Modulation Of Tight Junctions Research Articles

Inhibition of RhoA prevents Cryptococcus neoformans capsule glucuronoxylomannan-stimulated brain endothelial barrier disruption.

Cryptococcus neoformans (Cn) is an opportunistic fungus that causes severe central nervous system (CNS) disease in immunocompromised individuals. Brain parenchyma invasion requires fungal traversal of the blood-brain barrier. In this study, we describe that Cn alters the brain endothelium by activating small GTPase RhoA, causing reorganization of the actin cytoskeleton and tight junction modulation to regulate endothelial barrier permeability. We confirm that the main fungal capsule polysaccharide glucuronoxylomannan is responsible for these alterations. We reveal a therapeutic benefit of RhoA inhibition by CCG-1423 in vivo. RhoA inhibition prolonged survival and reduced fungal burden in a murine model of disseminated cryptococcosis, supporting the therapeutic potential targeting RhoA in the context of cryptococcal infection. We examine the complex virulence of Cn in establishing CNS disease, describing cellular components of the brain endothelium that may serve as molecular targets for future antifungal therapies to alleviate the burden of life-threatening cryptococcal CNS infection.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.

Cornea‐SELEX for aptamers targeting the surface of eyes and liposomal drug delivery

AbstractCornea is the major barrier to drug delivery to the eye, which results in low bioavailability and poor efficacy of topical eye treatment. In this work, we first select cornea‐binding aptamers using tissue‐SELEX on pig cornea. The top two abundant aptamers, Cornea‐S1 and Cornea‐S2, could bind to pig cornea, and their Kd values to human corneal epithelial cells (HCECs) were 361 and 174 nм, respectively. Aptamer‐functionalized liposomes loaded with cyclosporine A (CsA) were developed as a treatment for dry eye diseases. The Kd of Cornea‐S1‐ or Cornea‐S2‐functionalized liposomes reduces to 1.2 and 15.1 nм, respectively, due to polyvalent binding. In HCECs, Cornea‐S1 or Cornea‐S2 enhanced liposome uptake within 15 min and extended retention to 24 h. Aptamer CsA liposomes achieved similar anti‐inflammatory and tight junction modulation effects with ten times less CsA than a free drug. In a rabbit dry eye disease model, Cornea‐S1 CsA liposomes demonstrated equivalence in sustaining corneal integrity and tear break‐up time when compared to commercial CsA eye drops while utilizing a lower dosage of CsA. The aptamers obtained from cornea‐SELEX can serve as a general ligand for ocular drug delivery, suggesting a promising avenue for the treatment of various eye diseases and even other diseases.

Intestinal absorption of bioactive oligopeptides: paracellular transport and tight junction modulation.

Bioactive oligopeptides have gained increasing attention due to their diverse physiological functions, and these can be transported into the vasculature via transcellular and paracellular pathways. Among these, paracellular transport through the intercellular space is a passive diffusion process without energy consumption. It is currently the most frequently reported absorption route for food-derived bioactive oligopeptides. Previous work has demonstrated that paracellular pathways are mainly controlled by tight junctions, but the mechanism by which they regulate paracellular absorption of bioactive oligopeptides remains unclear. In this review, we summarized the composition of paracellular pathways across the intercellular space and elaborated on the paracellular transport mechanism of bioactive oligopeptides in terms of the interaction between oligopeptides and tight junction proteins, the protein expression level of tight junctions, the signaling pathways regulating intestinal permeability, and the properties of oligopeptides themselves. These findings contribute to a more profound understanding of the paracellular absorption of bioactive oligopeptides.

Structure-function analysis of tight junction-directed permeation enhancer PIP250

The intestinal paracellular route of absorption is modulated via tight junction (TJ) structures located at the apical neck of polarized intestinal epithelial cells to restrict solute movement through the intercellular space between them. Tight junctions open or close in response to changes in the phosphorylation status of light chain (MLC) at position Ser-19. This phosphorylation event is primarily controlled by MLC kinase (MLCK) and MLC phosphatase (MLCP), the latter being a holoenzyme that involves interaction between protein phosphatase 1 (PP1) and myosin targeting protein 1 (MYPT1). An entirely D-amino acid Permeant Inhibitor of Phosphatase (PIP) peptide sequence designed to disrupt PP1-MYPT1 interactions at the cytoplasmic surface of TJs, PIP250 (rrfkvktkkrk) localized at intracellular TJ structures, altered expression levels of specific TJ proteins, increased cellular phosphorylated MLC (pMLC) levels, binding to PP1, decreased epithelial barrier function, and significantly increased systemic uptake of the poorly absorbed antibiotic gentamicin in vivo. A series of PIP250 peptide analogues showed that positions phe3 and val5 were critical to its functional properties, with some providing opportunities to tune the dynamic actions of its TJ modulation properties. These data confirm the activity of PIP250 as a rationally designed oral permeation enhancer and validated key amino acids involved in its interaction with PP1 that define its overall actions; the magnitude and duration of these enhancing properties were associated with the MYPT1-mimetic properties of the PIP250 peptide analogues described.

