Publications by authors named "Sahil Adriouch"

55 Publications

A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy.

Nat Commun 2021 01 28;12(1):653. Epub 2021 Jan 28.

Université Côte d'Azur, CNRS, INSERM, IRCAN, Nice, France.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4 T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.
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http://dx.doi.org/10.1038/s41467-021-20912-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843983PMC
January 2021

Evaluation of P2X7 Receptor Function in Tumor Contexts Using rAAV Vector and Nanobodies (AAVnano).

Front Oncol 2020 11;10:1699. Epub 2020 Sep 11.

Normandie University, UNIROUEN, INSERM, U1234, Pathophysiology, Autoimmunity, Neuromuscular Diseases and Regenerative THERapies, Rouen, France.

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. Extracellular ATP is known to signal through plasma membrane receptors of the P2Y and P2X families. Among the P2X receptors, P2X7 has attracted increasing interest in the field of inflammation as well as in cancer. P2X7 is expressed by immune cells and by most malignant tumor cells where it plays a crucial yet complex role that remains to be clarified. P2X7 activity has been associated with production and release of pro-inflammatory cytokines, modulation of the activity and survival of immune cells, and the stimulation of proliferation and migratory properties of tumor cells. Hence, P2X7 plays an intricate role in the tumor microenvironment combining beneficial and detrimental effects that need to be further investigated. For this, we developed a novel methodology termed AAVnano based on the use of Adeno-associated viral vectors (AAV) encoding nanobodies targeting P2X7. We discuss here the advantages of this tool to study the different functions of P2X7 in cancer and other pathophysiological contexts.
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http://dx.doi.org/10.3389/fonc.2020.01699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518291PMC
September 2020

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

Arterioscler Thromb Vasc Biol 2020 07 14;40(7):1722-1737. Epub 2020 May 14.

From the Normandy University, UniRouen, Inserm (Institut National de la Santé et de la Recherche Médicale) UMR1096 (EnVI Laboratory), FHU REMOD-VHF, Rouen, France (H.M., A.D., V.T., I.B., J.P.H., S.R., J.R., S.F., V.R., P.M.).

Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-C therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4 and CD8 T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI.

Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-C. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.
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http://dx.doi.org/10.1161/ATVBAHA.120.314370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310303PMC
July 2020

Nanobody-Enhanced Targeting of AAV Gene Therapy Vectors.

Mol Ther Methods Clin Dev 2019 Dec 16;15:211-220. Epub 2019 Sep 16.

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

A limiting factor for the use of adeno-associated viruses (AAVs) as vectors in gene therapy is the broad tropism of AAV serotypes, i.e., the parallel infection of several cell types. Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allow easy reformatting into fusion proteins. Herein we show that a membrane protein-specific nanobody can be inserted into a surface loop of the VP1 capsid protein of AAV2. Using three structurally distinct membrane proteins-a multispan ion channel, a single-span transmembrane protein, and a glycosylphosphatidylinositol (GPI)-anchored ectoenzyme-we show that this strategy can dramatically enhance the transduction of specific target cells by recombinant AAV2. Moreover, we show that the nanobody-VP1 fusion of AAV2 can be incorporated into the capsids of AAV1, AAV8, and AAV9 and thereby effectively redirect the target specificity of other AAV serotypes. Nanobody-mediated targeting provides a highly efficient AAV targeting strategy that is likely to open up new avenues for genetic engineering of cells.
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http://dx.doi.org/10.1016/j.omtm.2019.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819893PMC
December 2019

Pyroglutamide-Based P2X7 Receptor Antagonists Targeting Inflammatory Bowel Disease.

J Med Chem 2020 03 27;63(5):2074-2094. Epub 2019 Sep 27.

CHRU de Lille, Faculté de Médecine-Pôle Recherche, Inserm U995, LIRIC, Université de Lille, Place Verdun, F-59045 Lille Cedex, France.

This report deals with the design, the synthesis, and the pharmacological evaluation of pyroglutamide-based P2X7 antagonists. A dozen were shown to possess improved properties, among which inhibition of YO-PRO-1/TO-PRO-3 uptake and IL1β release upon BzATP activation of the receptor and dampening signs of DSS-induced colitis on mice, in comparison with reference antagonist GSK1370319A. Docking study and biological evaluation of synthesized compounds has highlighted new SAR, and low toxicity profiles of pyroglutamides herein described are clues for the finding of a usable -P2X7 antagonist drug. Such a drug would raise the hope for a cure to many P2X7-dependent pathologies, including inflammatory, neurological, and immune diseases.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00584DOI Listing
March 2020

P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis.

Nat Commun 2019 06 20;10(1):2711. Epub 2019 Jun 20.

