289 Characterization of three-dimensional in vitro human skin culture model mimicking atopic dermatitis

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols

International Journal of Molecular Sciences ◽

10.3390/ijms21155335 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5335

Author(s):

Hana Barosova ◽

Bedia Begum Karakocak ◽

Dedy Septiadi ◽

Alke Petri-Fink ◽

Vicki Stone ◽

...

Keyword(s):

Prolonged Exposure ◽

Three Dimensional ◽

Adverse Outcomes ◽

Alveolar Epithelial Cells ◽

Proinflammatory Response ◽

Multiwalled Carbon ◽

Alveolar Epithelial ◽

Culture Model ◽

Air Liquid Interface

In vitro three-dimensional (3D) lung cell models have been thoroughly investigated in recent years and provide a reliable tool to assess the hazard associated with nanomaterials (NMs) released into the air. In this study, a 3D lung co-culture model was optimized to assess the hazard potential of multiwalled carbon nanotubes (MWCNTs), which is known to provoke inflammation and fibrosis, critical adverse outcomes linked to acute and prolonged NM exposure. The lung co-cultures were exposed to MWCNTs at the air-liquid interface (ALI) using the VITROCELL® Cloud system while considering realistic occupational exposure doses. The co-culture model was composed of three human cell lines: alveolar epithelial cells (A549), fibroblasts (MRC-5), and macrophages (differentiated THP-1). The model was exposed to two types of MWCNTs (Mitsui-7 and Nanocyl) at different concentrations (2–10 μg/cm2) to assess the proinflammatory as well as the profibrotic responses after acute (24 h, one exposure) and prolonged (96 h, repeated exposures) exposure cycles. The results showed that acute or prolonged exposure to different concentrations of the tested MWCNTs did not induce cytotoxicity or apparent profibrotic response; however, suggested the onset of proinflammatory response.

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