Étude de la perméabilité intestinale des médicaments par la reconstitution du transporteur BCRP/ABCG2 dans des protéoliposomes

La perméabilité membranaire des cellules régule l’absorption et la distribution des médicaments dans le corps. Des modèles in vitro comme le test cellulaire Caco-2 et le test non cellulaire PAMPA sont utilisés pour prédire la biodisponibilité des médicaments. Cependant, le premier est limité par sa reproductibilité et l’hétérogénéité des conditions de culture et le second ne permet que d’étudier le transport passif. Par ailleurs, aucun modèle ne permet l’analyse d’un transporteur membranaire spécifique de façon isolée. Il est proposé d’étudier la perméabilité membranaire en utilisant une nouvelle approche basée sur l’incorporation de transporteurs recombinants dans des liposomes. Le transporteur choisi pour ce projet est la protéine humaine ABCG2/BCRP qui a le potentiel de limiter la biodisponibilité orale des médicaments. BCRP a été exprimé dans la levure Pichia pastoris et a été purifié par chromatographie d’affinité. L’isoforme 1 de BCRP a été modifié par PCR et cloné dans le vecteur d’expression pJ902-15. Le plasmide résultant contient la région codante pour BCRP avec une étiquette de 10 histidines sous le contrôle du promoteur AOX1. La transformation dans P. pastoris est effectuée par électroporation avec le vecteur pJ902-ABCG2i-His10 linéarisé. Le clone possédant le plus haut niveau d’expression de BCRP a été déterminé par immunobuvardage à l’aide de l’anticorps monoclonal BXP-21. Ce clone a été inoculé dans 1 L de milieu MGY puis les cellules ont été induites dans le milieu MM contenant du méthanol. Les cellules ont été lysées à l’aide du Freezer/Mill et les microsomes ont été solubilisés et purifiés sur une colonne HisTrap HP par le système ÄKTA-FPLC. Un système d’expression a été établi pour la production de l’isoforme 1 de BCRP recombinant chez P. pastoris. L’immunobuvardage montre que BCRP-His10 a un poids apparent de 65 kDa. À partir d’une culture de levures de 1 L, environ 125 mg de BCRP a pu être partiellement purifié. La prochaine étape pour cette partie du projet est de confirmer que BCRP-His10 est fonctionnel en mesurant son activité ATPase en présence de la prasozine. Les protéoliposomes de BCRP recombinant serviront de preuve-de-concept pour la génération d’une librairie de transporteurs recombinants incorporé dans des liposomes. Ce modèle facilitera la compréhension des mécanismes structuraux et fonctionnels du transport des médicaments dans les cellules.Intestinal membrane transporters play a critical role in the pharmacokinetics of orally administered drugs. Membrane permeability of cells regulate the absorption of drugs by modulating their distribution in the body. In vitro models like the Caco-2 cell-based assay and the non-cellular PAMPA assay are used to predict the bioavailability of drugs. However, the Caco-2 assay is limited by its reproducibility and heterogeneity of the culture conditions while the PAMPA assay is only used to observe passive permeability. Thus, no model allows the study of isolated and specific membrane transporters. We propose to study membrane permeability with a new approach using a non-cellular lipid bilayer constituted of recombinant transporter. In this study, the isoform 1 of the human BCRP/ABCG2, which is known to limit the intestinal absorption of drug substrates, was expressed in the yeast Pichia pastoris and purified by affinity chromatography. The isoform 1 of BCRP cDNA was modified by PCR and cloned into the expression vector pJ902-15. The resulting construct contain the coding region of BCRP with a 10 histidines tag under the control of the AOX1 promoter. P. pastoris transformation was carried by electroporation with the linearized vector pJ902-ABCG2i-His10. The clone with the highest BCRP expression was determined immunoblotting using mAb BXP-21. This clone was inoculated in 1 L of MGY media and induced in the MM media containing methanol. The cells were lysed using a Freezer/Mill and the microsomes were solubilized and purified on an ÄKTA-FPLC system using a HisTrap HP column. We have established a P. pastoris expression system for the production of the recombinant transporter BCRP. Immunoblotting of whole cell lysates shows that the recombinant BCRP has an apparent weight of 65 kDa. From a 1 L yeast culture, approximately 125 mg of BCRP was partially purified. To confirm that BCRP-His10 is functional, further studies are necessary by measuring the ATPase activity in presence of prazosin. The proteoliposomes of the recombinant BCRP will serve as a proof-of-concept for the generation of a library of recombinant transporter proteins incorporated into liposomes. This model will facilitate the assessment and understanding of the bioavailability of drug candidates

Card9 Broadly Regulates Host Immunity against Experimental Pulmonary <i>Cryptococcus neoformans</i> 52D Infection

The ubiquitous soil-associated fungus Cryptococcus neoformans causes pneumonia that may progress to fatal meningitis. Recognition of fungal cell walls by C-type lectin receptors (CLRs) has been shown to trigger the host immune response. Caspase recruitment domain-containing protein 9 (Card9) is an intracellular adaptor that is downstream of several CLRs. Experimental studies have implicated Card9 in host resistance against C. neoformans; however, the mechanisms that are associated with susceptibility to progressive infection are not well defined. To further characterize the role of Card9 in cryptococcal infection, Card9em1Sq mutant mice that lack exon 2 of the Card9 gene on the Balb/c genetic background were created using CRISPR-Cas9 genome editing technology and intratracheally infected with C. neoformans 52D. Card9em1Sq mice had significantly higher lung and brain fungal burdens and shorter survival after C. neoformans 52D infection. Susceptibility of Card9em1Sq mice was associated with lower pulmonary cytokine and chemokine production, as well as reduced numbers of CD4+ lymphocytes, neutrophils, monocytes, and dendritic cells in the lungs. Histological analysis and intracellular cytokine staining of CD4+ T cells demonstrated a Th2 pattern of immunity in Card9em1Sq mice. These findings demonstrate that Card9 broadly regulates the host inflammatory and immune response to experimental pulmonary infection with a moderately virulent strain of C. neoformans

A scoping review of mHealth monitoring of pediatric bronchial asthma before and during COVID-19 pandemic

Dauletbaev N, Oftring Z, Akik W, et al. A scoping review of mHealth monitoring of pediatric bronchial asthma before and during COVID-19 pandemic. Paediatric Respiratory Reviews . 2022.Mobile (m) Health technology is well-suited for Remote Patient Monitoring (RPM) in a patient's habitual environment. In recent years there have been fast-paced developments in mHealth-enabled pediatric RPM, especially during the COVID-19 pandemic, necessitating evidence synthesis. To this end, we conducted a scoping review of clinical trials that had utilized mHealth-enabled RPM of pediatric asthma. MEDLINE, Embase and Web of Science were searched from September 1, 2016 through August 31, 2021. Our scoping review identified 25 publications that utilized synchronous and asynchronous mHealth-enabled RPM in pediatric asthma, either involving mobile applications or via individual devices. The last three years has seen the development of evidence-based, multidisciplinary, and participatory mHealth interventions. The quality of the studies has been improving, such that 40% of included study reports were randomized controlled trials. In conclusion, there exists high-quality evidence on mHealth-enabled RPM in pediatric asthma, warranting future systematic reviews and/or meta-analyses of the benefits of such RPM. Copyright © 2022 Elsevier Ltd. All rights reserved