Mélanie Samson

Characterization of Drug-Resistant Influenza Virus A(H1N1) and A(H3N2) Variants Selected In Vitro with Laninamivir

ABSTRACT Neuraminidase inhibitors (NAIs) play a major role for managing influenza virus infections. The widespread oseltamivir resistance among 2007-2008 seasonal A(H1N1) viruses and community outbreaks of oseltamivir-resistant A(H1N1)pdm09 strains highlights the need for additional anti-influenza virus agents. Laninamivir is a novel long-lasting NAI that has demonstrated in vitro activity against influenza A and B viruses, and its prodrug (laninamivir octanoate) is in phase II clinical trials in the United States and other countries. Currently, little information is available on the mechanisms of resistance to laninamivir. In this study, we first performed neuraminidase (NA) inhibition assays to determine the activity of laninamivir against a set of influenza A viruses containing NA mutations conferring resistance to one or many other NAIs. We also generated drug-resistant A(H1N1) and A(H3N2) viruses under in vitro laninamivir pressure. Laninamivir demonstrated a profile of susceptibility that was similar to that of zanamivir. More specifically, it retained activity against oseltamivir-resistant H275Y and N295S A(H1N1) variants and the E119V A(H3N2) variant. In vitro, laninamivir pressure selected the E119A NA substitution in the A/Solomon Islands/3/2006 A(H1N1) background, whereas E119K and G147E NA changes along with a K133E hemagglutinin (HA) substitution were selected in the A/Quebec/144147/2009 A(H1N1)pdm09 strain. In the A/Brisbane/10/2007 A(H3N2) background, a large NA deletion accompanied by S138A/P194L HA substitutions was selected. This H3N2 variant had altered receptor-binding properties and was highly resistant to laninamivir in plaque reduction assays. Overall, we confirmed the similarity between zanamivir and laninamivir susceptibility profiles and demonstrated that both NA and HA changes can contribute to laninamivir resistance in vitro.

Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line.

Estradiol (E2) plays a crucial role in all reproduction processes. In the placenta, it is well recognized that E2 is synthesized from fetal dehydroepiandrosterone sulfate (DHEAS). However, there is some controversy about the biosynthetic pathway involved, some authors suggest that E2 is produced by aromatization of testosterone (T), while others suggest that E2 is produced by the conversion of estrone (E1) into E2 by type 1 17beta-HSD, subsequent to the aromatization of 4-androstenedione (4-dione) into E1. In the present report, using the precursor [(14)C]DHEA, inhibitors of steroidogenic enzymes (chemical inhibitors and siRNA) and a choriocarcinoma (JEG-3) cell line that expresses all the enzymes necessary to transform DHEA into E2, we could determine the sequential steps and the specific steroidogenic enzymes involved in the transformation of DHEA into E2. Quantification of mRNA expression levels using real-time PCR, strongly suggests that type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1), aromatase and type 1 17beta-HSD (17beta-HSD1) that are highly expressed in JEG-3 cells are the enzymes responsible for the transformation of DHEA into E2. Analysis of the intermediates produced in the absence and presence of 3beta-HSD, aromatase and 17beta-HSD1 inhibitors permits to determine the following sequential steps: DHEA is transformed into 4-dione by 3beta-HSD1, then 4-dione is aromatized into E1 by aromatase and E1 is finally transformed into E2 by 17beta-HSD1. Our data are clearly in favor of the pathway in which the step of aromatization precedes the step of reduction by 17beta-HSD.

Intracellular trafficking and fate of chimeric adenovirus 5/F35 in human B lymphocytes

Investigation of the molecular processes that control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte‐associated pathogenesis. Adenovirus‐mediated gene transfer provides a powerful tool for investigating these processes. However, we observed variation in transgene expression among normal human peripheral blood B lymphocytes from different donors and at distinct stages of differentiation. It is recognized that efficient gene transfer is highly dependent on the intracellular route by which the viruses travel within the host cell. Thus, we aimed to examine this aspect in the present study.

Suppression of protein phosphatase 2A activity enhances Ad5/F35 adenovirus transduction efficiency in normal human B lymphocytes and in Raji cells

Investigation of the molecular processes which control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte associated pathogenesis. Adenovirus-mediated gene transfer provided a powerful tool to investigate these processes. We have previously demonstrated that adenoviral vector Ad5/F35 transduces plasma cell lines at a higher efficiency than primary B cells, owing to differences in intracellular trafficking. Given that phosphatases are effectors of intracellular trafficking, here we have analyzed the effects of a panel of phosphatase inhibitors on Ad5/F35 transduction efficiency in B lymphocytes in the present study. FACS analysis was conducted to determine Ad5/F35-EYFP transduction efficiency in lymphoid cells, including human primary B cells, following serine/threonine phosphatase (PSP) inhibitor treatment. We further used confocal microscopy to analyze intracellular trafficking and fate of CY3 labeled Ad5/F35 vectors, in PSP treated lymphoid cell. Finally, we analyzed the MAPK pathway by Western blot in PSP treated cells. Adenoviral transduction efficiency was unresponsive to inhibition of PP1 whereas inhibition of PP2A by cantharidic acid, or PP1 and PP2A by okadaic acid, substantially increased transduction efficiency. Importantly, confocal microscopy analyses revealed that inhibition of PP2A shut down adenovirus recycling. Moreover, inhibition of PP2A resulted in increased phosphorylation of AKT, ERK1/2 and MEK1/2. Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells following PP2A inhibition. Our results are in agreement with reports indicating that PP2A is involved in the formation of recycling vesicles and might be of interest for gene therapy applications.

