been thoroughly characterized in bone marrow-derived macrophages, but the PRRs repertoire and functionality in microglial cells remain largely unknown. Microglial cells express various Toll-like Receptors and the Nod1/2 receptors. Recently, a novel innate immune signalling pathway, the inflammasome pathway has been uncovered. Inflammasome activation leads to caspase-1 activation, release of the proinflammatory cytokines, IL-1 and IL-18 and cell death in a process termed pyroptosis. One inflammasome receptor, NLRP3, has been characterized in microglial cells and associated with response to infections and in the initiation of neuro-degeneration in an Alzheimer's disease model. Legionella pneumophila (L.pneumophila) is a flagellated bacterium replicating within macrophages. In bone marrow-derived macrophages, L. pneumophila is detected in a flagellin-dependent manner by the Naip5-NLRC4 (Ipaf) inflammasome pathway. In this study, we decided to use L. pneumophila to investigate the presence and the functionality of this inflammasome in primary murine microglial cells. We show that microglial cells detect L. pneumophila infection in a flagellin-dependent manner leading to caspase-1-mediated bacterial growth restriction, infected cell death and secretion of the proinflammatory cytokines IL-1 and IL18. Overall, our data demonstrate that microglial cells have a functional Naip5-NLRC4 inflammasome likely to be important to monitor and clear CNS infections by flagellated bacteria. (c) 2013 Wiley Periodicals, Inc. (c) 2013 Wiley Periodicals, Inc.   

APR       2013     

DOI : 10.1002/glia.22454             

Pubmed : 23355222

Abstract :

 Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. Considerable progress in deciphering the mechanisms controlling the activation or deletion of T cells has been made by using T cell receptor (TCR) transgenic mice. One such model is the F5 model in which CD8 T cells express a TCR specific for an epitope derived from the influenza NP68 protein. Our aim was to create transgenic mouse models expressing constitutively the NP68 epitope fused to enhanced green fluorescent protein (EGFP) in order to assess unambiguously the relative levels of NP68 epitope expressed by single cells. We used a lentiviral-based approach to generate two independent transgenic mouse strains expressing the fusion protein EGFP-NP68 under the control of CAG (CMV immediate early enhancer and the chicken -actin promoter) or spleen focus-forming virus (SFFV) promoters. Analysis of the pattern of EGFP expression in the hematopoietic compartment showed that CAG and SFFV promoters are differentially regulated during T cell development. However, both promoters drove high EGFP-NP68 expression in dendritic cells (pDCs, CD8+ cDCs, and CD8 cDCs) from spleen or generated in vitro following differentiation from bone-marrow progenitors. NP68 epitope was properly processed and successfully presented by dendritic cells (DCs) by direct presentation and cross-presentation to F5 CD8 T cells. The models presented here arevaluable tools to investigate the priming of F5 CD8 T cells by different subsets of DCs. genesis 51:193200, 2013. (c) 2012 Wiley Periodicals, Inc.       

MAR      2013     

DOI : 10.1002/dvg.22364            

Pubmed : 23281269

Abstract :

Nucleolin is a multifunctional protein that carries several post-translational modifications. We characterized nucleolin acetylation and developed antibodies specific to nucleolin K88 acetylation. Using this antibody we show that nucleolin is acetylated in vivo and is not localized in the nucleoli, but instead is distributed throughout the nucleoplasm. Immunofluorescence studies indicate that acetylated nucleolin is co-localized with the splicing factor SC35 and partially with Y12. Acetylated nucleolin is expressed in all tested proliferating cell types. Our findings show that acetylation defines a new pool of nucleolin which support a role for nucleolin in the regulation of mRNA maturation and transcription by RNA polymerase II. Structured summary of protein interactions: SC35 physically interacts with Nucleolin by anti bait coimmunoprecipitation (View interaction) Nucleolin and SC35 colocalize by fluorescence microscopy (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.         

