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Usually, duodenal barriers are investigated using intestinal cell lines like Caco-2, which in contrast to native tissue are limited in cell-type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation. Growing three-dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid-derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco-2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco-2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco-2. Transcriptomic data showed that Caco-2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco-2 cells differ mostly in transcellular properties, ODMs reflect trans- and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue-specific investigations., (© 2022 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)

Although functional and structural models for paracellular channels formed by claudins have been reported, mechanisms regulating charge and size selectivity of these channels are unknown in detail. Here, claudin-15 and claudin-10b cation channels showing high-sequence similarity but differing channel properties were analyzed. Mutants of pore-lining residues were expressed in MDCK-C7 cells. In claudin-15, proposed ion interaction sites (D55 and E64) conserved between both claudins were neutralized. D55N and E64Q substitutions decreased ion permeabilities, and D55N/E64Q had partly additive effects. D55N increased cation dehydration capability and decreased pore diameter. Additionally, residues differing between claudin-15 and -10b close to pore center were analyzed. Claudin-10b-mimicking W63K affected neither assembly nor function of claudin-15 channels. In contrast, in claudin-10b, corresponding (claudin-15b-mimicking) K64W and K64M substitutions disturbed integration into tight junction and slightly altered relative permeabilities for differently sized monovalent cations. Removal of claudin-10b-specific negative charge (D36A substitution) was without effect. The data suggest that a common tetra-aspartate ring (D55/D56) in pore center of claudin-15/-10b channels directly attracts cations, while E64/D65 may be at least partly shielded by W63/K64. Charge at position W63/K64 affects assembly and properties for claudin-10b but not for claudin-15 channels. Our findings add to the mechanistic understanding of the determinants of paracellular cation permeability., (© 2022 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)

Both nerve injury and complex regional pain syndrome (CRPS) can result in chronic pain. In traumatic neuropathy, the blood nerve barrier (BNB) shielding the nerve is impaired-partly due to dysregulated microRNAs (miRNAs). Upregulation of microRNA-21-5p (miR-21) has previously been documented in neuropathic pain, predominantly due to its proinflammatory features. However, little is known about other functions. Here, we characterized miR-21 in neuropathic pain and its impact on the BNB in a human-murine back translational approach. MiR-21 expression was elevated in plasma of patients with CRPS as well as in nerves of mice after transient and persistent nerve injury. Mice presented with BNB leakage, as well as loss of claudin-1 in both injured and spared nerves. Moreover, the putative miR-21 target RECK was decreased and downstream Mmp9 upregulated, as was Tgfb. In vitro experiments in human epithelial cells confirmed a downregulation of CLDN1 by miR-21 mimics via inhibition of the RECK/MMP9 pathway but not TGFB. Perineurial miR-21 mimic application in mice elicited mechanical hypersensitivity, while local inhibition of miR-21 after nerve injury reversed it. In summary, the data support a novel role for miR-21, independent of prior inflammation, in elicitation of pain and impairment of the BNB via RECK/MMP9., (© 2022 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)

The iron-binding glycoprotein lactoferrin (LF) is naturally present in human breast milk. Several studies suggest that LF contributes to infant health and development owing to a variety of protective effects, including antimicrobial and anti-inflammatory features. Therefore, we aimed to elucidate its protective properties on intestinal epithelial barrier dysfunction induced by infection or inflammation using the human epithelial cell culture models HT-29/B6 and T84. During barrier perturbation induced by the proinflammatory cytokine tumor necrosis factor α (TNF-α), bovine LF restored tight junction (TJ) morphometry and inhibited TNF-α-induced epithelial apoptosis. This resulted in an attenuation of the TNF-α-induced decrease in transepithelial resistance (TER) and increases in permeability of fluorescein and FITC-dextran (4 kDa) and was as effective as the apoptosis inhibitor Q-VD-Oph. The enteropathogenic bacterium Yersinia enterocolitica is a frequent cause of diarrhea in early childhood. This involves focal changes in TJ protein expression and localization. LF diminished the Y. enterocolitica-induced drop in TER in the present in vitro model, which was paralleled by an inhibition of the Yersinia-induced reduction of claudin-8 expression via c-Jun kinase signaling. In conclusion, LF exerts protective effects against inflammation- or infection-induced barrier dysfunction in human intestinal cell lines, supporting its relevance for healthy infant development., (© 2017 New York Academy of Sciences.)