Dark Chocolate Intake Positively Modulates Gut Permeability in Elite Football Athletes: A Randomized Controlled Study.

Gut barrier disruption can lead to enhanced intestinal permeability, which allows endotoxins, pathogens, and other proinflammatory substances to move through the intestinal barrier into circulation. Intense exercise over a prolonged period increases intestinal permeability, which can be further worsened by the increased production of reactive oxygen species (ROS) and pro-inflammatory cytokines. The aim of this study was to assess the degree of intestinal permeability in elite football players and to exploit the effect of cocoa polyphenols on intestinal permeability induced by intensive physical exercise. Biomarkers of intestinal permeability, such as circulating levels of zonulin, a modulator of tight junctions, occludin, a tight junction protein, and LPS translocation, were evaluated in 24 elite football players and 23 amateur athletes. Moreover, 24 elite football players were randomly assigned to either a dark chocolate (>85% cocoa) intake (n = 12) or a control group (n = 12) for 30 days in a randomized controlled trial. Biochemical analyses were performed at baseline and after 30 days of chocolate intake. Compared to amateur athletes, elite football players showed increased intestinal permeability as indicated by higher levels of zonulin, occludin, and LPS. After 30 days of dark chocolate intake, decreased intestinal permeability was found in elite athletes consuming dark chocolate. In the control group, no changes were observed. In vitro, polyphenol extracts significantly improved intestinal damage in the human intestinal mucosa cell line Caco-2. These results indicate that chronic supplementation with dark chocolate as a rich source of polyphenols positively modulates exercise-induced intestinal damage in elite football athletes.

Interlaboratory Variability in the Madin-Darby Canine Kidney Cell Proteome.

Madin-Darby canine kidney (MDCK) cells are widely used to study epithelial cell functionality. Their low endogenous drug transporter protein levels make them an amenable system to investigate transepithelial permeation and drug transporter protein activity after their transfection. MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratory variability in drug permeability assessments is observed. Consequently, in vitro-in vivo extrapolation (IVIVE) approaches using permeability and/or transporter activity data require calibration. A comprehensive proteomic quantification of 11 filter-grown parental or mock-transfected MDCK monolayers from 8 different pharmaceutical laboratories using the total protein approach (TPA) is provided. The TPA enables estimations of key morphometric parameters such as monolayer cellularity and volume. Overall, metabolic liability to xenobiotics is likely to be limited for MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporter linked to xenobiotic activity, while ABCC4 (MRP4) was the highest abundant ABC transporter. Our data supports existing findings that claudin-2 levels may be linked to tight junction modulation, thus impacting trans-epithelial resistance. This unique database provides data on more than 8000 protein copy numbers and concentrations, thus allowing an in-depth appraisal of the control monolayers used in each laboratory.

Recent Advances in Doxorubicin Formulation to Enhance Pharmacokinetics and Tumor Targeting.

Doxorubicin (DOX), a widely used drug in cancer chemotherapy, induces cell death via multiple intracellular interactions, generating reactive oxygen species and DNA-adducted configurations that induce apoptosis, topoisomerase II inhibition, and histone eviction. Despite its wide therapeutic efficacy in solid tumors, DOX often induces drug resistance and cardiotoxicity. It shows limited intestinal absorption because of low paracellular permeability and P-glycoprotein (P-gp)-mediated efflux. We reviewed various parenteral DOX formulations, such as liposomes, polymeric micelles, polymeric nanoparticles, and polymer-drug conjugates, under clinical use or trials to increase its therapeutic efficacy. To improve the bioavailability of DOX in intravenous and oral cancer treatment, studies have proposed a pH- or redox-sensitive and receptor-targeted system for overcoming DOX resistance and increasing therapeutic efficacy without causing DOX-induced toxicity. Multifunctional formulations of DOX with mucoadhesiveness and increased intestinal permeability through tight-junction modulation and P-gp inhibition have also been used as orally bioavailable DOX in the preclinical stage. The increasing trends of developing oral formulations from intravenous formulations, the application of mucoadhesive technology, permeation-enhancing technology, and pharmacokinetic modulation with functional excipients might facilitate the further development of oral DOX.

