An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins

Abstract

Human intestinal epithelial cell lines (T84, Caco-2, and HCT-8) grown on permeable Transwell™ filters serve as models of the gastrointestinal barrier. In this study, this in vitro model system was evaluated for effectiveness at distinguishing between hazardous and non-hazardous proteins. Indicators of cytotoxicity (LDH release, MTT conversion), monolayer barrier integrity ([(3)H]-inulin flux, horseradish peroxidase flux, trans-epithelial electrical resistance [TEER]), and inflammation (IL-8, IL-6 release) were monitored following exposure to hazardous or non-hazardous proteins. The hazardous proteins examined include streptolysin O (from Streptococcus pyogenes), Clostridium difficile Toxins A and B, heat-labile toxin from enterotoxigenic Escherichia coli, listeriolysin O (from Listeria monocytogenes), melittin (from bee venom), and mastoparan (from wasp venom). Non-hazardous proteins included bovine and porcine serum albumin, bovine fibronectin, and ribulose bisphosphate carboxylase/oxygenase (RuBisco) from spinach. Food allergenic proteins bovine milk β-lactoglobulin and peanut Ara h 2 were also tested as was the anti-nutritive food protein wheat germ agglutinin. Results demonstrated that this model system effectively distinguished between hazardous and non-hazardous proteins through combined analysis of multiple cells lines and assays. This experimental strategy may represent a useful adjunct to multi-component analysis of proteins with unknown hazard profiles.

Illuminating dynamic neutrophil trans-epithelial migration with micro-optical coherence tomography

“…For 3 H-inulin flux, 2.5 μCi/ml 3 H-inulin (PerkinElmer Inc. - NEN [New England Nuclear], Wellesley, Massachusetts, USA) was added to the apical surface and the amount of 3 H-inulin that reached the basolateral compartment after 24 hours in the presence or absence of EDTA was determined by sampling counts per minute (cpm) using an LS 6500 Multi-purpose Scintillation Counter from Beckman Coulter, Inc. (Brea, CA). Data is presented as % flux of 3 H-inulin where the amount measured in the basolateral compartment adjusted for volume is divided by the total amount added to the apical surface and multiplied by 10022.…”

Emergent food proteins – Towards sustainability, health and innovation

“…Proteins and peptides toxicity take a central part in the regulation of body functions. Protein chemical or non-chemical modifications could result in nutritional value changes, possible toxic peptides or amino acid derivatives formation, and contamination by toxic chemicals harmful for health (Hurley et al, 2016;Zimmermann et al, 2018). Analyses intending to establish the proteins' cytotoxicity properties have to consider a range of factors (e.g., proteins' effect on the gastrointestinal tract and susceptibility to digestion -see section 5.1).…”

“…Therefore, the application of in vitro assays as screening tools can assist in estimating the potential proteins toxicity prior to their test in animal models and clinical studies. The cytotoxic effects of proteins usually are evaluated using in vitro assays such as 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) test based on mitochondrial dehydrogenase activity detection in living cells and lactate dehydrogenase (LDH) released from dead cells (Hurley et al, 2016). Additionally, necrosis, apoptosis and/or cell cycle disturbances activities are assessed in many research works with the purpose to clarify about cell death mechanism stimulated by protein or peptides (Chalamaiah, Yu, & Wu, 2018).…”

Congenital Tufting Enteropathy-Associated Mutant of Epithelial Cell Adhesion Molecule Activates the Unfolded Protein Response in a Murine Model of the Disease

“…To ensure our ability to detect a UPR response, T84 human colonic adenocarcinoma cells were utilized. Though colonic, rather than small intestinal, T84 cells were utilized as they are a well-studied model for polarized intestinal epithelial cells [26][27][28]. The cells were cultured in 1:1 Dulbecco's modified Eagle's medium/F12 Ham's medium (Gibco, Waltham, MA, USA) with 15 mM l-glutamine (Gibco), 5% bovine calf serum (Sigma, St. Louis, MO, USA), 1% penicillin-streptomycin (Hyclone, Logan, UT, USA) and maintained as described earlier [25].…”