Publications by authors named "Christophe Klein"

45 Publications

Ileal immune tonus is a prognosis marker of proximal colon cancer in mice and patients.

Cell Death Differ 2020 Dec 1. Epub 2020 Dec 1.

Gustave Roussy Cancer Campus (GRCC), Villejuif, France.

Ileal epithelial cell apoptosis and the local microbiota modulate the effects of oxaliplatin against proximal colon cancer by modulating tumor immunosurveillance. Here, we identified an ileal immune profile associated with the prognosis of colon cancer and responses to chemotherapy. The whole immune ileal transcriptome was upregulated in poor-prognosis patients with proximal colon cancer, while the colonic immunity of healthy and neoplastic areas was downregulated (except for the Th17 fingerprint) in such patients. Similar observations were made across experimental models of implanted and spontaneous murine colon cancer, showing a relationship between carcinogenesis and ileal inflammation. Conversely, oxaliplatin-based chemotherapy could restore a favorable, attenuated ileal immune fingerprint in responders. These results suggest that chemotherapy inversely shapes the immune profile of the ileum-tumor axis, influencing clinical outcome.
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http://dx.doi.org/10.1038/s41418-020-00684-wDOI Listing
December 2020

Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer.

Nat Med 2020 06 25;26(6):919-931. Epub 2020 May 25.

Gustave Roussy Cancer Campus (GRCC), Villejuif, France.

The prognosis of colon cancer (CC) is dictated by tumor-infiltrating lymphocytes, including follicular helper T (T) cells and the efficacy of chemotherapy-induced immune responses. It remains unclear whether gut microbes contribute to the elicitation of T cell-driven responses. Here, we show that the ileal microbiota dictates tolerogenic versus immunogenic cell death of ileal intestinal epithelial cells (IECs) and the accumulation of T cells in patients with CC and mice. Suppression of IEC apoptosis led to compromised chemotherapy-induced immunosurveillance against CC in mice. Protective immune responses against CC were associated with residence of Bacteroides fragilis and Erysipelotrichaceae in the ileum. In the presence of these commensals, apoptotic ileal IECs elicited PD-1 T cells in an interleukin-1R1- and interleukin-12-dependent manner. The ileal microbiome governed the efficacy of chemotherapy and PD-1 blockade in CC independently of microsatellite instability. These findings demonstrate that immunogenic ileal apoptosis contributes to the prognosis of chemotherapy-treated CC.
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http://dx.doi.org/10.1038/s41591-020-0882-8DOI Listing
June 2020

Active Fluctuations of the Nuclear Envelope Shape the Transcriptional Dynamics in Oocytes.

Dev Cell 2019 10 10;51(2):145-157.e10. Epub 2019 Oct 10.

CIRB, Collège de France/CNRS-UMR7241/INSERM-U1050, PSL Research University, Equipe Labellisée FRM, Paris 75005, France. Electronic address:

Nucleus position in cells can act as a developmental cue. Mammalian oocytes position their nucleus centrally using an F-actin-mediated pressure gradient. The biological significance of nucleus centering in mammalian oocytes being unknown, we sought to assess the F-actin pressure gradient effect on the nucleus. We addressed this using a dedicated computational 3D imaging approach, biophysical analyses, and a nucleus repositioning assay in mouse oocytes mutant for cytoplasmic F-actin. We found that the cytoplasmic activity, in charge of nucleus centering, shaped the nucleus while promoting nuclear envelope fluctuations and chromatin motion. Off-centered nuclei in F-actin mutant oocytes were misshaped with immobile chromatin and modulated gene expression. Restoration of F-actin in mutant oocytes rescued nucleus architecture fully and gene expression partially. Thus, the F-actin-mediated pressure gradient also modulates nucleus dynamics in oocytes. Moreover, this study supports a mechano-transduction model whereby cytoplasmic microfilaments could modulate oocyte transcriptome, essential for subsequent embryo development.
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http://dx.doi.org/10.1016/j.devcel.2019.09.010DOI Listing
October 2019

CFH exerts anti-oxidant effects on retinal pigment epithelial cells independently from protecting against membrane attack complex.

Sci Rep 2019 09 25;9(1):13873. Epub 2019 Sep 25.

Centre de Recherche des Cordeliers, Université Pierre et Marie Curie - Paris6, UMRS 872, Paris, F-75006, France.