Tricellulin is a tight junction protein localized to tricellular contacts in many epithelial tissues, where it is required for full barrier control. Here, we present crystal structures of the tricellulin C-terminal coiled-coil domain, revealing a potential dimeric arrangement. By combining structural, biochemical, functional, and mutation analyses, we gain insight into the mode of tricellulin oligomerization and suggest a model where dimerization of its cytoplasmic C-terminus may play an auxiliary role in stabilizing homophilic and potentially also heterophilic cis-interactions within tight junctions., (© 2017 New York Academy of Sciences.)

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a β-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery., (© 2017 New York Academy of Sciences.)

Infections by Campylobacter species are the most common foodborne zoonotic disease worldwide. Campylobacter jejuni and C. coli are isolated most frequently from human stool samples, but severe infections by C. fetus (Cf), which can cause gastroenteritis, septicemia, and abortion, are also found. This study aims at the characterization of pathological changes in Cf infection using an intestinal epithelial cell model. The Cf-induced epithelial barrier defects appeared earlier than those of avian Campylobacter species like C. jejuni/C. coli. Two-path impedance spectroscopy (2PI) distinguished transcellular and paracellular resistance contributions to the overall epithelial barrier impairment. Both transcellular and paracellular resistance of Cf-infected HT-29/B6 monolayers were reduced. The latter was attributed to activation of active anion secretion. Western blot analysis showed no decrease in tight junction (TJ) protein expression (claudin-1, -2, -3, and -4) but showed redistribution of claudin-1 off the TJ domain. In addition, Cf induced epithelial cell death, cell detachment, and lesions (focal leaks), as the result of which macromolecule flux (10-kDa dextran) was increased in Cf-invaded cell monolayers. In conclusion, barrier dysfunction from Cf infection was due to TJ protein redistribution, cell death induction, and leak formation, resulting in bacterial translocation, ion leak flux, and antigen uptake (leaky gut)., (© 2017 New York Academy of Sciences.)

The tricellular tight junction (tTJ) is a potential weak point of the paracellular barrier. For solving the proportional contribution of the tTJ, ion conductances and macromolecule permeabilities were analyzed in cell lines of different leakiness. MDCK II, Caco-2, and HT-29/B6 cells were subjected to two-path impedance spectroscopy and morphological analyses in order to calculate the contribution of the tTJ to paracellular and total ion conductivity. The contribution to macromolecule permeability was evaluated by tricellulin overexpression or knockdown. Tricellulin-dependent macromolecule passage was comparably regulated in leaky and tight epithelia, but relative and absolute ion permeabilities of the tTJs were different. Assuming a minimal (50 pS) and maximal (146 pS) conductivity per single tTJ, the possible range of contribution of the tTJ to paracellular ion conductance amounted to only 0.3-1.1% in the leaky cell line MDCK II, but 3-25% in the moderately tight cell line Caco-2, and not less than 29% in the tight cell line HT-29/B6. In these cells, this resulted in a contribution to total epithelial conductance of 9-32%. In conclusion, in leaky epithelia the bicellular TJ accounts for nearly the entire paracellular ion conductance, whereas in tight epithelia the low bicellular TJ conductance has large impact on the tTJ., (© 2017 New York Academy of Sciences.)

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., (© 2012 New York Academy of Sciences.)

Pore-forming properties of claudins (Cld) are likely defined by residues of their first extracellular loop (ECL1). Detailed mechanisms are unclear. MDCK cells overexpressing FLAG-Cld-1 wild-type and mutants were characterized by transepithelial resistance (TER) and ion permeability measurements. Replacing ECL1 residues of sealing Cld-1 by corresponding Cld-2 residues we aimed to identify new determinants responsible for sealing and/or pore formation. We found that E48K and S53E substitutions in human Cld-1 strongly reduced TER and increased permeability for Na(+) and Cl(-) . In contrast, K65D, D68S, and other single substitutions showed no significant change of TER and permeability for Na(+) and Cl(-) . Double substitution S53E/K65D did not change TER and ion permeability, whereas S53E/D68S decreased TER, albeit weaker than S53E. Ratio of permeabilities for Na(+) and Cl(-) revealed no clear charge specificity of the pore induced by S53E or S53E/D68S in Cld-1, suggesting that primarily S53 and potentially D68 in Cld-1 are involved in sealing of the paracellular cleft and that charge-unselective pores may be induced by substituting S53E., (© 2012 New York Academy of Sciences.)