Unidad de Inflamación Molecular y Cirugía Experimental, Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, 30120, Spain.

Sepsis is characterized by a systemic inflammatory response followed by immunosuppression of the host. Metabolic defects and mitochondrial failure are common in immunocompromised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection.
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http://dx.doi.org/10.1038/s41467-019-10626-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586640PMC
June 2019

Novel biologics targeting the P2X7 ion channel.

Curr Opin Pharmacol 2019 08 12;47:110-118. Epub 2019 Apr 12.

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Normandie University, UNIROUEN, INSERM, U1234, Pathophysiology, Autoimmunity, Neuromuscular diseases and regenerative THERapies (PANTHER), 76000, Rouen, France. Electronic address:

Targeting the P2X7 ion channel, a danger sensor for extracellular nucleotides, improves outcomes in models of inflammation, cancer, and brain-diseases. Antibodies and nanobodies have been developed that antagonize or potentiate gating of P2X7. Their potential advantages over small-molecule drugs include high specificity, lower off-target effects, and tunable in vivo half-life. Genetic fusion of P2X7-specific biologics to binding modules may enable targeting of specific cell subsets. Besides directly modulating P2X7 function, antibodies can also initiate specific depletion of P2X7-expressing cells. Adeno-associated viral vectors (AAV) can be used to express P2X7-specific antibodies in vivo to achieve long-lasting biological effects. Furthermore, if successfully targeted to P2X7-expressing cells, AAVs may enable modulation of the function of P2X7-expressing immune cells via encoded transgenic RNA or proteins.
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http://dx.doi.org/10.1016/j.coph.2019.03.001DOI Listing
August 2019

Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity.

Sci Rep 2019 03 6;9(1):3631. Epub 2019 Mar 6.

Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV's low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV's intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV's in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8 T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.
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http://dx.doi.org/10.1038/s41598-019-40071-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403382PMC
March 2019

AMPK Activation of PGC-1α/NRF-1-Dependent SELENOT Gene Transcription Promotes PACAP-Induced Neuroendocrine Cell Differentiation Through Tolerance to Oxidative Stress.

Mol Neurobiol 2019 Jun 28;56(6):4086-4101. Epub 2018 Sep 28.

UNIROUEN, Inserm U1239, Neuronal and Neuroendocrine Differentiation and Communication Laboratory, Rouen-Normandie University, 76821, Mont-Saint-Aignan, France.

Several cues including pituitary adenylate cyclase-activating polypeptide (PACAP), which acts through cAMP stimulation, specify the conversion of sympathoadrenal (SA) precursors toward different cell phenotypes by promoting their survival and differentiation. Selenoprotein T (SELENOT) is a PACAP-stimulated ER oxidoreductase that exerts an essential antioxidant activity and whose up-regulation is associated with SA cell differentiation. In the present study, we investigated the transcriptional cascade elicited by PACAP/cAMP to trigger SELENOT gene transcription during the conversion of PC12 cells from SA progenitor-like cells toward a neuroendocrine phenotype. Unexpectedly, we found that PACAP/cAMP recruits the canonical pathway that regulates mitochondrial function in order to elicit SELENOT gene transcription and the consequent antioxidant response during PC12 cell differentiation. This cascade involves LKB1-mediated AMPK activation in order to stimulate SELENOT gene transcription through the PGC1-α/NRF-1 complex, thus allowing SELENOT to promote PACAP-stimulated neuroendocrine cell survival and differentiation. Our data reveal that a PACAP and cAMP-activated AMPK-PGC-1α/NRF-1 cascade is critical for the coupling of oxidative stress tolerance, via SELENOT gene expression, and mitochondrial biogenesis in order to achieve PC12 cell differentiation. The data further highlight the essential role of SELENOT in cell metabolism during differentiation.
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http://dx.doi.org/10.1007/s12035-018-1352-xDOI Listing
June 2019

Monitoring Expression and Enzyme Activity of Ecto-ARTCs.

Methods Mol Biol 2018 ;1813:167-186

Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Mammalian ARTCs are expressed as glycosylphosphatidylinositol (GPI)-anchored ectoenzymes (ARTC1-ARTC4) or secretory proteins (ARTC5) by different cell types. The ARTC2 enzymes catalyze mono-ADP-ribosylation of arginine residues in the extracellular domain of membrane proteins or secretory proteins. In this chapter we provide protocols to monitor the expression and activity of ARTCs on the cell membrane of living cells and in soluble form in biological fluids.
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http://dx.doi.org/10.1007/978-1-4939-8588-3_11DOI Listing
March 2019

Contribution of genotoxic anticancer treatments to the development of multiple primary tumours in the context of germline TP53 mutations.