Age Related Macular Degeneration (AMD) is the first cause of social blindness in people aged over 65 leading to atrophy of retinal pigment epithelial cells (RPE), photoreceptors and choroids, eventually associated with choroidal neovascularization. Accumulation of undigested cellular debris within RPE cells or under the RPE (Drusen), oxidative stress and inflammatory mediators contribute to the RPE cell death. The major risk to develop AMD is the Y402H polymorphism of complement factor H (CFH). CFH interacting with oxidized phospholipids on the RPE membrane modulates the functions of these cells, but the exact role of CFH in RPE cell death and survival remain poorly understood. The aim of this study was to analyze the potential protective mechanism of CFH on RPE cells submitted to oxidative stress. Upon exposure to oxidized lipids 4-HNE (4-hydroxy-2-nonenal) derived from photoreceptors, both the human RPE cell line ARPE-19 and RPE cells derived from human induced pluripotent stem cells were protected from death only in the presence of the full length human recombinant CFH in the culture medium. This protective effect was independent from the membrane attack complex (MAC) formation. CFH maintained RPE cells tight junctions' structure and regulated the caspase dependent apoptosis process. These results demonstrated the CFH anti-oxidative stress functions independently of its capacity to inhibit MAC formation.
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http://dx.doi.org/10.1038/s41598-019-50420-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761137PMC
September 2019

Sostdc1 is expressed in all major compartments of developing and adult mammalian eyes.

Graefes Arch Clin Exp Ophthalmol 2019 Nov 16;257(11):2401-2427. Epub 2019 Sep 16.

Centre de Recherches des Cordeliers, UMR_S INSERM 1138, Equipe 17, Université Paris Descartes, 15 rue de l'école de médecine, 75006, Paris, France.

Purpose: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes.

Methods: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues.

Results: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels.  CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/β-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.
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http://dx.doi.org/10.1007/s00417-019-04462-4DOI Listing
November 2019

Sustained Type I interferon signaling as a mechanism of resistance to PD-1 blockade.

Cell Res 2019 Oct 3;29(10):846-861. Epub 2019 Sep 3.

INSERM U1015, Gustave Roussy, 114 rue Edouard Vaillant, 94805, Villejuif Cedex, France.

PD-1 blockade represents a major therapeutic avenue in anticancer immunotherapy. Delineating mechanisms of secondary resistance to this strategy is increasingly important. Here, we identified the deleterious role of signaling via the type I interferon (IFN) receptor in tumor and antigen presenting cells, that induced the expression of nitric oxide synthase 2 (NOS2), associated with intratumor accumulation of regulatory T cells (Treg) and myeloid cells and acquired resistance to anti-PD-1 monoclonal antibody (mAb). Sustained IFNβ transcription was observed in resistant tumors, in turn inducing PD-L1 and NOS2 expression in both tumor and dendritic cells (DC). Whereas PD-L1 was not involved in secondary resistance to anti-PD-1 mAb, pharmacological or genetic inhibition of NOS2 maintained long-term control of tumors by PD-1 blockade, through reduction of Treg and DC activation. Resistance to immunotherapies, including anti-PD-1 mAb in melanoma patients, was also correlated with the induction of a type I IFN signature. Hence, the role of type I IFN in response to PD-1 blockade should be revisited as sustained type I IFN signaling may contribute to resistance to therapy.
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http://dx.doi.org/10.1038/s41422-019-0224-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796942PMC
October 2019

Metabolic enzymes expressed by cancer cells impact the immune infiltrate.

Oncoimmunology 2019;8(6):e1571389. Epub 2019 Mar 30.

Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France.

The expression of two metabolic enzymes, ., aldehyde dehydrogenase 7 family, member A1 (ALDH7A1) and lipase C, hepatic type (LIPC) by malignant cells, has been measured by immunohistochemical methods in non-small cell lung carcinoma (NSCLC) biopsies, and has been attributed negative and positive prognostic value, respectively. Here, we demonstrate that the protein levels of ALDH7A1 and LIPC correlate with the levels of the corresponding mRNAs. Bioinformatic analyses of gene expression data from 4921 cancer patients revealed that the expression of LIPC positively correlates with abundant tumor infiltration by myeloid and lymphoid cells in NSCLC, breast carcinoma, colorectal cancer and melanoma samples. In contrast, high levels of ALDH7A1 were associated with a paucity of immune effectors within the tumor bed. These data reinforce the notion that the metabolism of cancer cells has a major impact on immune and inflammatory processes in the tumor microenvironment, pointing to hitherto unsuspected intersections between oncometabolism and immunometabolism.
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http://dx.doi.org/10.1080/2162402X.2019.1571389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492976PMC
March 2019

Polyploidy spectrum: a new marker in HCC classification.

Gut 2020 02 12;69(2):355-364. Epub 2019 Apr 12.

Team Proliferation Stress and Liver Physiopathology, Genome and Cancer, Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, Paris, France.