Evaluation of Serum Zonulin and Occludin Levels in Bipolar Disorder.

The etiology of bipolar disorder (BD) is still not fully understood. Little is currently known about the relationship between the interaction the gastrointestinal system and brain function and BD. Zonulin is the only known physiological modulator of tight junctions and is a biomarker for intestinal permeability (IP). Occludin is an integral transmembrane tight junction protein involved in the maintenance and assembly of such junctions. The current study aims to determine whether zonulin and occludin levels are altered in BD and whether they can serve as clinical biomarkers of disease. Forty-four patients with BD and 44 healthy controls were included in this study. The Young Mania Rating Scale (YMRS) was used to determine the severity of manic symptoms, while the Hamilton Depression Rating Scale (HDRS) was used to determine the severity of depressive symptoms, and the Brief Functioning Rating Scale (FAST) to assess functionality. Venous blood samples were taken from all participants and serum zonulin and occludin levels were measured. The mean serum zonulin and occludin levels of the patients were significantly higher compared to the healthy control group. There was no difference between manic, depressive, and euthymic patients in terms of zonulin and occludin levels. There was no correlation between the total number of attacks, duration of disease, YMRS, HDRS, FAST scores, and zonulin and occludin levels in the patient group. The groups were divided into three according to body mass index as normal, overweight, and obese. Zonulin and occludin levels increased as body mass index increased and were highest in the obese group. The study shows that zonulin and occludin levels in BD increase independently of the disease stage. Consideration of the role of IP in the pathogenesis of BD may be helpful in determining the appropriate treatment modality.

Abstract 2754: Transwell-grown monolayers derived from small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs

Abstract Although oral dosing is the most convenient route of drug administration, most anti-neoplastic drugs are administered intravenously due to poor bioavailability because of the epithelial barriers. A major organ involved in oral drug absorption is the small intestine where enterocytes are linked together by tight junctions (TJs). TJs regulate paracellular transport by creating a selectively permeable barrier to small molecules, ions, and water, thereby restricting the uptake of drugs. Transient modulation of the TJs is considered a potential strategy to improve drug delivery. Human small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. However, their geometry prevents access to the apical compartment of the epithelium (the outer, or luminal, surface being located within the organoid with the basal surface on the outside), making them unsuitable for the study of orally-administered drugs. Here we describe a method to generate transwell-grown cell monolayers derived from normal human small intestine, that allows direct access to the apical surface. We demonstrate that the format provides an in vivo-like cell composition, comprising of all the intestinal cellular types, including stem cells, Paneth cells, goblet cells, enteroendocrine cells and enterocytes, and respond to treatment with ‘tool drug’ TJ modulators such as sodium caprate, chitosan, EGTA and various cytokines. Briefly, for the generation of transwell-grown cell monolayers, SI organoids were dissociated into single cells and plated in matrix-coated transwell inserts. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements. Treatments with TJ modulators were applied after TEER reached a plateau (8-10 days after plating), and effects on TJs were evaluated 24-72 hours after treatment by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. In conclusion, our models provide an innovative in vitro solution to study the effect of TJ modulators on increasing paracellular transport of orally-administered, anti-neoplastic drugs. Furthermore, as anti-neoplastic drugs can also have toxic effects on the intestinal epithelium by affecting TJs, thereby creating a leaky barrier, the model is useful to predict such effects and stratify drug candidates prior to in vivo studies. The model also provides a system to test the restorative potential of molecules on barrier integrity that could be used in combination or after the administration of anti-neoplastic drugs. Citation Format: Valentina Ubertini, Frida Ponthan, Aude-Marine Bonavita, James Wilson. Transwell-grown monolayers derived from small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2754.