Eur J Cancer 2018 09 30;101:254-262. Epub 2018 Jul 30.

Normandie Univ, UNIROUEN, Inserm U1245, Rouen University Hospital, Department of Genetics, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

Introduction: Li-Fraumeni syndrome (LFS), due to TP53 germline mutations, is characterised by a remarkably high incidence of multiple primary cancers (MPCs), and the key role of p53 in response to DNA damage questions the contribution of anticancer treatments to MPCs development.

Materials And Methods: We first evaluated genotoxicity of X-rays and different classes of conventional chemotherapies, thanks to genotoxicity assays, based on the measurement of transcriptional response to DNA damage and performed in murine splenocytes, either exposed ex vivo or extracted from exposed mice. We then exposed a total of 208 Trp53Δ/Δ, wt/Δ or wt/wt mice to clinical doses of X-rays or genotoxic or non-genotoxic chemotherapies. Tumour development was monitored using whole-body magnetic resonance imaging and pathological examination at death.

Results: X-rays and conventional chemotherapies, except mitotic spindle poisons, were found to be genotoxic in both p53 genotoxicity assays. Exposition to X-rays and the topoisomerase inhibitor etoposide, analysed as genotoxic anticancer treatment, drastically increase the tumour development risk in Trp53Δ/Δ and wt/Δ mice (hazard ration [HR] = 4.4, 95% confidence interval [CI] [2.2-8.8], p < 0.001*** and HR = 4.7, 95% CI [2.4-9.3], p < 0.001***, respectively). In contrast, exposure to the non-genotoxic mitotic spindle poison, docetaxel, had no impact on tumour development.

Conclusions: This study shows that radiotherapy and genotoxic chemotherapies significantly increase the risk of tumour development in a LFS mice model. These results strongly support the contribution of genotoxic anticancer treatments to MPC development in LFS patients. Therefore, to reduce the risk of MPCs in germline TP53 mutation carriers, radiotherapy should be avoided whenever possible, surgical treatment prioritised, and non-genotoxic treatments considered.
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http://dx.doi.org/10.1016/j.ejca.2018.06.011DOI Listing
September 2018

Receptor Activator of NF-κB Orchestrates Activation of Antiviral Memory CD8 T Cells in the Spleen Marginal Zone.

Cell Rep 2017 Nov;21(9):2515-2527

Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS, Marseille, France. Electronic address:

The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169 MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.
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http://dx.doi.org/10.1016/j.celrep.2017.10.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723674PMC
November 2017

Ecto-ADP-ribosyltransferase ARTC2.1 functionally modulates FcγR1 and FcγR2B on murine microglia.

Sci Rep 2017 11 28;7(1):16477. Epub 2017 Nov 28.

Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Mammalian ecto-ADP-ribosyltransferases (ecto-ARTs or also ARTCs) catalyze the ADP-ribosylation of cell surface proteins using extracellular nicotinamide adenine dinucleotide (NAD) as substrate. By this post-translational protein modification, ecto-ARTs modulate the function of various target proteins. A functional role of ARTC2 has been demonstrated for peripheral immune cells such as T cells and macrophages. Yet, little is known about the role of ecto-ARTs in the central nervous system and on microglia. Here, we identified ARTC2.1 as the major ecto-ART expressed on murine microglia. ARTC2.1 expression was strongly upregulated on microglia upon co-stimulation with LPS and an ERK1/2 inhibitor or upon IFNβ stimulation. We identified several target proteins modified by ARTC2.1 on microglia with a recently developed mass spectrometry approach, including two receptors for immunoglobulin G (IgG), FcγR1 and FcγR2B. Both proteins were verified as targets of ARTC2.1 in vitro using a radiolabeling assay with P-NAD as substrate. Moreover, ADP-ribosylation of both targets strongly inhibited their capacity to bind IgG. In concordance, ARTC2.1 induction in WT microglia and subsequent cell surface ADP-ribosylation significantly reduced the phagocytosis of IgG-coated latex beads, which was unimpaired in NAD/DTT treated microglia from ARTC2.1 mice. Hence, induction of ARTC2.1 expression under inflammatory conditions, and subsequent ADP-ribosylation of cell surface target proteins could represent a hitherto unnoticed mechanism to regulate the immune response of murine microglia.
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http://dx.doi.org/10.1038/s41598-017-16613-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705771PMC
November 2017

Clonal Proliferation and Stochastic Pruning Orchestrate Lymph Node Vasculature Remodeling.