Objectives: Polyploidy is a fascinating characteristic of liver parenchyma. Hepatocyte polyploidy depends on the DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. Here, we investigated whether a specific ploidy spectrum is associated with clinical and molecular features of HCC.

Design: Ploidy spectra were determined on surgically resected tissues from patients with HCC as well as healthy control tissues. To define ploidy profiles, a quantitative and qualitative in situ imaging approach was used on paraffin tissue liver sections.

Results: We first demonstrated that polyploid hepatocytes are the major components of human liver parenchyma, polyploidy being mainly cellular (binuclear hepatocytes). Across liver lobules, polyploid hepatocytes do not exhibit a specific zonation pattern. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumours. Remarkably, nuclear ploidy is specifically amplified in HCC tumours. In fact, nuclear ploidy is amplified in HCCs harbouring a low degree of differentiation and mutations. Finally, our results demonstrated that highly polyploid tumours are associated with a poor prognosis.

Conclusions: Our results underline the importance of quantification of cellular and nuclear ploidy spectra during HCC tumorigenesis.
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http://dx.doi.org/10.1136/gutjnl-2018-318021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6984053PMC
February 2020

Development of Tools for the Selective Visualization and Quantification of TLS-Immune Cells on Tissue Sections.

Methods Mol Biol 2018 ;1845:47-69

Sorbonne University, UMRS 1138, Cordeliers Research Center, Paris, France.

Tertiary lymphoid structures (TLS) are considered as genuine markers of inflammation. Their presence within inflamed tissues or within the tumor microenvironment has been associated with the local development of an active immune response. While high densities of TLS are correlated with disease severity in autoimmune diseases or during graft rejection, it has been associated with longer patient survival in many cancer types. Their efficient visualization and quantification within human tissues may represent new tools for helping clinicians in adjusting their therapeutic strategy. Some immunohistochemistry (IHC) protocols are already used in the clinic to appreciate the level of immune infiltration in formalin-fixed, paraffin-embedded (FFPE) tissues. However, the use of two or more markers may sometimes be useful to better characterize this immune infiltrate, especially in the case of TLS. Besides the growing development of multiplex labeling approaches, imaging can also be used to overcome some technical difficulties encountered during the immunolabeling of tissues with several markers.This chapter describes IHC methods to visualize in a human tissue (tumoral or not) the presence of TLS. These methods are based on the immunostaining of four TLS-associated immune cell populations, namely follicular B cells, follicular dendritic cells (FDCs), mature dendritic cells (mDCs), and follicular helper T cells (T), together with non-T T cells. Methodologies for subsequent quantification of TLS density are also proposed, as well as a virtual multiplexing method based on image registration using the open-source software ImageJ (IJ), aiming at co-localizing several immune cell populations from different IHC stainings performed on serial tissue sections.
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http://dx.doi.org/10.1007/978-1-4939-8709-2_4DOI Listing
May 2019

Pregnancy, child bearing and prevention of giving birth to the affected children in patients with primary immunodeficiency disease; a case-series.

BMC Pregnancy Childbirth 2018 Jul 11;18(1):299. Epub 2018 Jul 11.

Department of Pediatric, Children's Hospital, University Hospital, LMU, Munich, Germany.

Background: Patients with primary immunodeficiency disease (PID) who survive to adulthood and willing to have a child mostly are worried whether their disease affects their fertility and/or pregnancy and also if their child would be predisposed to PID.

Case Presentation: We report the outcome of conception, pregnancy and their management in 9 families with definite diagnosis of PID. A chronic granulomatous disease subject with an uneventful pregnancy developed fungal sacral osteomyelitis few weeks after delivery. A pregnant common variable immunodeficiency disease (CVID) patient with idiopathic thrombocytopenia had platelet count dropped before delivery. A sever neutropenic mother who refused to get IFNγ delivered two healthy children. A CVID case intolerant to IVIg with eclampsia and PTE delivered a baby. Another CVID female gave birth to a baby without being on any treatment since she was not diagnosed with immunodeficiency disease at that time. A healthy girl was implanted via preimplantation gender selection in a family who owned a Wiskott Aldrich-affected son. A family who had two children with Ataxia Telangiectasia used donated oocyte for their 3rd child. Prenatal genetic diagnosis was used to screen the fetus for the impaired BTK and CVID genes detected in sibling and father respectively in 2 separate families.

Conclusion: Pregnancy in PID patients is more complex than normal population. Because, not only it has the chance of being inherited by the offspring, but also there are some risks for the mother if she has any kind of immunity component defects. So consultation with a clinical geneticist is crucial to choose the best available approach. They also should be observed and followed by a clinical immunologist to take the best possible safe care.
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http://dx.doi.org/10.1186/s12884-018-1927-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042236PMC
July 2018

Involvement of neural crest and paraxial mesoderm in oral mucosal development and healing.

