Selective Uptake Into Inflamed Human Intestinal Tissue and Immune Cell Targeting by Wormlike Polymer Micelles.

Inflammatory bowel disease (IBD) has become a globally prevalent chronic disease with no causal therapeutic options. Targeted drug delivery systems with selectivity for inflamed areas in the gastrointestinal tract promise to reduce severe drug-related side effects. By creating three distinct nanostructures (vesicles, spherical, and wormlike micelles) from the same amphiphilic block copolymer poly(butyl acrylate)-block-poly(ethylene oxide) (PBA-b-PEO), the effect of nanoparticle shape on human mucosal penetration is systematically identified. An Ussing chamber technique is established to perform the ex vivo experiments on human colonic biopsies, demonstrating that the shape of polymeric nanostructures represents a rarely addressed key to tissue selectivity required for efficient IBD treatment. Wormlike micelles specifically enter inflamed mucosa from patients with IBD, but no significant uptake is observed in healthy tissue. Spheres (≈25 nm) and vesicles (≈120 nm) enter either both normal and inflamed tissue types or do not penetrate any tissue. According to quantitative image analysis, the wormlike nanoparticles localize mainly within immune cells, facilitating specific targeting, which is crucial for further increasing the efficacy of IBD treatment. These findings therefore demonstrate the untapped potential of wormlike nanoparticles not only to selectively target the inflamed human mucosa, but also to target key pro-inflammatory cells.

Colonic Epithelial Permeability to Ions Is Restored after Vedolizumab Treatment and May Predict Clinical Response in Inflammatory Bowel Disease Patients.

Vedolizumab (VDZ) is used for treating inflammatory bowel disease (IBD) patients. A study investigating colonic epithelial barrier function ex vivo following VDZ is lacking. This work aims to evaluate ex vivo the colonic epithelial barrier function in IBD patients at baseline and during VDZ treatment, and to investigate the relationships between barrier function and clinical parameters. Colonic specimens were obtained from 23 IBD patients before, and at 24 and 52 weeks after VDZ treatment, and from 26 healthy volunteers (HV). Transepithelial electrical resistance (TEER, permeability to ions) and paracellular permeability were measured in Ussing chambers. IBD patients showed increased epithelial permeability to ions (TEER, 13.80 ± 1.04 Ω × cm2 vs. HV 20.70 ± 1.52 Ω × cm2, p < 0.001) without changes in paracellular permeability of a 4 kDa probe. VDZ increased TEER (18.09 ± 1.44 Ω × cm2, p < 0.001) after 52 weeks. A clinical response was observed in 58% and 25% of patients at week 24, and in 62% and 50% at week 52, in ulcerative colitis and Crohn's disease, respectively. Clinical and endoscopic scores were strongly associated with TEER. TEER < 14.65 Ω × cm2 predicted response to VDZ (OR 11; CI 2-59). VDZ reduces the increased permeability to ions observed in the colonic epithelium of IBD patients before treatment, in parallel to a clinical, histological (inflammatory infiltrate), and endoscopic improvement. A low TEER predicts clinical response to VDZ therapy.

CFTR Modulators Rescue the Activity of CFTR in Colonoids Expressing the Complex Allele p.[R74W;V201M;D1270N]/dele22_24.

