Correction: Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

[This corrects the article DOI: 10.1371/journal.pone.0157822.]

Development of a specific tribometer for implementation in an environmental SEM

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Light sheet fluorescent microscopy versus confocal microscopy: in quest of a suitable tool to assess drug and nanomedicine penetration into multicellular tumor spheroids

Daniele Vinciguerra and Anna Balasso contributed equally to this work.International audienceWe recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the three-dimensional (3D) architecture of solid tumors. In this study, we used these hetero-type spheroids as a tool to assess the penetration of doxorubicin (used as a model drug) through the whole tumor mass either in a free form or loaded into polymer nanoparticles (NPs), and we investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM), would be best to provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to accurately monitor the diffusion of small molecules such as the doxorubicin. Indeed, it only allowed to scan a layer of 100 µm depth and no information on deeper layers could be available because of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type multicellular tumor spheroids (MCTS) was obtained by LSFM and multi-view image fusion which revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm in diameter. However, no doxorubicin-loaded polymer nanoparticles were detected in the spheroids, highlighting the challenge of nanomedicine delivery through biological barriers. Overall, the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in tumors. Accordingly, it could provide useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved

Mouse Basophils Reside in Extracellular Matrix-Enriched Bone Marrow Niches Which Control Their Motility

Made available in DSpace on 2015-06-08T14:01:47Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) wilson_sovino2etal_IOC_2014.pdf: 3263108 bytes, checksum: 9132ef912d83ec3454d9e1b5b492795b (MD5) Previous issue date: 2013Universidade Federal de Alagoas. Instituto de Ciências Biológicas e da Saúde. Laboratório de Biologia Celular.. Maceió, AL, Brasil.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. Cell Imaging Platform. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Basophils co-express FceRIa and CD49b, the a-2 chain of integrin-type receptor VLA-2 (a2b1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrowderived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (a-4, a-5 and a-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow

Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency

International audienceMutations in the a disintegrin and metalloproteinase with thrombospondin motif-like 2 ( ADAMTSL2) gene are responsible for the autosomal recessive form of geleophysic dysplasia, which is characterized by short stature, short extremities, and skeletal abnormalities. However, the exact function of ADAMTSL2 is unknown. To elucidate the role of this protein in skeletal development, we generated complementary knockout (KO) mouse models with either total or chondrocyte Adamtsl2 deficiency. We observed that the Adamtsl2 KO mice displayed skeletal abnormalities reminiscent of the human phenotype. Adamtsl2 deletion affected the growth plate formation with abnormal differentiation and proliferation of chondrocytes. In addition, a TGF-β signaling impairment in limbs lacking Adamtsl2 was demonstrated. Further investigations revealed that Adamtsl2 KO chondrocytes failed to establish a microfibrillar network composed by fibrillin1 and latent TGF-β binding protein 1 fibrils. Chondrocyte Adamtsl2 KO mice also exhibited dwarfism. These studies uncover the function of Adamtsl2 in the maintenance of the growth plate ECM by modulating the microfibrillar network.-Delhon, L., Mahaut, C., Goudin, N., Gaudas, E., Piquand, K., Le Goff, W., Cormier-Daire, V., Le Goff, C. Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

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AON-Mediated Exon Skipping to Bypass Protein Truncation in Retinal Dystrophies Due to the Recurrent <i>CEP290</i> c.4723A &gt; T Mutation. Fact or Fiction?

