Évaluation de la migration de cellules souches mésenchymateuses équines dans un caillot de fibrine riche en plaquette

Les lésions musculo-squelettiques sont fréquentes chez le cheval. La cicatrisation de ce type de lésion est souvent de mauvaise qualité ce qui entraîne un fort taux de récidive. Par exemple, c’est le cas pour la tendinite ou pour la dégénérescence du cartilage articulaire. Les traitements classiques sont parfois insuffisants pour guérir de telles affections. Ces dernières années, les traitements appartenant à la branche de la médecine régénérative ont fait leur apparition. La thérapie par les cellules souches mésenchymateuses (CSM) a déjà prouvé son efficacité dans le traitement des lésions ostéo-articulaires et l’utilisation des concentrés plaquettaires commence à se démocratiser. Le PRF (fibrine riche en plaquette) est un composé plaquettaire original, composé d’un réseau tridimensionnel de fibrine permettant l’enchevêtrement de plaquettes, leucocytes et facteurs de croissance en grande quantité. L’utilisation conjointe de CSM et de PRF pour répondre au traitement d’affection ostéo-articulaire et l’étude de leurs interactions sont très peu documentées dans la littérature. Nos expériences permettent d’affirmer que les CSM présentent un tropisme pour le PRF et que la migration cellulaire au sein de la fibrine est possible. Cet assemblage pourrait être un biomatériau de comblement idéal notamment pour des tissus dont la cicatrisation est incomplète et/ou de mauvaise qualité

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

<p>Abstract</p> <p>Background</p> <p>The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of <it>fut8 </it>gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat <it>fut8 </it>gene.</p> <p>Results</p> <p>The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat <it>fut8 </it>gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of <it>fut8 </it>in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 <it>fut8 </it>gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.</p> <p>Conclusion</p> <p>Altogether, these data contribute to a better knowledge of <it>fut8 </it>expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.</p

Climate change refugia for the flora and fauna of England

A variety of evidence suggests that species have, in the past, been able to withstand the effects of climatic change in localised environments known as refugia, where specific environmental conditions acted as a buffer against broader-scale climatic changes. Therefore, an important question for conservation is whether refugia might exist under current and future anthropogenic climate change. If there are areas that are likely to remain relatively climatically stable and so enable species to persist despite climate change making surrounding areas unsuitable, identifying and protecting these places will be an important part of future conservation strategies. This report is part of a project that is investigating this question. The report was commissioned to identify the characteristics of potential refugia, to investigate evidence for the existence of contemporary refugia by analysing patterns of local persistence and disappearance of over 1000 species across a range of taxa, and to identify sites in England with the potential to function as refugia for different taxonomic groups at a range of spatial scales

A Conformation-Sensitive Monoclonal Antibody against the A2 Domain of von Willebrand Factor Reduces Its Proteolysis by ADAMTS13

The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13

Cleavage of von Willebrand Factor by Granzyme M Destroys Its Factor VIII Binding Capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF