La vitrification en une seule étape d’embryons murins définit de nouveaux standards de cryopréservation

La cryopréservation d’embryons est un des outils les plus efficaces, économiques et utiles au niveau éthique pour préserver indéfiniment la génétique des animaux de laboratoire. Les bénéfices liés à son utilisation sont nombreux, et incluent la réduction des coûts associés à la pérennisation de lignées, la limitation de l’apparition et de la dissémination de mutations non voulues, la facilité et la sécurité pour les transferts internationaux, et la réduction du nombre d’animaux à élever en captivité. La vitrification a été démontrée comme étant plus efficace que la congélation lente en procréation médicalement assistée humaine, où elle constitue maintenant la méthode de référence. Il en va de même pour la cryopréservation des embryons de souris, où la vitrification préserve mieux l’intégrité de la chromatine, réduit la pénétration intracytoplasmique d’agents cryoprotecteurs potentiellement toxiques, et in fine permet une meilleure survie et un meilleur développement après réchauffement que la congélation lente. Cependant, pour être efficaces, les méthodes actuelles de vitrification nécessitent plusieurs étapes d’exposition des embryons à des solutions spécifiques avant le refroidissement, mais aussi au cours de leur réchauffement ultérieur. Cette relative complexité peut s’avérer difficile à gérer de manière optimale quand un grand nombre d’embryons doivent être traités au cours d’une même session. Nous avons développé et breveté une technologie de vitrification d’embryons en une étape (« one-step ») qui est aussi efficace que les meilleures méthodes de vitrification multi-étapes. De plus, nos milieux sont chimiquement définis (sans sérum ou autre composant biologique non défini), et des supports permettant la vitrification aseptique peuvent être utilisés sans perte de rendement. Notre technologie de vitrification one-step d’embryons de rongeurs répond ainsi aux problèmes d’ergonomie liés aux méthodes classiques de vitrification, et fournit ainsi aux scientifiques une solution efficace, biologiquement sûre et facile à utiliser pour la cryopréservation d’embryons de rongeurs. Elle rend ainsi l’efficacité de la vitrification plus aisément applicable aux rongeurs de laboratoire, contribuant ainsi à la réduction du nombre d’animaux nécessaires à la pérennisation et à la diffusion de lignées ou de colonies utiles.Vitricell: développement et valorisation de kits de vitrification de cellules en conditions aseptiques et chimiquement définie

Biobank quality management in the BBMRI.be network

From as early as 2005, different guidelines and quality standards covering biobank activities and sample handling methods have been developed to improve and guarantee the reproducibility of biomarker research. Ten years on, the BBMRI.be Quality working group wanted to gauge the current situation of these aspects in the biobanks of the BBMRI.be network. To this end, two online surveys were launched (fall 2017 and fall 2018) to the biobank quality managers in the BBMRI.be network to determine the status and setup of their current quality management system (QMS) and how their QMS and related practices have evolved over a 14 month time period. All biobanks addressed by the two surveys provided a complete response (12 and 13, respectively). A QMS was implemented in 85% of biobanks, with 4 standards emerging as primary basis. Supplementary guidelines were used, with a strong preference for the ISBER best practices for biobanks. The Standard Preanalytical Code-an indicator of the preanalytical lifecycle of a biospecimen impacting the downstream analysis results-was already implemented in 50% of the biobanks while the other half intends future implementation. To assess and maintain the quality of their QMS, 62% of biobanks used self-assessment tools and 71% participated in proficiency testing schemes. The majority of biobanks had implemented procedures for general and biobank specific activities. However, policies regarding the business and sustainability aspect of biobank were only implemented in a limited number of biobanks. A clear desire for a peer-review audit was expressed by 69% of biobanks, with over half of them intending to implement the recently published biobank standard ISO20387. Overall, the biobanks of the BBMRI.be network have actively implemented a solid quality approach in their practices. The implementation of ISO 20387 may bring further professionalization of activities. Based on the needs expressed in this survey, the Quality working group will be setting up an audit program for the BBMRI.be biobanks, to enhance, harmonize and streamline their activities. On the whole, the biobanks in the BBMRI.be network are able to substantially contribute to translational research, as a primary facilitator guaranteeing high quality standards and reproducibility

A phase I radiation dose-escalation study to determine the maximal dose of radiotherapy in combination with weekly gemcitabine in patients with locally advanced pancreatic adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>The primary objective of this study was to determine the maximum tolerated dose (MTD) of escalating doses of radiotherapy (RT) concomitantly with a fixed dose of gemcitabine (300 mg/m²/week) within the same overall treatment time.</p> <p>Methods</p> <p>Thirteen patients were included. Gemcitabine 300 mg/m²/week was administered prior to RT. The initial dose of RT was 45 Gy in 1.8 Gy fractions, escalated by adding 5 fractions of 1.8 Gy (one/week) to a dose of 54 Gy with a total duration kept at 5 weeks. All patients received a dynamic MRI to assess the pancreatic respiratory related movements. Toxicity was scored using the RTOG-EORTC toxicity criteria.</p> <p>Results</p> <p>Three of six patients experienced an acute dose limiting toxicity (DLT) at the 54 Gy dose level. For these patients a grade III gastro-intestinal toxicity (GI) was noted. Patients treated at the 45 Gy dose level tolerated therapy without DLT. The 54 Gy dose level was designated as the MTD and was deemed not suitable for further investigation.</p> <p>Between both dose levels, there was a significant difference in percentage weight loss (p = 0.006) and also in cumulative GI toxicity (p = 0.027). There was no grade 3 toxicity in the 45 Gy cohort versus 4 grade 3 toxicity events in the 54 Gy cohort. The mean dose to the duodenum was significantly higher in the 54 Gy cohort (38.45 Gy vs. 51.82 Gy; p = 0.001).</p> <p>Conclusion</p> <p>Accelerated dose escalation to a total dose of 54 Gy with 300 mg/m²/week gemcitabine was not feasible. GI toxicity was the DLT. Retrospectively, the dose escalation of 9 Gy by accelerated radiotherapy might have been to large. A dose of 45 Gy is recommended. Considering the good patient outcomes, there might be a role for the investigation of a fixed dose of gemcitabine and concurrent RT with small fractions (1.8 Gy/day) in borderline resectable or unresectable non-metastatic locally advanced pancreatic cancer.</p