An Italian, 46-year-old female patient carrying the complex allele p.[R74W;V201M;D1270N] in trans with CFTR dele22_24 was diagnosed at the Cystic Fibrosis (CF) Center of Verona as being affected by CF-pancreatic sufficient (CF-PS) in 2021. The variant V201M has unknown significance, while both of the other variants of this complex allele have variable clinical consequences, according to the CFTR2 database, with reported clinical benefits for treatment with ivacaftor + tezacaftor and ivacaftor + tezacaftor + elexacaftor in patients carrying the R74W-D1270N complex allele, which are currently approved (in USA, not yet in Italy). She was previously followed up by pneumologists in northern Italy because of frequent bronchitis, hemoptysis, recurrent rhinitis, Pseudomonas aeruginosa lung colonization, bronchiectasis/atelectasis, bronchial arterial embolization and moderately compromised lung function (FEV1: 62%). Following a sweat test with borderline results, she was referred to the Verona CF Center where she presented abnormal values in both optical beta-adrenergic sweat tests and intestinal current measurement (ICM). These results were consistent with a diagnosis of CF. CFTR function analyses were also performed in vitro by forskolin-induced swelling (FIS) assay and short-circuit currents (Isc) in the monolayers of the rectal organoids. Both of these assays showed significantly increased CFTR activity following treatment with the CFTR modulators. Western-blot analysis revealed increased fully glycosylated CFTR protein after treatment with correctors, in line with the functional analysis. Interestingly, tezacaftor, together with elexacaftor, rescued the total organoid area under steady-state conditions, even in the absence of the CFTR agonist forskolin. In conclusion, in ex vivo and in vitro assays, we measured a residual function that was significantly enhanced by in vitro incubation with CFTR modulators, especially by ivacaftor + tezacaftor + elexacaftor, suggesting this combination as a potentially optimal treatment for this case.

Role of CB1 receptors in the acute regulation of small intestinal permeability: effects of high-fat diet.

The endocannabinoid system of the gastrointestinal tract is involved in the control of intestinal barrier function. Whether the cannabinoid 1 (CB1) receptor is expressed on the intestinal epithelium and acutely regulates barrier function has not been determined. Here, we tested the hypothesis that ligands of the CB1 receptor acutely modulate small intestinal permeability and that this is associated with altered distribution of tight junction proteins. We examined the acute effects of CB1 receptor ligands on small intestinal permeability both in chow-fed and 2-wk high-fat diet (HFD)-fed mice using Ussing chambers. We assessed the distribution of CB1 receptor and tight junction proteins using immunofluorescence and the expression of CB1 receptor using PCR. A low level of CB1 expression was found on the intestinal epithelium. CB1 receptor was highly expressed on enteric nerves in the lamina propria. Neither the CB1/CB2 agonist CP55,940 nor the CB1 neutral antagonist AM6545 altered the flux of 4kDa FITC dextran (FD4) across the jejunum or ileum of chow-fed mice. Remarkably, both CP55,940 and AM6545 reduced FD4 flux across the jejunum and ileum in HFD-fed mice that have elevated baseline intestinal permeability. These effects were absent in CB1 knockout mice. CP55,940 reduced the expression of claudin-2, whereas AM6545 had little effect on claudin-2 expression. Neither ligand altered the expression of ZO-1. Our data suggest that CB1 receptor on the intestinal epithelium regulates tight junction protein expression and restores barrier function when it is increased following exposure to a HFD for 2 wk.NEW & NOTEWORTHY The endocannabinoid system of the gastrointestinal tract regulates homeostasis by acting as brake on motility and secretion. Here we show that when exposed to a high fat diet, intestinal permeability is increased and activation of the CB1 receptor on the intestinal epithelium restores barrier function. This work further highlights the role of the endocannabinoid system in regulating intestinal homeostasis when it is perturbed.

PCSK9 is not secreted from mature differentiated intestinal cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.

Changes in Intestinal Permeability Ex Vivo and Immune Cell Activation by Three Commonly Used Emulsifiers.

Food additives such as emulsifiers are used in increasing quantities in the food industry. The aim of this study was to compare three different emulsifiers (polysorbate 80 (P80), carboxymethyl cellulose (CMC), and ß-lactoglobulin (ß-lac) with regards to their effect on the stimulation of immune cells and intestinal permeability. The immune stimulatory effects were studied in the myeloid cell line MUTZ-3-cells, while the change in intestinal permeability was studied in the Caco-2 cell line and ex vivo in the Ussing chamber system using small intestinal fragments from rats. The tested concentrations of the emulsifiers ranged from 0.02% up to 1%, which are concentrations commonly used in the food industry. The results showed that P80 affected both the myeloid cells and the intestinal permeability more than CMC (p < 0.05) and ß-lac (p < 0.05) at the highest concentration. CMC was found to neither affect the permeability in the intestine nor the MUTZ-3 cells, while ß-lac changed the permeability in the total part of the small intestine in rats. These findings indicate that P80 might be more cytotoxic compared to the other two emulsifiers.

An Assessment of the Permeation Enhancer, 1-phenyl-piperazine (PPZ), on Paracellular Flux Across Rat Intestinal Mucosae in Ussing Chambers.

PURPOSE: 1-phenyl piperazine (PPZ) emerged from a Caco-2 monolayer screen as having high enhancement potential due to a capacity to increase permeation without significant toxicity. Our aim was to further explore the efficacy and toxicity of PPZ in rat ileal and colonic mucosae in order to assess its true translation potential. METHODS: Intestinal mucosae were mounted in Ussing chambers and apparent permeability coefficient (Papp) values of [(14)C]-mannitol and FITC-dextran 4 kDa (FD-4) and transepithelial electrical resistance (TEER) values were obtained following apical addition of PPZ (0.6-60 mM). Exposed issues were assessed for toxicity by histopathology and lactate dehydrogenase (LDH) release. Mucosal recovery after exposure was also assessed using TEER readings. RESULTS: PPZ reversibly increased the Papp of both agents across rat ileal and distal colonic mucosae in concentration-dependent fashion, accompanied by TEER reduction, with acceptable levels of tissue damage. The complex mechanism of tight junction opening was part mediated by myosin light chain kinase, stimulation of transepithelial electrogenic chloride secretion, and involved activation of 5-HT4 receptors. CONCLUSIONS: PPZ is an efficacious and benign intestinal permeation enhancer in tissue mucosae. However, its active pharmacology suggest that potential for further development in an oral formulation for poorly permeable molecules will be difficult.

Esophageal Epithelial Resistance and Lower Esophageal Sphincter Muscle Contraction Increase in a Chronic Diabetic Rabbit Model.

BACKGROUND: Esophageal motility disorders and possibly gastroesophageal reflux disease are common in patients with diabetes mellitus. AIMS: We aimed to investigate both the electrophysiological characteristics of the esophageal epithelium and the contractility of the lower esophageal sphincter (LES) muscle in alloxane-induced diabetic rabbits. METHODS: Electrophysiological properties were measured using an Ussing chamber method. An acid-pepsin model was employed with pH 1.7 or weakly acidic (pH 4) Ringer and/or pepsin. Smooth muscle strips of the LES were mounted in an isolated organ bath. Contractile responses to an electrical field stimulation and cumulative concentrations of acetylcholine were recorded. Contractility of the muscle strips were tested in the presence of Rho-kinase inhibitor (Y-27632) and nonspecific nitric oxide inhibitor (L-NAME). RESULTS: The resistance of diabetic tissue perfused in the pH 1.7 Ringer decreased 17 %; pepsin addition decreased it by 49 %. The same concentrations caused a more distinct loss of resistance in the control tissues (22 and 76 %, p < 0.05). The perfusion of tissues in increased concentrations of luminal and serosal glucose did not change the tissue resistance and voltage. Diabetes significantly increased both the electrical field stimulation and acetylcholine-induced contractions in the LES muscle strips (p < 0.01). Incubation with Y-27632 significantly decreased the acetylcholine-induced contractions in a concentration-dependent manner (p < 0.01). CONCLUSIONS: The acid-pepsin model in the diabetic rabbit esophageal tissue had less injury compared with the control. The diabetic rabbit LES muscle had higher contractility, possibly because of the activation of the Rho-Rhokinase pathway. Our results show that in a chronic diabetic rabbit model the esophagus resists reflux by activating mechanisms of mucosal defense and increasing the contractility of the LES.

Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

Modulation of intestinal calcium and phosphate transport in young goats fed a nitrogen- and/or calcium-reduced diet.

Feeding ruminants a reduced N diet is a common approach to reduce N output based on rumino-hepatic circulation. However, a reduction in N intake caused massive changes in Ca and inorganic phosphate (Pi) homoeostasis in goats. Although a single dietary Ca reduction stimulated intestinal Ca absorption in a calcitriol-dependent manner, a concomitant reduction of Ca and N supply led to a decrease in calcitriol, and therefore a modulation of intestinal Ca and Pi absorption. The aim of this study was to examine the potential effects of dietary N or Ca reduction separately on intestinal Ca and Pi transport in young goats. Animals were allocated to a control, N-reduced, Ca-reduced or combined N- and Ca-reduced diet for about 6-8 weeks, whereby N content was reduced by 25 % compared with recommendations. In Ussing chamber experiments, intestinal Ca flux rates significantly decreased in goats fed a reduced N diet, whereas Pi flux rates were unaffected. In contrast, a dietary Ca reduction stimulated Ca flux rates and decreased Pi flux rates. The combined dietary N and Ca reduction withdrew the stimulating effect of dietary Ca reduction on Ca flux rates. The expression of Ca-transporting proteins decreased with a reduced N diet too, whereas Pi-transporting proteins were unaffected. In conclusion, a dietary N reduction decreased intestinal Ca transport by diminishing Ca-transporting proteins, which became clear during simultaneous N and Ca reduction. Therefore, N supply in young ruminant nutrition is of special concern for intestinal Ca transport.

Formulation approaches to improving the delivery of an antiviral drug with activity against seasonal flu.

The main objective of the present study was to develop formulations of noscapine hydrochloride hydrate with enhanced solubility and bioavailability using co-solvent- and cyclodextrin-based approaches. Different combinations of co-solvents, which were selected on the basis of high-throughput solubility screening, were subjected to in vitro intestinal drug permeability studies conducted with Ussing chambers. Vitamin E tocopherol polyethylene glycol succinate and propylene glycol based co-solvent formulations provided the maximum permeability coefficient for the drug. Inclusion complexes of the drug were prepared using hydroxypropyl-ß-cyclodextrin and sulphobutylether cyclodextrins. Pharmacokinetic studies were carried out in male Sprague-Dawley rats for the selected formulations. The relative bioavailabilities of the drug with the co-solvent- and cyclodextrin-based formulations were found to be similar.

The effect of Campylobacter jejuni and Campylobacter coli colonization on the gut morphology, functional integrity, and microbiota composition of female turkeys.

BACKGROUND: Campylobacter (C.) species are the most common bacterial cause of foodborne diarrhea in humans. Despite colonization, most animals do not show clinical signs, making recognition of affected flocks and disruption of the infection chain before slaughter challenging. Turkeys are often cocolonized with C. jejuni and C. coli. To understand the pathogen-host-interaction in the context of two different Campylobacter species, we compared the colonization patterns and quantities in mono- and co-colonized female commercial turkeys. In three repeated experiments we investigated the impact on gut morphology, functional integrity, and microbiota composition as parameters of gut health at seven, 14, and 28 days post-inoculation. RESULTS: Despite successful Campylobacter colonization, clinical signs or pathological lesions were not observed. C. coli persistently colonized the distal intestinal tract and at a higher load compared to C. jejuni. Both strains were isolated from livers and spleens, occurring more frequently in C. jejuni- and co-inoculated turkeys. Especially in C. jejuni-positive animals, translocation was accompanied by local heterophil infiltration, villus blunting, and shallower crypts. Increased permeability and lower electrogenic ion transport of the cecal mucosa were also observed. A lower relative abundance of Clostridia UCG-014, Lachnospiraceae, and Lactobacillaceae was noted in all inoculated groups compared to controls. CONCLUSIONS: In sum, C. jejuni affects gut health and may interfere with productivity in turkeys. Despite a higher cecal load, the impact of C. coli on investigated parameters was less pronounced. Interestingly, gut morphology and functional integrity were also less affected in co-inoculated animals while the C. jejuni load decreased over time, suggesting C. coli may outcompete C. jejuni. Since a microbiota shift was observed in all inoculated groups, future Campylobacter intervention strategies may involve stabilization of the gut microbiota, making it more resilient to Campylobacter colonization in the first place.

Osteopontin (OPN)/Secreted Phosphoprotein 1 (SPP1) Binds Integrins to Activate Transport of Ions Across the Porcine Placenta.

BACKGROUND: Fetal-placental development depends on a continuous and efficient supply of nutrients from maternal blood that are acquired by exchange through the placenta. However, the placenta is a low permeability barrier, and effective transport of substances depends on specific transport mechanisms. Active transport requires that ions or nutrients be moved against an electrical and/or concentration gradient. In pigs, active transport of ions occurs across the chorioallantois placenta to produce an electrochemical gradient that changes throughout gestation. The aim of this study was to utilize Ussing chambers to detect regulation of ion transport across the porcine chorioallantois by a factor(s) within the uterine-placental environment of pigs. METHODS: For the measurement of transchorioallantoic voltage potential as an index of ion transport across the placenta, pieces of chorioallantoic tissue from Day 60 of gestation were mounted into the cassettes of Ussing chambers, and treatments were added to the mucasal side of the tissue. Treatments included: (1) media incubated with Day 60 chorioallantois (placenta-conditioned media); (2) osteopontin/secreted phosphoprotein 1 (OPN/SPP1) purified from cow's milk; (3) placenta-conditioned media from which OPN/SPP1 was removed; and (4) recombinant rat OPN with an intact RGD integrin binding sequence or a mutated RAD sequence. Ouabain was added to both sides of the chamber. Immunofluorescence was utilized to localize beta 3 integrin, aquaporin 8 and OPN/SPP1 in porcine placental tissues, and OPN/SPP1 within porcine lung, kidney and small intestine. RESULTS: Day 60 chorioallantoic membranes had greater transepithelial voltage in the presence of porcine placenta-conditioned media, indicating that a molecule(s) released from the placenta increased ion transport across the placenta. OPN/SPP1 purified from cow's milk increased ion transport across the placenta. When OPN/SPP1 was removed from placenta-conditioned media, ion transport across the placenta did not increase. Recombinant rat OPN/SPP1 with a mutated RGD sequence that does not bind integrins (RAD) did not increase ion transport across the placenta. Ouabain, an inhibiter of the sodium-potassium ion pump, ablated ion transport across the placenta. CONCLUSIONS: The present study documents a novel pericellular matrix role for OPN/SPP1 to bind integrins and increase ion transport across the porcine chorioallantoic placenta.

Experimental evidence that polystyrene nanoplastics cross the intestinal barrier of European seabass.

Plastic pollution in marine ecosystems constitutes an important threat to marine life. For vertebrates, macro/microplastics can obstruct and/or transit into the airways and digestive tract whereas nanoplastics (NPs; < 1000 nm) have been observed in non-digestive tissues such as the liver and brain. Whether NPs cross the intestinal epithelium to gain access to the blood and internal organs remains controversial, however. Here, we show directly NP translocation across the intestinal barrier of a fish, the European seabass, Dicentrarchus labrax, ex vivo. The luminal side of median and distal segments of intestine were exposed to fluorescent polystyrene NPs (PS-NPs) of 50 nm diameter. PS-NPs that translocated to the serosal side were then detected quantitatively by fluorimetry, and qualitatively by scanning electron microscopy (SEM) and pyrolysis coupled to gas chromatography and high-resolution mass spectrometry (Py-GC-HRMS). Fluorescence intensity on the serosal side increased 15-90 min after PS-NP addition into the luminal side, suggesting that PS-NPs crossed the intestinal barrier; this was confirmed by both SEM and Py-GC-HRMS. This study thus evidenced conclusively that NPs beads translocate across the intestinal epithelium in this marine vertebrate.

Osmoregulatory Plasticity of Juvenile Greater Amberjack (Seriola dumerili) to Environmental Salinity.

Osmotic costs in teleosts are highly variable, reaching up to 50% of energy expenditure in some. In several species, environmental salinities close to the isosmotic point (~15 psu) minimize energy demand for osmoregulation while enhancing growth. The present study aimed to characterize the physiological status related to osmoregulation in early juveniles of the greater amberjack, Seriola dumerili, acclimated to three salinities (15, 22, and 36 psu). Our results indicate that plasma metabolic substrates were enhanced at the lower salinities, whereas hepatic carbohydrate and energetic lipid substrates decreased. Moreover, osmoregulatory parameters, such as osmolality, muscle water content, gill and intestine Na+-K+-ATPase activities, suggested a great osmoregulatory capacity in this species. Remarkably, electrophysiological parameters, such as short-circuit current (Isc) and transepithelial electric resistance (TER), were enhanced significantly at the posterior intestine. Concomitantly, Isc and TER anterior-to-posterior intestine differences were intensified with increasing environmental salinity. Furthermore, the expression of several adeno-hypophyseal genes was assessed. Expression of prl showed an inverse linear relationship with increasing environmental salinity, while gh mRNA enhanced significantly in the 22 psu-acclimated groups. Overall, these results could explain the better growth observed in S. dumerili juveniles kept at salinities close to isosmotic rather than in seawater.

Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine.

An increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls.

Claudin-18 Loss Alters Transcellular Chloride Flux but not Tight Junction Ion Selectivity in Gastric Epithelial Cells.

BACKGROUND & AIMS: Tight junctions form a barrier to the paracellular passage of luminal antigens. Although most tight junction proteins reside within the apical tight junction complex, claudin-18 localizes mainly to the basolateral membrane where its contribution to paracellular ion transport is undefined. Claudin-18 loss in mice results in gastric neoplasia development and tumorigenesis that may or may not be due to tight junction dysfunction. The aim here was to investigate paracellular permeability defects in stomach mucosa from claudin-18 knockout (Cldn18-KO) mice. METHODS: Stomach tissue from wild-type, heterozygous, or Cldn18-KO mice were stripped of the external muscle layer and mounted in Ussing chambers. Transepithelial resistance, dextran 4 kDa flux, and potential difference (PD) were calculated from the chambered tissues after identifying differences in tissue histopathology that were used to normalize these measurements. Marker expression for claudins and ion transporters were investigated by transcriptomic and immunostaining analysis. RESULTS: No paracellular permeability defects were evident in stomach mucosa from Cldn18-KO mice. RNAseq identified changes in 4 claudins from Cldn18-KO mice, particularly the up-regulation of claudin-2. Although claudin-2 localized to tight junctions in cells at the base of gastric glands, its presence did not contribute overall to mucosal permeability. Stomach tissue from Cldn18-KO mice also had no PD versus a lumen-negative PD in tissues from wild-type mice. This difference resulted from changes in transcellular Cl- permeability with the down-regulation of Cl- loading and Cl- secreting anion transporters. CONCLUSIONS: Our findings suggest that Cldn18-KO has no effect on tight junction permeability in the stomach from adult mice but rather affects anion permeability. The phenotype in these mice may thus be secondary to transcellular anion transporter expression/function in the absence of claudin-18.

A review of reductionist methods in fish gastrointestinal tract physiology.

A holistic understanding of a physiological system can be accomplished through the use of multiple methods. Our current understanding of the fish gastrointestinal tract (GIT) and its role in both nutrient handling and osmoregulation is the result of the examination of the GIT using multiple reductionist methods. This review summarizes the following methods: in vivo mass balance studies, and in vitro gut sac preparations, intestinal perfusions, and Ussing chambers. From Homer Smith's initial findings of marine fish intestinal osmoregulation in the 1930s through to today's research, we discuss the methods, their advantages and pitfalls, and ultimately how they have each contributed to our understanding of fish GIT physiology. Although in vivo studies provide substantial information on the intact animal, segment specific functions of the GIT cannot be easily elucidated. Instead, in vitro gut sac preparations, intestinal perfusions, or Ussing chamber experiments can provide considerable information on the function of a specific tissue and permit the delineation of specific transport pathways through the use of pharmacological agents; however, integrative inputs (e.g. hormonal and neuronal) are removed and only a fraction of the organ system can be studied. We conclude with two case studies, i) divalent cation transport in teleosts and ii) nitrogen handling in the elasmobranch GIT, to highlight how the use of multiple reductionist methods contributes to a greater understanding of the organ system as a whole.