Mars-01 2013    

DOI : 10.1016/j.febslet.2013.01.035       

Pubmed : 23353999

Abstract :

Type I interferons induce a complex transcriptional program that leads to a generalized antiviral response against a large panel of viruses, including human immunodeficiency virus type 1 (HIV-1). However, despite the fact that interferons negatively regulate HIV-1 ex vivo, a chronic interferon state is linked to the progression of AIDS and to robust viral replication, rather than protection, in vivo. To explain this apparent contradiction, we hypothesized that HIV-1 may have evolved a partial resistance to interferon, and to test this hypothesis, we analyzed the effects of alpha interferon (IFN-alpha) on the infectivity of HIV-1, human immunodeficiency virus type 2 (HIV-2), and rhesus monkey simian immunodeficiency virus (SIVmac). The results we obtained indicate that HIV-1 is more resistant to an IFN-alpha-induced response than are HIV-2 and SIVmac. Our data indicate that the accumulation of viral DNA is more compromised following the infection of IFN-alpha-treated cells with HIV-2 and SIVmac than with HIV-1. This defect correlates with a faster destabilization of HIV-2 viral nucleoprotein complexes (VNCs), suggesting a link between VNC destabilization and impaired viral DNA (vDNA) accumulation. The differential susceptibilities to IFN-alpha of the primate lentiviruses tested here do not map to the capsid protein (CA), excluding de facto a role for human tripartite motif protein isoform 5 alpha (Trim5 alpha) in this restriction; this also suggests that an additional restriction mechanism differentially affects primate lentivirus infection. The different behaviors of HIV-1 and HIV-2 with respect to IFN-alpha responses may account at least in part for the differences in pathogenesis observed between these two virus types.      

MAR      2013     

DOI : 10.1128/JVI.02553-12        

Pubmed : 23255800

Abstract :

Dendritic cells initiate adaptive immune responses, leading either to control cancer by effector T cells or to exacerbate cancer by regulatory T cells that inhibit IFN-gamma-mediated Th1-type response. Dendritic cells can also induce Th17-type immunity, mediated by IL-17A. However, the controversial role of this cytokine in cancer requires further investigations. We generated dendritic cells from peripheral blood monocytes to investigate lifespan, phenotype and chemoresistance of dendritic cells, treated with IL-17A with or without IFN-gamma. Studying the expression of Bcl-2 family members, we demonstrated that dendritic cells constitutively express one pro-survival Bcl-2 member: MCL1. Immature dendritic cells were CD40(low)HLADR(low) CD1a(+) MCL1(+), did not express CD14, CD68 or BCL2A1, and displayed a short 2-day lifespan. IL-17A-treated DC exhibited a semi-mature (CD40(high)HLADR(low)) pre-M2 (CCL22(+) CD206(+) CD163(+) IL1RN(+) IL-10(-) CXCL10(-) IL-12(-)) mixed (CD1a(+) CD14+ CD68(+)) macrophage-dendritic cell phenotype. They efficiently exerted mannose receptor-mediated endocytosis and did not produce superoxide anions, in the absence of TLR engagement. Interestingly, IL-17A promoted a long-term survival of dendritic cells, beyond 12 days, that correlated to BCL2A1 induction, a pro-survival Bcl-2 family member. BCL2A1 transcription was activated by NF-kappa B, downstream of IL-17A transduction. Thus, immature dendritic cells only express MCL1, whereas IL-17A-treated dendritic cells concomitantly expressed two pro-survival Bcl-2 family members: MCL1 and BCL2A1. These latter developed chemoresistance to 11 of the 17 chemotherapy agents tested. However, high doses of either vinblastine or cytarabine decreased MCL1 expression and induced dendritic cell death. When IL-17A is produced in vivo, administration of anti-IL-17A biotherapy may impair dendritic cell survival by targeting BCL2A1 expression. Consequently, depending on the effector or regulatory role of dendritic cells, blocking IL-17A, may be either dangerous or beneficial for cancer outcomes, thus contributing to the apparent controversy around the role of IL-17A in cancer.          