Increased β-Cell Mass by Islet Transplantation and PLAG1 Overexpression Causes Hyperinsulinemic Normoglycemia and Hepatic Insulin Resistance in Mice

OBJECTIVE-It is believed that an organism remains normoglycemic despite an increase in the beta-cell mass because of decreased insulin production by beta-cells on a per-cell basis However, some transgenic mouse models with beta-cell hyperplasia suggest that insulin production remains excessive and that normoglycemia is maintained by insulin resistance METHODS-Here, we investigated the effect of an increased beta-cell mass on glycemia and insulin resistance by grafting excess normal islets in normoglycemic mice, as well as using targeted PLAG1 expression in beta-cells, which leads to beta-cell expansion. RESULTS-In both models, fasting plasma insulin levels were increased, even though animals were normoglycemic. After an intraperitoneal glucose tolerance test, plasma insulin levels increased, which was associated with improved glucose clearing Under these conditions, normoglycemia is maintained by hepatic insulin resistance as demonstrated by hyperinsulinemic euglycemic clamp experiments. CONCLUSIONS-In conclusion, we demonstrate that when excess beta-cells are grafted, insulin production on a per beta-cell basis is not sufficiently decreased, leading to hyperinsulinemia and hepatic insulin resistance. This observation might be important for the design of stem cell-based islet replacement therapies. Diabetes 59:1957-1965, 2010Diabetes mellitus: pathophysiological changes and therap

GPR101 drives growth hormone hypersecretion and gigantism in mice via constitutive activation of Gs and Gq/11

peer reviewedGrowth hormone (GH) is a key modulator of growth and GH over-secretion can lead to gigantism. One form is X-linked acrogigantism (X-LAG), in which infants develop GH-secreting pituitary tumors over-expressing the orphan G-protein coupled receptor, GPR101. The role of GPR101 in GH secretion remains obscure. We studied GPR101 signaling pathways and their effects in HEK293 and rat pituitary GH3 cell lines, human tumors and in transgenic mice with elevated somatotrope Gpr101 expression driven by the rat Ghrhr promoter (GhrhrGpr101). Here, we report that Gpr101 causes elevated GH/prolactin secretion in transgenic GhrhrGpr101 mice but without hyperplasia/tumorigenesis. We show that GPR101 constitutively activates not only Gs, but also Gq/11 and G12/13, which leads to GH secretion but not proliferation. These signatures of GPR101 signaling, notably PKC activation, are also present in human pituitary tumors with high GPR101 expression. These results underline a role for GPR101 in the regulation of somatotrope axis function.GOLIATH

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts

Infectious disorders of the duodenum

E La comparaison entre les animaux axéniques et ceux porteurs d'une flore conventionnelle a montré que la « cellularité physiologique » de la lamina propria et le développement du tissu lymphoïde associé à l'intestin est en général une réponse à cette flore. L'inflammation du duodénum peut survenir dans un certain nombre de situations incluant la maladie coeliaque, la maladie de Crohn, des maladies infectieuses spécifiques ou parasitaires et enfin suite à une pathologie pancréatique ou des voies biliaires. Dans la plupart des infections virales connues de l'intestin grêle, on n'observe que des modifications histopathologiques non spécifiques (c'est-à-dire: altération épithéliale, villosités émoussées, et inflammation chronique). Des bactéries de types très différents contaminent le tractus digestif humain et entraînent diverses sortes de gastro-entérites et/ou entérocolites. Le diagnostic spécifique d'infection bactériologique recourt en général à la culture et la sérologie puisque les syndromes cliniques tentent au chevauchement et les caractéristiques pathologiques sont le plus souvent non spécifiques. Les exceptions confirment la règle et c'est le cas de la duodénite à Helicobacter pylori et de la maladie de Whipple. La giardiase, infection protozoaire, est une parasitose commune dans de nombreux pays d'Europe et aux USA. Des infections par helminthes sont rares dans les zones non-endémiques

Lymphomas of the gastro-intestinal tract: uncommon types

Bien que, comparés aux carcinomes, les lymphomes du tractus digestif soient extrêmement rares, leur incidence est croissante. Cette croissance peut s'expliquer par une augmentation de l'incidence des perturbations liées à l'immunodéficience, elles-mêmes associées à une augmentation du risque de développer des lymphomes gastro-intestinaux. Les lymphomes gastro-intestinaux sont principalement des lymphomes B de type diffus à grandes cellules et des lymphomes de la zone marginale. Malgré la rareté des autres types de lymphomes tels les lymphomes du manteau, les lymphomes folliculaires ou les lymphomes T associés a une entéropathie, la classification REAL tout comme la classification publiée récemment par l'OMS, considèrent ces lymphomes comme des entités distinctes en se basant sur leur morphologie, l'immunohistochimie, la cytogénétique et leur présentation clinique