Immunity 2016 10;45(4):877-888

Aix-Marseille Université, CNRS, INSERM, CIML, 13288 Marseille, France. Electronic address:

Lymph node (LN) expansion during an immune response relies on the transient remodeling of its vasculature. Although the mechanisms driving LN endothelial cell division are beginning to be understood, a comprehensive view of LN endothelial cell dynamics at the single-cell level is lacking. Here, we used multicolored fluorescent fate-mapping models to track the behavior of blood endothelial cells during LN expansion upon inflammation and subsequent return to homeostasis. We found that expansion of the LN vasculature relied on the sequential assembly of endothelial cell proliferative units. This segmented growth was sustained by the clonal proliferation of high endothelial venule (HEV) cells, which act as local progenitors to create capillaries and HEV neo-vessels at the periphery of the LN. Return to homeostasis was accompanied by the stochastic death of pre-existing and neo-synthesized LN endothelial cells. Thus, our fate-mapping studies unravel-at a single-cell level-the complex dynamics of vascular-tree remodeling during LN expansion and contraction.
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http://dx.doi.org/10.1016/j.immuni.2016.09.017DOI Listing
October 2016

Induction of Hematopoietic Microchimerism by Gene-Modified BMT Elicits Antigen-Specific B and T Cell Unresponsiveness toward Gene Therapy Products.

Front Immunol 2016 15;7:360. Epub 2016 Sep 15.

Normandie University, UNIROUEN, Pathophysiology and Biotherapy of Inflammatory and Autoimmune Diseases, INSERM, CHU Rouen , Rouen , France.

Background: Gene therapy is a promising treatment option for hemophilia and other protein deficiencies. However, immune responses against the transgene product represent an obstacle to safe and effective gene therapy, urging for the implementation of tolerization strategies. Induction of a hematopoietic chimerism bone marrow transplantation (BMT) is a potent means for inducing immunological tolerance in solid organ transplantation.

Objectives: We reasoned, here, that the same viral vector could be used, first, to transduce BM cells for inducing chimerism-associated transgene-specific immune tolerance and, second, for correcting protein deficiencies by vector-mediated systemic production of the deficient coagulation factor.

Methods: Evaluation of strategies to induce B and T cell tolerance was performed using gene transfer with lentiviral (LV) vectors encoding coagulation factor IX (FIX) or the SIINFEKL epitope of ovalbumin. Following induction of microchimerism BMT, animals were challenged with gene transfer with LV vectors.

Results: The experimental approach prevented humoral immune response against FIX, resulting in persistence of therapeutic levels of circulating FIX, after LV-mediated gene transfer . In an ovalbumin model, we also demonstrated that this approach effectively tolerized the CD8 T cell compartment in an antigen-specific manner.

Conclusion: These results provide the proof-of-concept that inducing a microchimerism by gene-modified BMT is a powerful tool to provide transgene-specific B and T cell tolerance in a gene therapy setting.
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http://dx.doi.org/10.3389/fimmu.2016.00360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023671PMC
September 2016

CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations.

Mol Cell 2016 08 21;63(3):526-38. Epub 2016 Jul 21.

Normandie Univ, UNIROUEN, INSERM, DC2N, 76000 Rouen, France; Institute for Research and Innovation in Biomedicine (IRIB), 76000 Rouen, France. Electronic address:

Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer.
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http://dx.doi.org/10.1016/j.molcel.2016.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537739PMC
August 2016

HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis and is down-regulated by both deletion and epigenetic alterations.

Leuk Res 2016 06 11;45:90-100. Epub 2016 Apr 11.

Henri Becquerel Center, INSERM U918, Institut de Recherche et d'Innovation Biomédicale (iRiB) Rouen, France.

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1, HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in many human tumors. Here, we report HACE1 as a candidate tumor suppressor gene down-regulated by a combination of deletion and epigenetic mechanisms. HACE1 deletions were observed in 40% of B-cell lymphoma tumors. Hypermethylation of the HACE1 promoter CpG177 island was found in 60% (68/111) of cases and in all tested B-cell lymphoma lines. Using HDAC inhibitors, we observed predominantly inactive chromatin conformation (methylated H3 histones H3K9me2) in HACE1 gene promoter region. We demonstrated in Ramos and Raji cells that down-regulation of HACE1 expression was associated with a significant decrease in apoptosis and an accumulation of cells in the S and G2/M phases. Our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and can be downregulated by deacetylation of its promoter region chromatin, which makes HACE1 a potential target for HDAC inhibitors.
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http://dx.doi.org/10.1016/j.leukres.2016.04.007DOI Listing
June 2016

Prophylactic Injection of Recombinant Alpha-Enolase Reduces Arthritis Severity in the Collagen-Induced Arthritis Mice Model.

PLoS One 2015 24;10(8):e0136359. Epub 2015 Aug 24.