FEB 18   2013     

DOI : 10.1371/journal.pone.0056865    

Pubmed : 23441221

Abstract :

Background: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms. Results: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems. Conclusions: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

2013     

DOI : 10.1186/gb-2013-14-7-r82      

 Pubmed : 23902802     

Abstract :

Laminin 332, composed of the alpha 3, beta 3 and gamma 2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external forces, including skin and stratified squamous mucosa. After being secreted and deposited in the extracellular matrix, laminin 332 undergoes physiological maturation processes consisting in the proteolytic processing of domains located within the alpha 3 and the gamma 2 chains. These maturation events are essential for laminin 332 integration into the basement membrane where it plays an important function in the nucleation and maintenance of anchoring structures. Studies in normal and pathological situations have revealed that laminin 332 can trigger distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and structural characteristics of laminin 332 domains are presented and we discuss whether they trigger specific functions.                 

JAN-FEB              2013      

DOI : 10.4161/cam.23132                    

Pubmed : 23263634

Abstract :

Permanent activation of the NF-kappa B pathway by the human T cell leukemia virus type 1 (HTLV-1) Tax (Tax1) viral transactivator is a key event in the process of HTLV-1-induced T lymphocyte immortalization and leukemogenesis. Although encoding a Tax transactivator (Tax2) that activates the canonical NF-kappa B pathway, HTLV-2 does not cause leukemia. These distinct pathological outcomes might be related, at least in part, to distinct NF-kappa B activation mechanisms. Tax1 has been shown to be both ubiquitinated and SUMOylated, and these two modifications were originally proposed to be required for Tax1-mediated NF-kappa B activation. Tax1 ubiquitination allows recruitment of the IKK-gamma/NEMO regulatory subunit of the IKK complex together with Tax1 into centrosome/Golgi-associated cytoplasmic structures, followed by activation of the IKK complex and RelA/p65 nuclear translocation. Herein, we compared the ubiquitination, SUMOylation, and acetylation patterns of Tax2 and Tax1. We show that, in contrast to Tax1, Tax2 conjugation to endogenous ubiquitin and SUMO is barely detectable while both proteins are acetylated. Importantly, Tax2 is neither polyubiquitinated on lysine residues nor ubiquitinated on its N-terminal residue. Consistent with these observations, Tax2 conjugation to ubiquitin and Tax2-mediated NF-kappa B activation is not affected by overexpression of the E2 conjugating enzyme Ubc13. We further demonstrate that a nonubiquitinable, non-SUMOylable, and nonacetylable Tax2 mutant retains a significant ability to activate transcription from a NF-kappa B-dependent promoter after partial activation of the IKK complex and induction of RelA/p65 nuclear translocation. Finally, we also show that Tax2 does not interact with TRAF6, a protein that was shown to positively regulate Tax1-mediated activation of the NF-kappa B pathway. 

JAN        2013     

DOI :    10.1128/JVI.01792-12    

Pubmed :   23135727

Abstract :

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (D Delta Np53). Historically, D Delta Np53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that D Delta Np53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and D Delta Np53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of D Delta Np53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to D Delta Np53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that D Delta Np53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology. Cell Death and Differentiation (2013) 20, 108-116; doi:10.1038/cdd.2012.100; published online 17 August 2012

JAN        2013     

DOI :     10.1038/cdd.2012.100   

Pubmed :  22898807

Abstract :

A versatile approach is introduced for the synthesis of well-defined, biocompatible conjugates combining two-photon chromophores and hydrophilic multifunctional polymers synthesized by RAFT controlled radical polymerization. As an illustration, two different classes of conjugates carrying multiple fluorophores (based on an anthracene moiety, Anth) or photosensitizers (based on a dibromobenzene moiety, DBB) along the polymer chain were elaborated for bioimaging and photodynamic therapy (PDT) applications, respectively. In both cases, the polymer greatly improved the solubility in biorelevant media as well as the cell uptake. Anth conjugates provided high quality fluorescence microscopy images using both one- and two-photon excitation. DBB conjugates potently induced the death of cancer cells upon photoactivation.             

2013     

DOI :   10.1039/c2py20565c