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In drug development, estimating fraction absorbed (F a) in man for permeability-limited compounds is important but challenging. To model F a of such compounds from apparent permeabilities (P app) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the P app across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The P app values of acamprosate and of several paracellular marker molecules were measured. These P app values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (P modeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate P app across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate P app, 1.56 ± 0.28 × 10 -7 cm·s -1, is similar to the P app of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10 -7 cm·s -1), lucifer yellow (1.80 ± 0.35 × 10 -7 cm·s -1), and fluorescein (2.10 ± 0.28 × 10 -7 cm·s -1), and lower than that of atenolol (7.32 ± 0.60 × 10 -7 cm·s -1 mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Q monolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the P app of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The P modeled for all the paracellular markers and acamprosate was dominated by the P para component and matched the experimentally obtained P app. Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B 0,+AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the P modeled estimated well P para, and the paracellular route appears to be the predominant mechanism for acamprosate P app across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.

Dextran sulfate sodium (DSS)-induced colitis is the most commonly used animal model for inflammatory bowel diseases. However, the precise molecular action of DSS, in particular its initial effect on the epithelial tissue permeability, is still poorly understood. In the present work, organ culture of mouse – and pig colon explants were performed for 1–2 h in the presence/absence of 2% DSS together with polar- and lipophilic fluorescent probes. Probe permeability was subsequently assessed by fluorescence microscopy. DSS rapidly increased paracellular permeability of 70-kDa dextran without otherwise affecting the overall epithelial integrity. FITC-conjugated DSS likewise permeated the epithelial barrier and strongly accumulated in nuclei of cells scattered in the lamina propria. By immunolabeling, plasma cells, T cells, macrophages, mast cells, and fibroblasts were identified as possible targets for DSS, indicating that accumulation of the polyanion in nuclei was not confined to a particular type of cell in the lamina propria. In contrast, colonocytes were rarely targeted by DSS, but as visualized by transmission electron microscopy, it induced the formation of vacuole-like structures in the intercellular space between adjacent epithelial cells. Nuclei of various cell types in the lamina propria, including both cells of the innate and adaptive immune system, are novel targets for a rapid action of DSS, and from previous in vitro studies, polyanions like DSS are known to disrupt nucleosomes by binding to the histones. We therefore propose that nuclear targeting is one way whereby DSS exerts its inflammatory action as a colitogen in animal models of inflammatory bowel diseases.

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly “from blood to gut” and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase protein involved in the host's response to endotoxin and mainly synthesized and secreted to the blood by the liver. But in addition, LBP is also made by extrahepatic cells, including the enterocyte-like cell line Caco-2. To study in closer detail the synthesis and storage of LBP in the intestinal mucosal epithelium, we performed an immunolocalization of LBP in mouse small intestine. By immunofluorescence microscopy, an antibody recognizing the 58-60 kDa protein of LBP distinctly labeled a small population of cells located deep into the crypts. This cell population was also positive for lysozyme and alpha-defensin 4, identifying Paneth cells as the main intestinal LBP-producing cells. By immunogold electron microscopy, intense labeling was observed in the secretory granules of these cells. We conclude that Paneth cells express LBP together with other proteins acting in the innate immune response of the gut, such as lysozyme, defensins and intelectin.

Absorption of dietary fat in the small intestine involves epithelial exposure to potentially harmful molecules such as bile salts and free fatty acids. We used organ culture of porcine jejunal explants incubated with a pre-digested mixture of fat (plant oil), bile and pancreatin to mimick the physiological process of dietary fat absorption, and short exposures to the fat mixture caused fat droplet accumulation within villus enterocytes. Lucifer yellow (LY), a fluorescent membrane-impermeable polar tracer was included to monitor epithelial integrity. Both in controls and during fat absorption LY penetrated the epithelium and accumulated in the basal lamina and the lamina propria. LY was also seen in the paracellular space, whereas villus enterocytes were generally only weakly labeled except for small amounts taken up by apical endocytosis. In the crypts, however, fat absorption induced cell permeabilization with LY accumulating in the cytosol and nucleus. Morphologically, both apical and basolateral membranes appeared intact, indicating that the leakiness was caused by minor lesions in the membrane. Albeit to a lesser extent, bile alone was capable of permeabilizing crypt cells, implying that the surfactant properties of bile salts are involved in the process. In addition to LY, crypt enterocytes also became permeable for albumin, ovalbumin and insulin. In conclusion, during fat absorption the permeability of the gut epithelium is increased mainly in the crypts. A possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.

The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross-linking galectins and intelectin, but little is known about the dynamic properties of this highly specialized membrane. Here, we probed the endocytic membrane trafficking from the brush border of organ-cultured pig intestinal mucosal explants by use of a fixable, lipophilic FM dye. The fluorescent dye readily incorporated into the brush border, and by 15 min faint but distinct punctae were detectable ∼1 μm beneath the brush border, indicative of a constitutive endocytosis. The punctae represented a subpopulation of early endosomes confined to the actomyosin-rich terminal web region, and their number and intensity increased by 1 h, but trafficking further into the enterocyte was not observed except in immature epithelial cells of the crypts. A powerful ligand for receptor-mediated endocytosis, cholera toxin B subunit, increased apical endocytosis and caused membrane trafficking to proceed to compartments localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell, contributes to the overall permeability barrier of the gut.

The current study assessed the effects of fixed-time reinforcement schedules on problem behavior of students with emotional-behavioral disorders in a clinical day-treatment classroom setting. Three elementary-aged students with a variety of emotional and behavioral problems participated in the study. Initial functional assessments indicated that social attention was the maintaining reinforcer for their verbally disruptive behavior. Baseline phases were alternated with phases in which attention was provided on fixed-time schedules in the context of an ABAB design. The results indicated that the provision of attention on fixed-time schedules substantially reduced the participants' rate of verbal disruptions. These decreases were maintained during initial thinning of the schedules. The results provide one of the first examples that such an intervention can be successfully implemented in a classroom setting.