Inhibition of human-type 1 3β-hydroxysteroid deshydrogenase/Δ5-Δ4-isomerase expression using siRNA ☆

Abstract Specific inhibition of type 1 3β-HSD is of particular interest since it will allow us to control the formation of androgens and estrogens in peripheral target tissues without affecting type 2 3β-HSD, which is responsible for the biosynthesis of glucocorticoids and mineralocorticoids in the adrenals. The high homology between types 1 and 2 3β-HSD is a major difficulty in the development of specific inhibitors through classical chemical synthesis. In this report, we describe the use of small interference RNA (siRNA) to specifically inhibit human type 1 3β-HSD. We have constructed three DNA vector-based RNAi vectors that allow us to produce three RNA duplexes of 21 nucleotides targeting three different coding regions of human type 1 3β-HSD mRNA. The resulting constructs were co-transfected into HEK-293 cells with a vector expressing type 1 3β-HSD. The results indicate that while the two duplexes that target sequences in the 5′-region do not have a strong inhibitory effect, the duplex that targets the 3′-region efficiently inhibits 3β-HSD activity. Up to 98% inhibition has been observed. To our knowledge, this is the first report showing successful inhibition of steroidogenic enzymes using siRNA technology.

Revisiting Qúebec's Jus Commune in the Era of the Human Rights Charters

Quebec is a distinct society because of its history, its legal system, and its values. Our analysis examines the delicate issue of the relationship between the Canadian Charter of Rights and Freedoms, the Quebec Charter of Human Rights and Freedoms, and the Civil Code of Quebec, the primary expression of Quebecs jus commune, as noted in its Preliminary Provision. As of the nineteenth century, a doctrinal trend born of the desire to protect the integrity of the civil law system grew worried about the “disruptive” influence of the common law on the civil law and, more specifically, on the Civil Code of Lower Canada. The doctrine later expressed reluctance as to the entry of fundamental rights into Quebec private law. The charters of rights were, and are sometimes still, perceived as disruptive elements, capable of distorting the Civil Code. We want to show that the influence of human rights philosophy on Quebecs jus commune is not only inevitable but desirable. The Civil Code and, more broadly, Quebecs jus commune, can only be enriched by respect for fundamental rights.

Le dialogue du droit civil et des droits de la personne au Québec : l’exemple de la protection juridique des personnes aînées

La protection de la personne âgee et vulnerable exige une cooperation interdisciplinaire et, sur le plan juridique, elle sollicite differentes branches du droit. Notre etude porte sur l’interaction du droit civil et des droits de la personne en matiere de protection des personnes âgees au Quebec. Elle concerne plus specifiquement le caractere autonome mais convergent des garanties qu’offrent a ces personnes la Charte des droits et libertes de la personne et le Code civil du Quebec. Nous demontrerons qu’en tant que loi quasi constitutionnelle de protection des droits de la personne, la Charte offre a la personne âgee vulnerable des garanties specifiques qui excedent celles prevues par le Code et que, d’autre part, elle oriente l’interpretation de l’ensemble des dispositions du Code civil de facon a proteger les droits des aines.Protection juridique des aines – vulnerabilite - exploitation - Charte des droits et libertes de la personne – Code civil du Quebec – interpretation des lois – loi quasi constitutionnelle

Le rayonnement des droits de la personne en droit privé québécois: Que de chemin parcouru… mais que de chemin à parcourir!

With the adoption of the Canadian Charter of Rights and Freedoms and the Quebec Charter of Human Rights and Freedoms, civil law and human rights influence each other and must be contemplated as a whole. The Preliminary Provision of the Civil Code of Quebec establishes a close relationship between the Charter and the Civil Code, by indicating that the latter must be interpreted « in harmony » with the former. In our presentation, we describe the ways in which the Quebec Charter and the Civil Code work together to ensure a maximum protection of human rights. We measure the influence of human rights on contract law, civil liability rules, property law and family lawὑ.

Les conflits de compétence entre tribunaux spécialisés : une question de textes ou de contextes?

[1] L’histoire contemporaine atteste que « le progrès des idéologies égalitaires et démocratiques a amené l’État à dispenser une série de biens et de services et à régler luimême des relations qu’il avait longtemps abandonnées à la volonté autonome des citoyens ». Sur le plan juridique, ces interventions se sont traduites par la prolifération de lois et de mesures gouvernementales visant à assurer la prise en charge, par l’État, de diverses responsabilités, tantôt pour assurer la protection de la population, tantôt pour rehausser son bien-être et favoriser ainsi l’harmonie entre ses membres et l’équité sociale. Dans les faits, rares sont les domaines « où l’État n’est pas intervenu pour surveiller, contrôler ou réglementer ». Cette multiplication des normes étatiques a « suscité la constitution, à l’extérieur du réseau des ministères et des cours de justice, de nombreux