Immunohistochemical and molecular detection of equine herpesvirus 1 in Uruguay

Equine herpesvirus 1 (EHV-1) is a major cause of epidemic abortion, neonatal mortality, respiratory disease and neurological disorders in horses. In South America, the virus has been isolated in Brazil, Argentina and Colombia. In Chile pathological findings from one aborted foetus have been reported, and in Uruguay only serological data about EHV-1 activity have been found. Some pathological findings were reported in Uruguay several years ago, but these data have never been officially confirmed. The present work describes the relevant findings of a study of EHV-1 infections in the Uruguayan equine population using polymerase chain reaction (PCR) and histological and immunohistochemical analysis techniques. The sequence analysis of a portion of the glycoprotein C gene amplified by PCR confirmed EHV-1 activity. The real-time PCR revealed the association of the virus with the non-neuropathogenic genotype. This study describes for the first time the immunohistochemical and molecular detection of EHV-1 in Uruguay.Facultad de Ciencias Veterinaria

Immunohistochemical and molecular detection of equine herpesvirus 1 in Uruguay

Equine herpesvirus 1 (EHV-1) is a major cause of epidemic abortion, neonatal mortality, respiratory disease and neurological disorders in horses. In South America, the virus has been isolated in Brazil, Argentina and Colombia. In Chile pathological findings from one aborted foetus have been reported, and in Uruguay only serological data about EHV-1 activity have been found. Some pathological findings were reported in Uruguay several years ago, but these data have never been officially confirmed. The present work describes the relevant findings of a study of EHV-1 infections in the Uruguayan equine population using polymerase chain reaction (PCR) and histological and immunohistochemical analysis techniques. The sequence analysis of a portion of the glycoprotein C gene amplified by PCR confirmed EHV-1 activity. The real-time PCR revealed the association of the virus with the non-neuropathogenic genotype. This study describes for the first time the immunohistochemical and molecular detection of EHV-1 in Uruguay.Facultad de Ciencias Veterinaria

Analysis of the mass transfers in an artificial kidney microchip

International audienceIn this communication we demonstrate a conception of an artificial microkidney using pertinent microtools that more accurately mimic organ functions in vitro. We present a technique to integrate polyethersulfone (PES) membranes usually used in hemodialysis inside a polydimethylsiloxane (PDMS) microchip. The purpose of the microchip is to model glomerular filtration "on-chip". Mass transfer of urea (60 Da), vitamin B12 (1355 Da) and albumin (70,000 Da) are investigated by using two types of membranes (cut-off at 500,000 Da and 40,000 Da) in co-current and counter-current flow conditions. The time of urea, vitamin B12 and albumin removal, and the mechanisms of mass transfer, are controlled either by controlling the pore size of the membranes or by controlling the pressure profiles along the membrane via the flow conditions. An analytical model, which is supported by our data, is put forth. The model allows the extraction of the diffusion coefficients of each molecule through the various membranes studied. Due to the downscaling, the model and the experiments demonstrate that the dialysance in the microchip is expressed by the sum of the diffusion and convection mass transfer components. The results of this work support an analytical model which describes the mass transfer in a microchip modelling a glomerular unit. Coupled with the advantages of the microfluidic biochip (high surface/volume ratio, reduction of the fluid volumes), our data will complete the integration of further cellular functions for the utilisation of the present microchip as a future in vitro model of a miniaturized bio artificial kidney

Flow and mass transfer modeling for tissue engineering applications.

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An appropriate selection of 3D alginate culture model for hepatic Huh-7 cell line encapsulation intended for viral studies

International audienceThree-dimensional (3D) culture systems have been introduced to provide cells with a biomimetic environment similar to in vivo conditions. Among the polymeric molecules available, sodium alginate salt (Na-alg) is a material currently employed in different areas of drug delivery and tissue engineering because it offers biocompatibility, optimal chemical properties and its gelation with calcium chloride provides calcium alginate (Ca-alg) scaffolds with mechanical stability and relative permeability. In this work, four different preparations of Ca-alg beads with varying Na-alg viscosity and concentration were used for a human hepatoma cell line (Huh-7) encapsulation. The effects of Ca-alg bead preparation on structural cell organization, liver-specific functions, and specific receptors expression implicated in hepatotropic virus permissivity were evaluated. Hepatic cells were cultured in 500Am diameter Ca-alg beads during 7 days under dynamic condition. For all culture systems, cell viability reached almost 100% at day 7. Cell proliferation was concomitantly followed by hepatocyte organization in aggregates, which adopted two different morphologies (spheroïd aggregates or multicellular channel-like structures), depending on Ca-alg bead preparation. These cellular organizations established real 3D hepatocyte architecture with cell polarity, cell junctions and abundant bile canaliculi possessing microvillus-lined channels. The functionality of these 3D cultures was confirmed by the production of albumin and the exhibition of activity CYP1A over culture time, which were variable, according to Ca-alg bead condition. The expression of specific receptors of Hepatitis C Virus (HCV) by Huh-7 cells suggests encouraging data for further development of a new viral culture system in Ca-alg beads. In summary, this 3D hepatic cell culture represents a promising physiologically relevant system for further in vitro studies and demonstrates that adequate encapsulation condition can be selected for each target application in liver tissue engineering, specifically in viral studies

Long term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drugs metabolisms when compared to Petri cultures.

International audienceHuman primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture

Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated C3A cells in bioartificial liver applications

Liver-assist devices have been developed in the last few decades to support patients with liver failure on the road to recovery or transplantation. Fluidised bed bio-artificial livers – where liver cells are encapsulated within alginate beads – appear to be a valuable alternative to hollow fibre devices for improving mass transfers and enhancing treatment efficacy. This approach nevertheless deserves optimization in terms of bead production. The aim of this study was to investigate the impact of alginate type and of two bead diameters (1000 µm and 600 µm) on mass transfers within beads and on the biological functions of encapsulated C3A cells.After assessing the effect of the encapsulation process on bead quality, we investigated cell viability and metabolic activities (ammonia, albumin, alpha-fetoprotein synthesis and glucose consumption). They were successfully maintained over 48 h within fluidised bed bioreactors, independently of alginate type and bead diameter. Mass transfers were not significantly influenced by the latter parameters. Finally, suggestions are made for improving the entrapment process as a means of enhancing the treatment efficiency of the fluidizsed bed bioartificial liver