INSERM, U905, Rouen, France; Normandy University, Institute for Research and Innovation in Biomedicine (IRIB), Rouen, France; Rouen University Hospital, Department of Rheumatology, Rouen, France.

Objective: To evaluate the ability of the glycolytic enzyme alpha-enolase (ENO1) or its immunodominant peptide (pEP1) to reduce the severity of CIA in DBA/1 mice when injected in a prophylactic way.

Methods: Mice were treated with mouse ENO1 or pEP1 one day prior to collagen II immunization. Clinical assessment was evaluated using 4 parameters (global and articular scores, ankle thickness and weight). Titers of serum anti-ENO1, anti-cyclic citrullinated peptides (anti-CCP) and anti-CII (total IgG and IgG1/IgG2a isotypes) antibodies were measured by ELISA at different time-points. Disease activity was assessed by histological analysis of both anterior and hind paws at the end of experimentation.

Results: Prophylactic injection of 100 μg of ENO1 reduced severity of CIA. Serum levels of anti-CII antibodies were reduced in ENO1-treated mice. Concordantly, ENO1-treated mice joints presented less severe histological signs of arthritis. ENO1 did not induce a shift toward a Th2 response since IgG1/IgG2a ratio of anti-CII antibodies remained unchanged and IL-4 serum levels were similar to those measured in the control group.

Conclusions: Pre-immunization with ENO1 or its immunodominant peptide pEP1 reduces CIA severity at the clinical, immunological and histological levels. Effects of pEP1 were less pronounced. This immunomodulatory effect is associated with a reduction in anti-CII antibodies production but is not due to a Th1/Th2 shift.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136359PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547710PMC
May 2016

Oral-tolerization Prevents Immune Responses and Improves Transgene Persistence Following Gene Transfer Mediated by Adeno-associated Viral Vector.

Mol Ther 2016 Feb 12;24(1):87-95. Epub 2015 Aug 12.

Inserm, U905, Rouen, France.

Gene therapy represents a feasible strategy to treat inherited monogenic diseases and intramuscular (i.m.) injection of recombinant adeno-associated viral (AAV) vector is now recognized as a convenient and safe method of gene transfer. However, this approach is hampered by immune responses directed against the vector and against the transgenic protein. We used here to reproduce this situation a mouse model where robust immune responses are induced following injection of an AAV vector coding for an immunogenic transgenic protein. We show that prophylactic oral administration of the immunogenic protein before AAV-mediated gene transfer completely prevented antibody formation and cytotoxic CD8(+) T-cell response. Consistently, prophylactic oral-tolerization considerably improved long-term transgene persistence and expression. Mechanistically, inhibition of the cytotoxic immune response involved abortive proliferation of antigen-specific cytotoxic CD8(+) T cells, upregulation of the PD-1 immunoregulatory molecule, downregulation of the Bcl-2 antiapoptotic factor, and their deletion in the context of AAV-mediated gene transfer. Hence, gene therapy may represent an ideal situation where oral-tolerization can be adopted before or at the same time as vector injection to efficiently prevent deleterious immune responses directed against the transgenic protein.
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http://dx.doi.org/10.1038/mt.2015.146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754539PMC
February 2016

P2X7 on Mouse T Cells: One Channel, Many Functions.

Front Immunol 2015 19;6:204. Epub 2015 May 19.

U905, INSERM , Rouen , France ; Institute for Research and Innovation in Biomedicine (IRIB), Normandy University , Rouen , France.

The P2X7 receptor is an adenosine triphosphate (ATP)-gated cation channel that is expressed by several cells of the immune system. P2X7 is best known for its proinflammatory role in promoting inflammasome formation and release of mature interleukin (IL)-1β by innate immune cells. Mounting evidence indicates that P2X7 is also an important regulatory receptor of murine and human T cell functions. Murine T cells express a sensitive splice variant of P2X7 that can be activated either by non-covalent binding of ATP or, in the presence of nicotinamide adenine dinucleotide, by its covalent ADP-ribosylation catalyzed by the ecto-ADP-ribosyltransferase ARTC2.2. Prolonged activation of P2X7 by either one of these pathways triggers the induction of T cell death. Conversely, lower concentrations of ATP can activate P2X7 to enhance T cell proliferation and production of IL-2. In this review, we will highlight the molecular and cellular consequences of P2X7 activation on mouse T cells and its versatile role in T cell homeostasis and activation. Further, we will discuss important differences in the function of P2X7 on human and murine T cells.
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http://dx.doi.org/10.3389/fimmu.2015.00204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436801PMC
June 2015

Tuning IL-2 signaling by ADP-ribosylation of CD25.

Sci Rep 2015 Mar 10;5:8959. Epub 2015 Mar 10.