Biomaterials 2018 07 21;172:41-53. Epub 2018 Apr 21.

Cordeliers Research Center, Laboratory of Molecular Oral Physiopathology, INSERM UMRS 1138, 15 rue de l'école de médecine, 75006 Paris, France; Paris-Descartes and Paris-Diderot Universities, UFR Odontology, 75006 Paris, France; Reference Center for Dental Rare Disease, Rothschild Hospital, 75012 Paris, France. Electronic address:

Tissue engineering therapies using adult stem cells derived from neural crest have sought accessible tissue sources of these cells because of their potential pluripotency. In this study, the gingiva and oral mucosa and their associated stem cells were investigated. Biopsies of these tissues produce neither scarring nor functional problems and are relatively painless, and fresh tissue can be obtained readily during different chairside dental procedures. However, the embryonic origin of these cells needs to be clarified, as does their evolution from the perinatal period to adulthood. In this study, the embryonic origin of gingival fibroblasts were determined, including gingival stem cells. To do this, transgenic mouse models were used to track neural crest derivatives as well as cells derived from paraxial mesoderm, spanning from embryogenesis to adulthood. These cells were compared with ones derived from abdominal dermis and facial dermis. Our results showed that gingival fibroblasts are derived from neural crest, and that paraxial mesoderm is involved in the vasculogenesis of oral tissues during development. Our in vitro studies revealed that the neuroectodermal origin of gingival fibroblasts (or gingival stem cells) endows them with multipotential properties as well as a specific migratory and contractile phenotype which may participate to the scar-free properties of the oral mucosa. Together, these results illustrate the high regenerative potential of neural crest-derived stem cells of the oral mucosa, including the gingiva, and strongly support their use in cell therapy to regenerate tissues with impaired healing.
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http://dx.doi.org/10.1016/j.biomaterials.2018.04.036DOI Listing
July 2018

Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.

Science 2018 Jan 2;359(6371):91-97. Epub 2017 Nov 2.

Gustave Roussy Cancer Campus (GRCC), Villejuif, France.

Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis induce sustained clinical responses in a sizable minority of cancer patients. We found that primary resistance to ICIs can be attributed to abnormal gut microbiome composition. Antibiotics inhibited the clinical benefit of ICIs in patients with advanced cancer. Fecal microbiota transplantation (FMT) from cancer patients who responded to ICIs into germ-free or antibiotic-treated mice ameliorated the antitumor effects of PD-1 blockade, whereas FMT from nonresponding patients failed to do so. Metagenomics of patient stool samples at diagnosis revealed correlations between clinical responses to ICIs and the relative abundance of Oral supplementation with after FMT with nonresponder feces restored the efficacy of PD-1 blockade in an interleukin-12-dependent manner by increasing the recruitment of CCR9CXCR3CD4 T lymphocytes into mouse tumor beds.
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http://dx.doi.org/10.1126/science.aan3706DOI Listing
January 2018

ROCK-1 mediates diabetes-induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy.

Sci Rep 2017 08 18;7(1):8834. Epub 2017 Aug 18.

Inserm UMR_S 1138, Team 17: From physiopathology of retinal diseases to clinical advances, Centre de Recherche des Cordeliers, Paris, France.

In diabetic retinopathy, the exact mechanisms leading to retinal capillary closure and to retinal barriers breakdown remain imperfectly understood. Rho-associated kinase (ROCK), an effector of the small GTPase Rho, involved in cytoskeleton dynamic regulation and cell polarity is activated by hyperglycemia. In one year-old Goto Kakizaki (GK) type 2 diabetic rats retina, ROCK-1 activation was assessed by its cellular distribution and by phosphorylation of its substrates, MYPT1 and MLC. In both GK rat and in human type 2 diabetic retinas, ROCK-1 is activated and associated with non-apoptotic membrane blebbing in retinal vessels and in retinal pigment epithelium (RPE) that respectively form the inner and the outer barriers. Activation of ROCK-1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. In RPE cells, actin cytoskeleton remodeling and membrane blebs in RPE cells contributes to outer barrier breakdown. Intraocular injection of fasudil, significantly reduces both retinal hypoxia and RPE barrier breakdown. Diabetes-induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.
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http://dx.doi.org/10.1038/s41598-017-07329-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562711PMC
August 2017

Expression of Phenotypic Astrocyte Marker Is Increased in a Transgenic Mouse Model of Alzheimer's Disease versus Age-Matched Controls: A Presymptomatic Stage Study.

Int J Alzheimers Dis 2016 8;2016:5696241. Epub 2016 Sep 8.

Centre de Recherche des Cordeliers, INSERM, Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Université Pierre et Marie Curie Université Paris 06, Sorbonne Universités, Paris, France.

Recent mouse studies of the presymptomatic stage of Alzheimer's disease (AD) have suggested that proinflammatory changes, such as glial activation and cytokine induction, may occur already at this early stage through unknown mechanisms. Because TNFα contributes to increased Aβ production from the Aβ precursor protein (APP), we assessed a putative correlation between APP/Aβ and TNFα during the presymptomatic stage as well as early astrocyte activation in the hippocampus of 3-month-old APPswe/PS1dE9 mice. While Western blots revealed significant APP expression, Aβ was not detectable by Western blot or ELISA attesting that 3-month-old, APPswe/PS1dE9 mice are at a presymptomatic stage of AD-like pathology. Western blots were also used to show increased GFAP expression in transgenic mice that positively correlated with both TNFα and APP, which were also mutually correlated. Subregional immunohistochemical quantification of phenotypic (GFAP) and functional (TSPO) markers of astrocyte activation indicated a selective and significant increase in GFAP-immunoreactive (IR) cells in the dentate gyrus of APPswe/PS1dE9 mice. Our data suggest that subtle morphological and phenotypic alterations, compatible with the engagement of astrocyte along the activation pathway, occur in the hippocampus already at the presymptomatic stage of AD.
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http://dx.doi.org/10.1155/2016/5696241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031839PMC
September 2016

Contribution of RIP3 and MLKL to immunogenic cell death signaling in cancer chemotherapy.

Oncoimmunology 2016 Jun 10;5(6):e1149673. Epub 2016 Mar 10.

Equipe 11 labellisée Ligue contre le Cancer, Center de Recherche des Cordeliers, Institut National de la Santé Et de la Recherche Medicale (INSERM) U 1138, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; Université Pierre et Marie Curie, Paris, France; Institut de Cancérologie Gustave Roussy Cancer Campus (GRCC), Villejuif, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Comprehensive Cancer Institute, Villejuif, France; Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France; Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden.

Chemotherapy can reinstate anticancer immunosurveillance through inducing tumor immunogenic cell death (ICD). Here, we show that anthracyclines and oxaliplatin can trigger necroptosis in murine cancer cell lines expressing receptor-interacting serine-threonine kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL). Necroptotic cells featured biochemical hallmarks of ICD and stimulated anticancer immune responses in vivo. Chemotherapy normally killed Rip3 (-/-) and Mlkl (-/-) tumor cells and normally induced caspase-3 activation in such cells, yet was unable to reduce their growth in vivo. RIP3 or MLKL deficiency abolished the capacity of dying cancer cells to elicit an immune response. This could be attributed to reduced release of ATP and high mobility group box 1 (HMGB1) by RIP3 and MLKL-deficient cells. Measures designed to compensate for deficient ATP and HMGB1 signaling restored the chemotherapeutic response of Rip3 (-/-) and Mlkl (-/-) cancers. Altogether, these results suggest that RIP3 and MLKL can contribute to ICD signaling and tumor immunogenicity.
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http://dx.doi.org/10.1080/2162402X.2016.1149673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938314PMC
June 2016

Targeting iron-mediated retinal degeneration by local delivery of transferrin.

Free Radic Biol Med 2015 Dec 8;89:1105-21. Epub 2015 Oct 8.

INSERM, UMRS 1138, team Behar-Cohen, From physiopathology of ocular diseases to clinical development, Centre de Recherche des Cordeliers, Paris, France; Université Pierre et Marie Curie-Paris 6, Centre de Recherche des Cordeliers UMRS 1138, Paris, France; Université René Descartes, Centre de Recherche des Cordeliers UMRS 1138, Paris, France; Jules Gonin Ophthalmic Hospital, Lausanne, Switzerland.

Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.08.018DOI Listing
December 2015

The cellular prion protein PrPc is a partner of the Wnt pathway in intestinal epithelial cells.

Mol Biol Cell 2015 Sep 29;26(18):3313-28. Epub 2015 Jul 29.