Investigations of Piperazine Derivatives as Intestinal Permeation Enhancers in Isolated Rat Intestinal Tissue Mucosae.

A limiting factor for oral delivery of macromolecules is low intestinal epithelial permeability. 1-Phenylpiperazine (PPZ), 1-(4-methylphenyl) piperazine (1-4-MPPZ) and 1-methyl-4-phenylpiperazine (1-M-4-PPZ) have emerged as potential permeation enhancers (PEs) from a screen carried out by others in Caco-2 monolayers. Here, their efficacy, mechanism of action and potential for epithelial toxicity were further examined in Caco-2 cells and isolated rat intestinal mucosae. Using high-content analysis, PPZ and 1-4-MPPZ decreased mitochondrial membrane potential and increased plasma membrane potential in Caco-2 cells to a greater extent than 1-M-4-PPZ. The Papp of the paracellular marker, [14C]-mannitol, and of the peptide, [3H]-octreotide, was measured across rat colonic mucosae following apical addition of the three piperazines. PPZ and 1-4-MPPZ induced a concentration-dependent decrease in transepithelial electrical resistance (TEER) and an increase in the Papp of [14C]-mannitol without causing histological damage. 1-M-4-PPZ was without effect. The piperazines caused the Krebs-Henseleit buffer pH to become alkaline, which partially attenuated the increase in Papp of [14C]-mannitol caused by PPZ and 1-4-MPPZ. Only addition of 1-4-MPPZ increased the Papp of [3H]-octreotide. Pre-incubation of mucosae with two 5-HT4 receptor antagonists, a loop diuretic and a myosin light chain kinase inhibitor, reduced the permeation enhancement capacity of PPZ and 1-4-MPP for [14C]-mannitol. 1-4-MPPZ holds most promise as a PE, but intestinal physiology may also be impacted due to multiple mechanisms of action.

Intestinal permeability enhancement of benzopyran HP1-loaded nanoemulsions.

The benzopyran HP1, a compound isolated from Hypericum polyanthemum, has demonstrated significant opioid-mediated antinociceptive activity after its oral administration. Despite the pharmacological potential, the poor aqueous solubility limits the oral absorption of this compound. For this reason, HP1 has been alternatively incorporated in lipid-based drug delivery systems. Given that nanoemulsions showed higher antinociceptive action than the free compound in a previous report, in this study, the main objective was to investigate the intestinal transport mechanisms of this system. The Ussing chamber model and rat jejunum were selected for this purpose. The apparent permeability coefficient of HP1 increased approximately 5.3 times after its incorporation in nanoemulsions. Considering that the absorptive transport of HP1 was significantly higher than the secretory transport, the participation of active transporters was suggested. The amount of HP1 in the acceptor chamber was reduced during permeability assays performed at 4 °C, supporting the hypothesis that active transporters are involved in the intestinal transport of this compound. The amount of free fatty acids released from nanoemulsion was approximately 60% after 90 min, demonstrating that part of this system is disassembled before absorption. Nanoemulsion constituents would be able to form new structures with biological constituents, leading to a rapid solubilization of HP1. A mucoadhesion rate of 50% was achieved by nanoemulsion after 30 min, which would also contribute to explain the higher absorption of this system. The particle size of the nanoemulsion is also compatible with endocytosis-mediated transport. Taken together, these results suggest that nanoemulsions containing HP1 could be efficiently delivered to humans considering that different absorption mechanisms are exploited.