1] Institute of Immunology, University Medical Center, 20246 Hamburg, Germany [2] Normandy University, Institute for Research and Innovation in Biomedicine, 76183 Rouen, France.

Control of immunologic tolerance and homeostasis rely on Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5, and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.
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http://dx.doi.org/10.1038/srep08959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354014PMC
March 2015

Evaluation and comparison of three different separation techniques for analysis of retroamide enantiomers and their biological evaluation against h-P2X7 receptor.

J Chromatogr B Analyt Technol Biomed Life Sci 2015 Apr 9;986-987:35-43. Epub 2015 Feb 9.

Inserm U995, Université de Lille, Laboratoire de Chimie Analytique, UFR Pharmacie, F-59000 Lille, France. Electronic address:

The P2X receptors are seven-transmembrane domain G protein-coupled receptors and the 7 subtypes of P2X receptors identified in humans, and named P2X1 to P2X7, are channel receptors whose endogenous ligand is ATP. New antagonists of the P2X7 receptor were developed, since this purinergic receptor was highlighted to be involved in many diseases such as different types of pain, cancer, ischemia, neurodegenerative diseases (including Parkinson's and Alzheimer's diseases) characterized by inflammatory processes. With the aim of evaluate the impact of chirality on the pharmacological activity of a new P2X7R antagonist, a semi-preparative method was developed in supercritical fluid chromatography (SFC). Among four polysaccharide based chiral stationary phases: Chiralcel OD-H and OJ-H and Chiralpak AS-H and AD-H, the last one namely amylose tris (3,5-dimethylphenylcarbamate) with a mobile phase consisted of carbon dioxide-ethanol (80:20, v/v), led to the successful separation of the enantiomers in short run time and with good resolution. Limits of detection and quantification were calculated and were found equal for compound 1, to 1.37 μM and 4.57 μM respectively, for peak 1 and were equal to 1.60 μM and 5.30 μM respectively, for peak 2 at λ=210 nm. Before carrying out the pharmacological evaluation of each enantiomer, two complementary methodologies, e.g. liquid chromatography and capillary electrophoresis were performed in parallel to improve the limits of detection and quantification to assess the enantiomeric purity. HPLC using a Chiralpak AD stationary phase led to four times lower limits of detection and quantification with regard to SFC. In the same time, capillary electrophoresis involving dual cyclodextrins system constituted of a SBE-β-CD and a MM-β-CD mixture enhanced the signal-to-noise ratio and led to similar limits of detection and quantification with regard to SFC. No trace of the other enantiomer was found in the isolated one. Biological activities of individual enantiomers were then evaluated and revealed no cytotoxicity against cell lines and a significant difference in terms of their IC50 values with respect to the investigated racemate (6.43 μM): 3.49 μM for the (R)-enantiomer and >10(-4)μM for the (S)-enantiomer, for compound 1, showing that, this antagonist activity is stereospecific.
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http://dx.doi.org/10.1016/j.jchromb.2015.02.001DOI Listing
April 2015

Chromosomal instability but lack of transformation in human myoblast preparations.

Cell Transplant 2014 ;23(12):1475-87

Inserm, U905, Rouen, France.

Genetic alterations have recently been described as emerging during the culture of embryonic stem cells or induced pluripotent stem cells, raising concerns about their safety in future clinical use. Myoblasts are adult stem cells with important therapeutic potential that have been used in clinical trials for almost 20 years, but their genome integrity has not yet been established. Here we produced 10 human myoblast preparations and investigated their genomic stability. At the third passage, half of the preparations had a normal karyotype and half showed one to four alterations/30 metaphases. Chromosome 2 trisomy was found in 1-2/30 metaphases and/or 2/100 nuclei by FISH in 3/10 samples, and there was no other recurrent anomaly. When prolonging cultures, these erratic abnormalities were never associated with a growth advantage. Cellular senescence was manifested in all samples by growth arrest before passage 15. Expression of TERT was always negative. Molecular analysis of individual p53 transcripts did not reveal tumorigenic mutations. CGH array (10 samples) and exome sequencing (one sample) failed to detect copy number variations or accumulation of mutations, respectively. Myoblasts did not grow either in soft agar or in vivo after injection in immunodeficient mice. Hence, occasional genomic abnormalities may occur during myoblast culture but are not associated with risk of transformation.
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http://dx.doi.org/10.3727/096368913X670192DOI Listing
September 2015

Genetic and pharmacological inactivation of the purinergic P2RX7 receptor dampens inflammation but increases tumor incidence in a mouse model of colitis-associated cancer.

Cancer Res 2015 Mar 6;75(5):835-45. Epub 2015 Jan 6.