Sorbonne Universités, Université Pierre et Marie Curie, Université Paris 06, UMRS 1138, Centre de Recherche des Cordeliers, F-75006 Paris, France Institut National de la Santé et de la Recherche Médicale, UMRS 1138, Centre de Recherche des Cordeliers, F-75006 Paris, France Université Paris Descartes, Sorbonne Paris Cité, UMRS 1138, Centre de Recherche des Cordeliers, F-75006 Paris, France Ecole Pratique des Hautes Etudes, PSL Research University, Laboratoire de Pharmacologie Cellulaire et Moléculaire, F-75006 Paris, France

We reported previously that the cellular prion protein (PrP(c)) is a component of desmosomes and contributes to the intestinal barrier function. We demonstrated also the presence of PrP(c) in the nucleus of proliferating intestinal epithelial cells. Here we sought to decipher the function of this nuclear pool. In human intestinal cancer cells Caco-2/TC7 and SW480 and normal crypt-like HIEC-6 cells, PrP(c) interacts, in cytoplasm and nucleus, with γ-catenin, one of its desmosomal partners, and with β-catenin and TCF7L2, effectors of the canonical Wnt pathway. PrP(c) up-regulates the transcriptional activity of the β-catenin/TCF7L2 complex, whereas γ-catenin down-regulates it. Silencing of PrP(c) results in the modulation of several Wnt target gene expressions in human cells, with different effects depending on their Wnt signaling status, and in mouse intestinal crypt cells in vivo. PrP(c) also interacts with the Hippo pathway effector YAP, suggesting that it may contribute to the regulation of gene transcription beyond the β-catenin/TCF7L2 complex. Finally, we demonstrate that PrP(c) is required for proper formation of intestinal organoids, indicating that it contributes to proliferation and survival of intestinal progenitors. In conclusion, PrP(c) must be considered as a new modulator of the Wnt signaling pathway in proliferating intestinal epithelial cells.
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http://dx.doi.org/10.1091/mbc.E14-11-1534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569320PMC
September 2015

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

Mol Vis 2014 23;20:908-20. Epub 2014 Jun 23.

Inserm, U1138, Team 17, Physiopathology of ocular diseases : Threrapeutic innovations, Université René Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, Paris, France ; Department of Ophtahlmology of Lausanne University, Jules Gonin Ophthalmic hospital, Lausanne, Switzerland.

Purpose: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation.

Methods: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment.

Results: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina.

Conclusions: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067232PMC
September 2014

Evaluating in vivo delivery of riboflavin with coulomb-controlled iontophoresis for corneal collagen cross-linking: a pilot study.

Invest Ophthalmol Vis Sci 2014 Apr 28;55(4):2731-8. Epub 2014 Apr 28.

Ophthalmic Biophysics Center, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States.

Purpose: To evaluate the efficacy of coulomb-controlled iontophoresis (CCI) for delivery of riboflavin prior to corneal collagen cross-linking (CXL).

Methods: The eyes of 20 8-week-old Lewis rats, subject to epithelium-ON (epi-ON, n = 20 eyes) or epithelium-OFF (epi-OFF, n = 20 eyes) conditions, were used to evaluate the in vivo delivery of two riboflavin solutions: 0.1% riboflavin-20% dextran T500 solution (riboflavin-dextran) and 0.1% riboflavin 5'-phosphate (riboflavin-phosphate). After systemic intramuscular anesthesia, 0.25 mL of the photosensitizing agent was delivered by either instillation or CCI (2.11 mA/cm(2) for 4 or 10 minutes) into either epithelial condition. The CCI probe on the eye without current served as control. Confocal microscopy of flat-mounted corneas was used to analyze intracorneal penetration and fluorometry was used to quantify riboflavin in the aqueous within 30 minutes of treatment.

Results: Instillation and CCI allowed for uniform delivery of riboflavin-dextran throughout the stroma after epithelial debridement. Transepithelial delivery of riboflavin-dextran was not efficacious. Riboflavin-phosphate was successfully delivered in both epithelium conditions. Complete saturation of the cornea was achieved using CCI after removing the epithelium, the epi-ON case allowed for limited diffusion. Increasing the time from 4 to 10 minutes greatly increased the amount of riboflavin detected in the cornea and aqueous humor.

Conclusions: Coulomb-controlled iontophoresis is an effective technique for transepithelial delivery of riboflavin-phosphate into the cornea. This drug delivery method would allow clinicians to significantly shorten the time required for the CXL procedure, with or without epithelial debridement. Whether efficient crosslinking can be achieved through an intact epithelium remains to be demonstrated.
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http://dx.doi.org/10.1167/iovs.14-13931DOI Listing
April 2014

Dendritic cells in tumor-associated tertiary lymphoid structures signal a Th1 cytotoxic immune contexture and license the positive prognostic value of infiltrating CD8+ T cells.

Cancer Res 2014 Feb 23;74(3):705-15. Epub 2013 Dec 23.

