In-Fah M Lee

Charité Universitätsmedizin Berlin, Berlín, Berlin, Germany

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Publications (5)30.89 Total impact

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    ABSTRACT: Background Total parenteral nutrition (TPN), a necessary treatment for patients who cannot receive enteral nutrition, is associated with infectious complications due in part to a loss of intestinal epithelial barrier function (EBF). Using a mouse model of TPN, with enteral nutrient deprivation, we previously demonstrated an increase in mucosal interferon-γ and tumor necrosis factor-α; these cytokine changes are a major mediator driving a reduction in epithelial tight junction (TJ) protein expression. However, the exact ultrastructural changes to the intestinal epithelial barrier have not been previously described. Aim We hypothesized that TPN dependence results in ultrastructural changes in the intestinal epithelial TJ meshwork. Methods C57BL/6 mice underwent internal jugular venous cannulation and were given enteral nutrition or TPN with enteral nutrient deprivation for 7 days. Freeze-fracture electron microscopy was performed on ileal tissue to characterize changes in TJ ultrastructure. EBF was measured using transepithelial resistance and tracer permeability, while TJ expression was measured via Western immunoblotting and immunofluorescence staining. Results While strand density, linearity, and appearance were unchanged, TPN dependence led to a mean reduction in one horizontal strand out of the TJ compact meshwork to a more basal region, resulting in a reduction in meshwork depth. These findings were correlated with the loss of TJ localization of claudin-4 and tricellulin, reduced expression of claudin-5 and claudin-8, and reduced ex vivo EBF. Conclusion Tight junction ultrastructural changes may contribute to reduced EBF in the setting of TPN dependence.
    No preview · Article · Dec 2015 · Digestive Diseases and Sciences
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    ABSTRACT: Infection with Yersinia enterocolitica causes acute diarrhea in early childhood. A mouse infection model presents new findings on pathological mechanisms in the colon. Symptoms involve diarrhea with watery feces and weight loss that have their functional correlates in decreased transepithelial electrical resistance and increased fluorescein permeability. Y. enterocolitica was present within the murine mucosa of both ileum and colon. Here, the bacterial insult was of focal nature and led to changes in tight junction protein expression and architecture. These findings are in concordance with observations from former cell culture studies and suggest a leak flux mechanism of diarrhea.
    Full-text · Article · Nov 2015 · The Journal of Infectious Diseases
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    ABSTRACT: α-Haemolysin (HlyA) influences host cell ionic homeostasis and causes concentration-dependent cell lysis. As a consequence, HlyA-producing Escherichia coli is capable of inducing 'focal leaks' in colon epithelia, through which bacteria and antigens translocate. This study addressed the role of HlyA as a virulence factor in the pathogenesis of colitis according to the 'leaky gut' concept. To study the action of HlyA in the colon, we performed oral administration of HlyA-expressing E coli-536 and its isogenic α-haemolysin-deficient mutant (HDM) in three mouse models: wild type, interleukin-10 knockout mice (IL-10(-/-)) and monoassociated mice. Electrophysiological properties of the colonised colon were characterised in Ussing experiments. Inflammation scores were evaluated and focal leaks in the colon were assessed by confocal laser-scanning microscopy. HlyA quantity in human colon biopsies was measured by quantitative PCR. All three experimental mouse models infected with HlyA-producing E coli-536 showed an increase in focal leak area compared with HDM. This was associated with a decrease in transepithelial electrical resistance and an increase in macromolecule uptake. As a consequence, inflammatory activity index was increased to a higher degree in inflammation-prone mice. Mucosal samples from human colon were E coli HlyA-positive in 19 of 22 patients with ulcerative colitis, 9 of 9 patients with Crohn's disease and 9 of 12 healthy controls. Moreover, focal leaks were found together with 10-fold increased levels of HlyA in active ulcerative colitis. E coli HlyA impairs intestinal barrier function via focal leak induction in the epithelium, thereby intensifying antigen uptake and triggering intestinal inflammation in vulnerable mouse models. Therefore, HlyA-expressing E coli strains should be considered as potential cofactors in the pathogenesis of intestinal inflammation.
    Full-text · Article · Feb 2014 · Gut
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    ABSTRACT: Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line's initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.
    Full-text · Article · Nov 2013 · PLoS ONE
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    ABSTRACT: Claudins are the main determinants of barrier properties of the tight junction. Many claudins have been shown to act by tightening the paracellular pathway, but several function as paracellular channels. While some depend on the endogenous claudin background of the analyzed cell line, for other claudins, a distinct charge-selectivity has been shown. This paper portrays cation-selective (claudin-2, claudin-10b, claudin-15) and anion-selective (claudin-10a, claudin-17) claudins and claudins with debatable channel properties (claudin-4, claudin-7, claudin-16). It also describes molecular properties determining the observed charge-selectivity and pore properties in general. In leaky tissues, they widely determine overall transport characteristics by providing paracellular ion-selective pathways. In small intestine, claudin-2 and claudin-15 replace each other in the developing gut. In kidney proximal tubules, claudin-2, claudin-10, and claudin-17 allow for paracellular reabsorption of sodium, chloride, and water.
    No preview · Article · Jun 2012 · Annals of the New York Academy of Sciences