Institute for Research on Cancer and Aging, Nice, France; IRCAN U1081 UMR CNRS 7284, Nice Cedex, France. University of Nice-Sophia Antipolis, Nice, France. Centre Antoine Lacassagne, Nice, France.

Colitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD). Binding of extracellular ATP to the purinergic receptor P2RX7 has emerged as a critical event in controlling intestinal inflammation, acting to limit elevation of proinflammatory mast cells and cytokines and promote survival of regulatory T cells (Treg) and enteric neurons. In this study, we investigated the effect of P2RX7 blockade in an established mouse model of CAC. Using genetic and pharmacologic tools, we found unexpectedly that while P2RX7 mediated inflammatory responses, it also acted at an early time to suppress CAC development. P2RX7 blockade enhanced proliferation of intestinal epithelial cells and protected them from apoptosis. The proliferative effects of P2RX7 blockade were associated with an increased production of TGFβ1 that was sufficient to stimulate the proliferation of intestinal epithelial cells. Finally, P2RX7 blockade also altered immune cell infiltration and promoted Treg accumulation within lesions of the digestive system. Taken together, our findings reveal an unexpected role for P2RX7 in preventing CAC, suggesting cautions in the use of P2RX7 inhibitors to treat IBD given the possibility of increasing risks CAC as a result.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1778DOI Listing
March 2015

Role of M2-like macrophage recruitment during angiogenic growth factor therapy.

Angiogenesis 2015 Apr 24;18(2):191-200. Epub 2014 Dec 24.

UFR Médecine-Pharmacie, Inserm (Institut National de la Santé et de la Recherche Médicale) UMR1096, 22 Boulevard Gambetta, 76183, Rouen Cedex, France.

Therapeutic angiogenesis has yet to fulfill its promise for the clinical treatment of ischemic diseases. Given the impact of macrophages during pathophysiological angiogenesis, we asked whether macrophages may similarly modulate vascular responses to targeted angiogenic therapies. Mouse matrigel plug assay and rat myocardial infarction (MI) model were used to assess angiogenic therapy with either VEGF-A or FGF-2 with HGF (F+H) delivered locally via albumin-alginate microcapsules. The infiltration of classical M1-type and alternative M2-like macrophages was assessed. Clodronate was used to prevent macrophage recruitment, and the VEGFR2 blocking antibody, DC101, to prevent VEGF-A signaling. At 3 weeks after matrigel implantation, the combination therapy (F+H) led to increased total, and specifically M2-like, macrophage infiltration versus control and VEGF-A plugs, correlating with the angiogenic response. In contrast, VEGF-A preferential recruited M1-type macrophages. In agreement with a direct role of M2-like macrophages in F+H-induced vessel growth, clodronate radically decreased angiogenesis. Further, DC101 reduced F+H-induced angiogenesis, without altering macrophage infiltration, revealing macrophage-derived VEGF-A as a crucial determinant of tissue responsiveness. Similarly, increased cardiac M2-like macrophage infiltration was found following F+H therapy post-MI, with strong correlation between macrophage levels and angiogenic and arteriogenic responses. In conclusion, M2-like macrophages play a decisive role, linked to VEGF-A production, in regulation of tissue responsiveness to angiogenic therapies including the combination of F+H. Our data suggest that future attempts at therapeutic revascularization in ischemic patients might benefit from coupling targeted growth factor delivery with either direct or indirect approaches to recruit pro-angiogenic macrophages in order to maximize therapeutic angiogenic/arteriogenic responses.
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http://dx.doi.org/10.1007/s10456-014-9456-zDOI Listing
April 2015

Intrinsic transgene immunogenicity gears CD8(+) T-cell priming after rAAV-mediated muscle gene transfer.

Mol Ther 2015 Apr 10;23(4):697-706. Epub 2014 Dec 10.

INSERM U1151, Institut Necker Enfants Malades, CNRS, UMR8253, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.
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http://dx.doi.org/10.1038/mt.2014.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395773PMC
April 2015

ADP-ribosylation of P2X7: a matter of life and death for regulatory T cells and natural killer T cells.

Curr Top Microbiol Immunol 2015 ;384:107-26

Institute of Immunology, University Medical Center, Hamburg-Eppendorf, Germany.