Authors' Affiliations: Laboratory Immune Microenvironment and Tumors, INSERM U872, Cordeliers Research Center; University Pierre et Marie Curie; University Paris Descartes, UMRS 872; Departments of Pathology and Thoracic Surgery, Hôtel Dieu Hospital, AP-HP; Department of Pathology, Institut Mutualiste Montsouris; Department of Immunology, European Georges Pompidou Hospital, AP-HP, Paris, France; and Oncology Research, MedImmune LLC, Gaithersburg, Maryland.

Tumor-infiltrating T cells, particularly CD45RO(+)CD8(+) memory T cells, confer a positive prognostic value in human cancers. However, the mechanisms that promote a protective T-cell response in the tumor microenvironment remain unclear. In chronic inflammatory settings such as the tumor microenvironment, lymphoid neogenesis can occur to create local lymph node-like structures known as tertiary lymphoid structures (TLS). These structures can exacerbate a local immune response, such that TLS formation in tumors may help promote an efficacious immune contexture. However, the role of TLS in tumors has yet to be investigated carefully. In lung tumors, mature dendritic cells (DC) present in tumor-associated TLS can provide a specific marker of these structures. In this study, we evaluated the influence of TLS on the characteristics of the immune infiltrate in cohorts of prospective and retrospective human primary lung tumors (n = 458). We found that a high density of mature DC correlated closely to a strong infiltration of T cells that are predominantly of the effector-memory phenotype. Moreover, mature DC density correlated with expression of genes related to T-cell activation, T-helper 1 (Th1) phenotype, and cytotoxic orientation. Lastly, a high density of TLS-associated DC correlated with long-term survival, which also allowed a distinction of patients with high CD8(+) T-cell infiltration but a high risk of death. Taken together, our results show how tumors infiltrated by TLS-associated mature DC generate a specific immune contexture characterized by a strong Th1 and cytotoxic orientation that confers the lowest risk of death. Furthermore, our findings highlight the pivotal function of TLS in shaping the immune character of the tumor microenvironment, in promoting a protective immune response mediated by T cells against cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1342DOI Listing
February 2014

Whole blood clots are more resistant to lysis than plasma clots--greater efficacy of rivaroxaban.

Thromb Res 2013 Mar 11;131(3):e100-9. Epub 2013 Jan 11.

UMRS 872 INSERM, Université Pierre et Marie Curie Paris VI and Université René Descartes, Paris, France.

Introduction: Defective thrombolysis, a thrombotic risk factor, can be attributed to the formation of a compact clot poorly accessible to fibrinolytic enzymes. Venous thrombi, rich in red blood cells (RBCs), and arterial thrombi containing various amounts of RBCS, plasma and whole blood (WB) clot permeability and degradability were compared. The effect of rivaroxaban, a potent direct factor Xa inhibitor, was also evaluated.

Materials And Methods: Fibrin permeability was determined by flow measurement through the clot. Clot degradability was evaluated by the amount of D-dimer generated by clot perfusion with plasminogen and tissue plasminogen activator. Fibrin clot structure was assessed by confocal microscopy.

Results: WB clot permeability (KS) and degradability were 6.7- and 38-fold lower, respectively, compared with plasma clots. This is attributed to 1) occlusion of fibrin pores by RBCs and 2) a consistent increase in thrombin generation due to platelets and RBCs inducing formation of a tighter clot. Rivaroxaban added to plasma or WB before clotting, in reducing thrombin generation, led to the formation of a looser clot that is more degradable by fibrinolytic enzymes. Permeability and degradability of whole blood clots formed in the presence of rivaroxaban were very similar to those of plasma clots.

Conclusion: The resistance to fibrinolysis of WB clots was reduced considerably when clots were formed with rivaroxaban. These results may have implications for the development of antithrombotic agents.
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http://dx.doi.org/10.1016/j.thromres.2012.11.029DOI Listing
March 2013

DsRed-mediated oligomerization stabilizes HMGB1 on chromatin in vivo and on DNA in vitro.

Biochimie 2013 Apr 14;95(4):962-6. Epub 2012 Nov 14.

Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Université Paris Descartes, INSERM, UMRS 872, Paris F-75006, France.

High-mobility group box-1 (HMGB1) is remarkably mobile in living cells, which reflects its ability to interact only transiently with both DNA and protein. This property is likely essential for HMGB1 nuclear activities. Nonetheless the weak interaction of HMGB1 with DNA and/or protein partners has also been a major limitation for investigating HMGB1 subnuclear localisation and for the identification of HMGB1 containing complexes by conventional biochemical approaches. In the present study, FRAP experiments demonstrated that DsRed-mediated oligomerization strongly reduces HMGB1 mobility due to an increased affinity for cellular chromatin. Moreover, oligomerized DsRed-HMGB1 exhibited a higher affinity for supercoiled DNA in vitro compared to its monomeric counterpart. These results indicate that DsRed-meditated oligomerization is prone to stabilize labile interactions involving HMGB1 both in vivo and in vitro.
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http://dx.doi.org/10.1016/j.biochi.2012.11.001DOI Listing
April 2013

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.