Mutations in CEP290 encoding a centrosomal protein important to cilia formation cause a spectrum of diseases, from isolated retinal dystrophies to multivisceral and sometimes embryo&#8211;lethal ciliopathies. In recent years, endogenous and/or selective non-canonical exon skipping of mutant exons have been documented in attenuated retinal disease cases. This observation led us to consider targeted exon skipping to bypass protein truncation resulting from a recurrent mutation in exon 36 (c.4723A &gt; T, p.Lys1575*) causing isolated retinal ciliopathy. Here, we report two unrelated individuals (P1 and P2), carrying the mutation in homozygosity but affected with early-onset severe retinal dystrophy and congenital blindness, respectively. Studying skin-derived fibroblasts, we observed basal skipping and nonsense associated&#8211;altered splicing of exon 36, producing low (P1) and very low (P2) levels of CEP290 products. Consistent with a more severe disease, fibroblasts from P2 exhibited reduced ciliation compared to P1 cells displaying normally abundant cilia; both lines presented however significantly elongated cilia, suggesting altered axonemal trafficking. Antisense oligonucleotides (AONs)-mediated skipping of exon 36 increased the abundance of the premature termination codon (PTC)-free mRNA and protein, reduced axonemal length and improved cilia formation in P2 but not in P1 expressing higher levels of skipped mRNA, questioning AON-mediated exon skipping to treat patients carrying the recurrent c.4723A &gt; T mutation

DCs from PC61-depleted mice and CD4 T cells reduce the percentages of CD8 T cells expressing high level of PD-1.

<p>(A) Histograms show cell surface expression of PD-1 on infiltrating CD8 T cells from treated and untreated tumours resected at day 14 (Left) and mean Fluorescence Intensity (MFI) of PD-1 in each group (Right). (B) CD11c^hiMHC II^hi tumour DCs were sorted from treated and untreated mice and cultured 3 days with naïve CD8 T cells labelled with CellTrace violet and 0.1μg/ml of anti-CD3 mAb. (C,D) Flow cytometry analysing the expression of PD-1 on dividing cells. CD11c^hiMHC II^hi DCs from Tumours (C) and CD11c^hi DCs from DLNs (D) were purified from untreated and PC61-treated mice and co-cultured 3 days with CellTrace violet-labelled CD8 T cells and in the presence or not with anti-CD3 mAb or CD4 T cells. Percentages of cells expressing PD-1 are represented in the middle of the graphs. (E,F) Histograms represent the level of PD-1 expression gated on all dividing cells according to indicated cultures. Results represent two independent experiments.</p

Treg depletion results in increased infiltration of CD8+CD11c+ effector T cells.

<p>(A) Representative dot plots show tumour infiltrating lymphocytes analysed by flow cytometry for cell surface CD4 and CD8β expression gated on CD3 T cells in untreated and treated tumour infiltrates (day 14). (B) Percentages of tumour infiltrating CD8 T cells in untreated (n = 4) and treated (n = 4) mice at different time points after tumour inoculation. (C) Representative dot plots of cell surface expression of CD8⁺CD11c⁺ gated on CD8 T cells. (D) Histogram shows frequencies of CD8⁺CD11c⁺ T cells in indicated groups of mice (n = 3). (E) Flow cytometry analysing proliferating cells (KI67⁺) in CD8⁺CD11c⁺ and CD8⁺CD11c^- T cells in the indicated groups of mice. A representative experiment of at least two independent experiments is shown, n = 3 mice per group. (F,left) Graph shows percentages of CD8⁺CD11c⁺ T cells expressing each gene and sorted from both group of untreated (119 cells) and PC61-treated (107 cells). (F,right) Graph shows percentages of CD8⁺CD11c⁺ T cells co-expressing the four genes (right). Data are obtained from two different cell-sorting (G) Frozen tumor sections obtained 14 days after tumor injection from treated and untreated mice were examined by confocal microscopy after staining with CD8β (red), CD11c (green) specific antibodies and Dapi (gray, left panels). Direct photography acquired with a 63x objective show co-staining of CD8 and CD11c antibodies on CD8 T cells. Cell surface co-expression of CD8β and CD11c (circles) and contact between CD8 T cells and dendritic cells (rectangle) are shown in white color. Data are representative of at least two independent experiments. (H) The tumour vasculature was examined by confocal microscopy following staining with CD31 specific antibody. Representative tumour sections from indicated group are shown by (A) 20x magnification; scale bar: 100 μm and (B) 63x magnification; scale bar: 100 μm. These data are representative of three independent experiments. *,p<0.05 **,p<0.001. P values were calculated using Student’s t test and (a) by Fisher’s exact test p<0.05.</p