In epithelia and endothelia, overall resistance (TER) is determined by all ion-conductive structures, such as membrane channels, tight junctions, and the intercellular space, whereas the epithelial capacitance is due to the hydrophobic phase of the plasma membrane. Impedance means alternating current resistance and, in contrast to ohmic resistance, takes into account that, e.g., capacitors become increasingly conductive with increasing frequency. Impedance spectroscopy uses the association of the capacitance with the transcellular pathway to distinguish between this capacitive pathway and purely conductive components (tight junctions, subepithelium). In detail, one-path impedance spectroscopy distinguishes the resistance of the epithelium from the resistance of subepithelial tissues. Beyond that, two-path impedance spectroscopy allows for the separation of paracellular resistance (governed by tight junctional properties) from transcellular resistance (determined by conductive structures residing in the cell membranes). The present paper reviews the basic principles of these techniques, some historic milestones, as well as recent developments in epithelial physiology., (© 2012 New York Academy of Sciences.)

Tight junctions form the paracellular barrier for ions and uncharged solutes not only in "tight" but also in "leaky" epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin-1 and many others, strengthen the barrier, while a few claudins, such as claudin-2 or -10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin-2 represents the prototype of a paracellular, channel-forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin-5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin-8, another barrier builder, was demonstrated to be regulated by Na(+) uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na(+) absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na(+) by upregulating claudin-8.

During the past decade, claudins have been established as major determinants of paracellular permeablilty in epithelia. In the kidney, each nephron segment expresses a distinct pattern of claudins. Cells of the thick ascending limb of Henle's loop (TAL), which is characterized by high paracellular cation permeability, co-express an unusually large number of different claudins: claudin-10, -16, and -19 and, depending on the species, also claudin-3, -4, -8, and/or -11. The function of most of these claudins has been investigated in vitro. We present a summary of their function with special emphasis on claudin-16 and -19. Mutations in the corresponding human genes lead to severely impaired renal Ca(2+) and Mg(2+) handling. To date, 42 different claudin-16 mutations and three claudin-19 mutations have been reported. These mutations prevent the claudins from reaching the surface membrane, decrease membrane residence time, or render them functionless. In spite of the clear clinical symptoms such as hypomagnesemia, hypercalciuria, nephrocalcinosis, and renal insufficiency, mechanisms that link claudin-16 and -19 to these symptoms are still unknown. Depending on the cell type used in overexpression studies, claudin-16 appears to cause a mild increase in paracellular Mg(2+)-permeability or a pronounced increase in Na(+) permeability. Claudin-19 selectively decreases Cl(-) permeability, thus synergistically increasing relative cation permeability, or indiscriminately decreases paracellular permeability. In the light of these results it is hypothesized that the renal Mg(2+)/Ca(2+) waste may not be solely due to reduced resorption in the TAL but at least in part to paracellular back-leak of Mg(2+)/Ca(2+) into the tubular lumen of the distal convoluted tubule.

High-resolution analysis of epithelial barrier function adds substantial information to that provided by conventional transepithelial electrical resistance (TER) measurements. This chapter describes three high-resolution techniques. First, two variants of impedance spectroscopy are delineated. One-path impedance spectroscopy discriminates vertically between serial pathways, namely resistances of the epithelial cell layer and of subepithelial tissues. As a typical application, measurements on human sigmoid colon biopsies from patients suffering from Crohn's disease are reported. Two-path impedance spectroscopy allows to discriminate between trans- and paracellular resistance, and the general principle of this technique is outlined. Second, the conductance scanning technique is presented, which discriminates horizontally between optically distinct parallel pathways over a wide range of spatial resolutions. Using this technique, it was shown that occludin--in contrast to the then prevailing opinion--is not irreplaceable to barrier function. Third, three-dimensional confocal fluorescence imaging for depicting transepithelial transport processes is introduced. Using this method the transepithelial translocation of bacteria which generate focal leaks was discovered.