Angiogenesis 2012 Dec 7;15(4):609-22. Epub 2012 Aug 7.

Centre de Recherche des Cordeliers, INSERM, UMR S 872, 75006, Paris, France.

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.
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http://dx.doi.org/10.1007/s10456-012-9290-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496480PMC
December 2012

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta-induced retinal degeneration.

Aging Cell 2012 Aug 11;11(4):683-93. Epub 2012 Jun 11.

Centre de Recherche des Cordeliers, Université Paris Descartes, UMR S 872, F-75006 Paris, France.

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.
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http://dx.doi.org/10.1111/j.1474-9726.2012.00834.xDOI Listing
August 2012

Live-cell imaging reveals multiple interactions between Epstein-Barr virus nuclear antigen 1 and cellular chromatin during interphase and mitosis.

J Virol 2012 May 15;86(9):5314-29. Epub 2012 Feb 15.

UPMC Université Paris 6, UMRS 872, Paris, France.

Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.
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http://dx.doi.org/10.1128/JVI.06303-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347338PMC
May 2012

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics.

Biol Cell 2011 Nov;103(11):499-517

Université Pierre et Marie Curie-Paris 6, UMR S 872, Les Cordeliers, Paris 75006, France.

Background Information: Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.

Results: Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.

Conclusions: We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
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http://dx.doi.org/10.1042/BC20110024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181828PMC
November 2011

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy.

PLoS One 2011 Mar 7;6(3):e17462. Epub 2011 Mar 7.

Institut National pour la Santé Et la Recherche Médicale U872, Physiopathology of ocular diseases: Therapeutic innovations, Paris, France.

Objective: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1).

Materials And Methods: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis.

Results: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation.

Conclusion: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049767PMC
March 2011

Oncologic trogocytosis with Hospicells induces the expression of N-cadherin by breast cancer cells.

Int J Oncol 2010 Dec;37(6):1453-61

INSERM U563, Centre de Physiopathologie de Toulouse-Purpan, Toulouse F-31300, France.

In breast cancers, the appearance of metastasis is synonymous with poor prognosis. The metastatic process is usually associated with epithelial-mesenchymal transition (EMT) which is often induced by several soluble factors produced either by the tumour cells themselves or by cells constituting the tumour microenvironment. The aim of the present study was to determine whether the mesenchymal properties given by some molecules such as N-cadherin, for instance, could be acquired by cancer cells via the trogocytosis process with cells of the tumour microenvironment. Hospicells are stromal cells which were first isolated from cancer cell aggregates of patients with ovarian cancer. We recently showed that these cells are immunosuppressive for T lymphocyte functions and confer chemoresistance to cancer cells by the transfer of the MDR protein via trogocytosis. In this study, we showed that a mammary cancer cell line (MDA-MB-231) acquires patches of membrane via oncologic trogocytosis with Hospicells. This unidirectional and active process depends on actin polymerization and can be increased via inhibition of the Src family and decreased via inhibition of PI3K. Trogocytosis between Hospicells and MDA-MB-231 does not lead to the direct acquisition of N-cadherin but rather it leads to the production of soluble factor(s) which induce de novo expression of N-cadherin by the cancer cells. The novelty here is that this factor is produced only if cancer cells interact and undergo trogocytosis with Hospicells. This new expression could confer a more invasive phenotype to the cancer cells and thus can explain the correlation of the presence of Hospicells with the number of invaded lymph nodes in patients with mammary adenocarcinoma.
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http://dx.doi.org/10.3892/ijo_00000797DOI Listing
December 2010

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects.

J Lipid Res 2010 May 10;51(5):945-56. Epub 2009 Nov 10.

Centre de Recherche des Cordeliers, INSERM, U872, Université Pierre et Marie Curie - Paris 6, France.

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability.
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http://dx.doi.org/10.1194/jlr.M001016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853462PMC
May 2010

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

J Ocul Pharmacol Ther 2009 Feb;25(1):9-21

INSERM, U872 Physiopathology of Ocular Disease, Therapeutic Innovations, Paris, France.

Purpose: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU).

Methods: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence.

Results: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma.

Conclusions: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.
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http://dx.doi.org/10.1089/jop.2008.0074DOI Listing
February 2009