ADP-ribosyltransferases comprise a family of enzymes originally discovered as bacterial toxins and later characterised also in mammals. In mice, the ADP-ribosyltransferase ARTC2.2 is expressed at the surface of T lymphocytes and has been studied extensively. In the presence of extracellular NAD(+), ARTC2.2 ADP-ribosylates several cell surface target proteins and thereby regulates their function. P2X7, an ATP-gated cation channel, has been discovered as a prominent ARTC2.2 target at the surface of mouse T cells. ADP-ribosylation of P2X7 in the presence of low micromolar extracellular NAD(+) induces long-lasting P2X7 activation and triggers cell death. Regulatory T cell subsets (Tregs and NKT cells) are remarkably sensitive to NAD(+)-induced cell death (NICD). Thus, liberation of endogenous NAD(+) by stressed cells is now viewed as a danger signal promoting immune responses by hindering regulatory T cells. This review will highlight the recent discoveries on the in vivo role of the ARTC2.2/P2X7 pathway triggered by the endogenous release of extracellular NAD(+), the relative sensitivity of lymphocytes subsets to this regulatory pathway and its pharmacological manipulation using camelid-derived ARTC2.2-blocking nanobodies.
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http://dx.doi.org/10.1007/82_2014_420DOI Listing
March 2015

Role of Toll-like receptors 2 and 4 in mediating endothelial dysfunction and arterial remodeling in primary arterial antiphospholipid syndrome.

Arthritis Rheumatol 2014 Nov;66(11):3210-20

Rouen University Hospital, INSERM U1096, University of Rouen, and Centre d'Investigation Clinique, INSERM 1404, Rouen, France.

Objective: To assess the role of Toll-like receptors (TLRs) in antiphospholipid antibody (aPL)-mediated vascular abnormalities in patients with primary arterial antiphospholipid syndrome (APS).

Methods: Forty-eight subjects participated in the study. Arterial function and structure and TLR pathway activation were determined in patients with primary arterial APS and matched controls. The pathogenic effects of aPL isolated from patients were assessed in wild-type (WT) and TLR-knockout mice.

Results: APS patients had endothelial dysfunction, arterial stiffening, and hypertrophy, as evidenced by decreased brachial artery endothelium-dependent flow-mediated dilation (FMD) and increased aortic pulse wave velocity and carotid intima-media thickness (IMT), as compared with controls. Plasma samples from APS patients revealed decreased nitric oxide (NO) availability and a pro-oxidative, proinflammatory, and prothrombotic state illustrated by a decrease in nitrite and an increase in lipid peroxidation, tumor necrosis factor α levels, and tissue factor (TF) levels. Furthermore, TLR pathway activation was found in APS patients with increased TLR-2 and TLR-4 messenger RNA expression and increased protein levels of the activated TLR transduction protein interleukin-1 receptor-associated kinase 1 in peripheral blood mononuclear cells. Moreover, agonist-stimulated cell-surface expression of TLR-2 and TLR-4 in circulating monocytes was higher in APS patients than in controls. These changes were positively associated with IMT and negatively associated with FMD. Finally, aPL injection decreased mesenteric endothelium-dependent relaxation and increased TF expression in WT mice but not in TLR-2- or TLR-4-knockout mice.

Conclusion: This translational study supports the notion that TLR-2 and TLR-4 play a role in mediating vascular abnormalities in patients with primary arterial APS. TLRs thus constitute a promising pharmacologic target for preventing cardiovascular complications in APS.
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http://dx.doi.org/10.1002/art.38785DOI Listing
November 2014

Mechanical-stretch of C2C12 myoblasts inhibits expression of Toll-like receptor 3 (TLR3) and of autoantigens associated with inflammatory myopathies.

PLoS One 2013 4;8(11):e79930. Epub 2013 Nov 4.

Department of Anatomy, Southern Medical University, GuangZhou, China.

Recent studies in patients suffering from inflammatory autoimmune myopathies suggested that moderate exercise training improves or at least stabilizes muscle strength and function without inducing disease flares. However, the precise mechanisms involved in this beneficial effect have not been extensively studied. Here we used a model of in vitro stretched C2C12 myoblasts to investigate whether mechanical stretch could influence myoblast proliferation or the expression of proinflammatory genes. Our results demonstrated that cyclic mechanical stretch stimulated C2C12 cell cycling and early up-regulation of the molecules related to mechanical-stretch pathway in muscle (calmodulin, nNOS, MMP-2, HGF and c-Met). Unexpectedly, mechanical stretch also reduced the expression of TLR3 and of proteins known to represent autoantigens in inflammatory autoimmune myopathies (Mi-2, HRS, DNA-PKcs, U1-70). Interestingly, stimulation or inhibition of calmodulin, NOS, HGF or c-Met molecules in vitro affected the expression of autoantigens and TLR3 proteins confirming their role in the inhibition of autoantigens and TLR3 during mechanical stretch. Overall, this study demonstrates for the first time that mechanical stretch could be beneficial by reducing expression of muscle autoantigens and of pro-inflammatory TLR3 and may provide new insight to understand how resistance training can reduce the symptoms associated with myositis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079930PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817111PMC
June 2014