1.
Gullberg, Elisabet, et al.
(author)
Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis
2008
In: Laboratory investigation; a journal of technical methods and pathology. - New York, USA : Elsevier BV. - 1530-0307 .- 0023-6837. ; 88:11, s. 1215-26
Journal article (peer-reviewed) abstract
Crohn's disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv+ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P<0.01) and ileal mucosa (268+/-47% of control; P<0.01), whereas inv bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size- and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of beta1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of beta1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.
2.
Hayeshi, Rose, et al.
(author)
Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories
2008
In: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987 .- 1879-0720. ; 35:5, s. 383-396
Journal article (peer-reviewed) abstract
Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.
3.
Pedersen, Jenny, et al.
(author)
Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2)
2008
In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 51:11, s. 3275-3287
Journal article (peer-reviewed) abstract
The chemical space of registered oral drugs was explored for inhibitors of the human multidrug-resistance associated protein 2 (MRP2, ABCC2), using a data set of 191 structurally diverse drugs and drug-like compounds. The data set included a new reference set of 75 compounds, for studies of hepatic drug interactions with transport proteins, CYP enzymes, and compounds associated with liver toxicity. The inhibition of MRP2-mediated transport of estradiol-17 beta-D-glucuronide was studied in inverted membrane vesicles from Sf9 cells overexpressing human MRP2. A total of 27 previously unknown MRP2 inhibitors were identified, and the results indicate an overlapping but narrower inhibitor space for MRP2 compared with the two other major ABC efflux transporters P-gp (ABCB1) and BCRP (ABCG2). In addition, 13 compounds were shown to stimulate the transport of cstradiol-17 beta-D-glucuronide. The experimental results were used to develop a computational model able to discriminate inhibitors from noninhibitors according to their molecular structure, resulting in a predictive power of 86% for the training set and 72% for the test set. The inhibitors were in general larger and more lipophilic and presented a higher aromaticity than the noninhibitors. The developed computational model is applicable in an early stage of the drug discovery process and is proposed as a tool for prediction of MRP2-mediated hepatic drug interactions and toxicity.
4.
Strand, Sabina P, et al.
(author)
Tailoring of chitosans for gene delivery : novel self-branched glycosylated chitosan oligomers with improved functional properties
2008
In: Biomacromolecules. - : American Chemical Society (ACS). - 1526-4602 .- 1525-7797. ; 9:11, s. 3268-76
Journal article (peer-reviewed) abstract
Chitosan is a promising biomaterial with an attractive safety profile; however, its application potential for gene delivery is hampered by poor compatibility at physiological pH values. Here we have tailored the molecular architecture of chitosan to improve the functional properties and gene transfer efficacy of chitosan oligomers and have developed self-branched glycosylated chitosan oligomer (SB-TCO) substituted with a trisaccharide containing N-acetylglucosamine, AAM. SB-TCO was prepared by controlled depolymerization of chitosan, followed by simultaneous branching and AAM substitution. The product was fully soluble at physiological pH and complexed plasmid DNA into polyplexes of high colloidal and physical stability. SB-TCO displayed high transfection efficacy in HEK293 cells, reaching transfection efficiencies of up to 70%, and large amounts of transgene were produced. Gene transfer efficacy was confirmed in HepG2 cells, where gene expression levels mediated by SB-TCO were up to 10 and 4 times higher than those obtained with unsubstituted and substituted linear oligomers, respectively. The rapid onset of transgene expression in both cell lines indicates efficient DNA release and transcription from SB-TCO polyplexes. In comparison with 22 kDa linear PEI-based transfection reagent used as the control, SB-TCO possessed higher gene transfer efficacy, significantly lower cytotoxicity, and improved serum compatibility.