Understanding the mechanisms of ultrasound-targeted microbubble cavitation-mediated blood brain barrier opening

Ultrasound-targeted microbubble cavitation (UTMC) transiently opens the blood brain barrier (BBB). We previously determined that UTMC induces BBB hyperpermeability through an influx of calcium. As activation of RhoA is a calcium-dependent pathway that causes cytoskeletal reorganization, leading to the breakdown of tight junctions, we tested the hypothesis that UTMC-induced activation of RhoA leads to BBB hyperpermeability. We utilized a transwell model with brain endothelial cells and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 250 kPa, 10 μs pulse duration, 10 ms pulse interval) was applied in the presence of lipid microbubbles for 20 s. BBB permeability was assessed using dextran flux and transendothelial electrical resistance (TEER) measured across the membrane. Integrity of tight junctions was evaluated by staining for ZO-1. UTMC reduced TEER (p < 0.05), confirming reduced barrier integrity. One hour after UTMC, there was a significant decrease in ZO-1 mean pixel intensity (p < 0.05). Treatment of cells with Rho inhibitor II (Y16) significantly reduced UTMC-induced dextran flux across the BBB (p < 0.05) and UTMC-induced decrease in TEER. In our co-culture model of the BBB, UTMC induces hyperpermeability through modulation of tight junctions, at least in part through a RhoA-dependent mechanism.

Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: Focus on tight junction modulation.

Oxidative stress and inflammation lead by dietary oxidised lipids, as oxysterols, have been linked to the loss of intestinal barrier integrity, a crucial event in the initiation and progression of intestinal disorders. In the last decade, probiotic lactobacilli have emerged as an interesting tool to improve intestinal health, thanks to their antioxidant and anti-inflammatory properties. The aim of the present study was to evaluate the ability of two commercial probiotic strains of lactobacilli (Lactiplantibacillus plantarum 299v® (DMS 9843) and Lacticaseibacillus casei DG® (CNCMI-1572)), both as live bacteria and intracellular content, to attenuate the oxysterols-induced alteration of intestinal epithelial Caco-2cell monolayer permeability. Our investigation was focused on the modulation of tight junctions (TJs) proteins, occludin, ZO-1 and JAM-A, in relation to redox-sensitive MAPK p38 activation. Obtained results provided evidence on the ability of the two probiotics to counteract the alteration of monolayer permeability and loss of TJs proteins, at least in part, through the modulation of p38 pathway. The protective action was exerted by live bacteria, whose adhesion to Caco-2cells was not altered by oxysterols, and bacterial intracellular components equally able to interact with the signaling pathway.

Intestinal permeability assessment using lactulose and mannitol in celiac disease.

Alterations in intestinal permeability can lead to increased uptake of luminal antigens, which has been linked to several intestinal diseases, such as inflammatory bowel diseases, celiac disease, and irritable bowel syndrome, but also to extra-intestinal diseases. Promising therapies that target intestinal permeability could be developed, for instance tight junction modulators. Consequently, permeability assays are increasingly being used as treatment endpoints in clinical studies. Therefore, reliable, reproducible, and feasible methods for measuring intestinal permeability in the clinical setting are necessary. Currently, a variety of in vivo, ex vivo, and in vitro tests are available, some of which are only applicable to basic research. Despite the various options available to measure gut permeability, their use in clinical setting is still limited because of their heterogeneity. Here, we describe a clinical method to measure intestinal permeability using two non-metabolizable sugars.

Genetically edited T-cell membrane coated AIEgen nanoparticles effectively prevents glioblastoma recurrence

Glioblastoma stem cells (GSCs) are subpopulations of tumor-initiating cells responsible for glioblastoma (GBM) tumorigenesis and recurrence. Dual inhibition of vascular endothelium and GSCs is still a challenge due to their different pathological features. Here we present a combined all-in-control strategy to realize a local photothermal therapy (PTT). We designed T-cell-mimic nanoparticles with aggregation-induced emission (AIE) characteristics by coating the genetically engineered T cell membrane (CM) onto AIE nanoparticles (CM@AIE NPs). The CM shell was designed against CD133 and epidermal growth factor receptor (EGFR) which provides the possibility to target both GBM cells and GSCs for cancer therapy. CM@AIE NPs can serve as the tight junction (TJ) modulators to trigger an intracellular signaling cascade, causing TJ disruption and actin cytoskeleton reorganization to allow CM@AIE NPs to cross the blood-brain barrier (BBB) silently. The 980 nm excitation-triggered PTT can completely inhibit tumorigenesis and recurrence. The combination of CM-coating nanotechnology and genetic editing technique can inspire further development of synergetic techniques for preventing GBM recurrence.

Molecular weight-dependent hyaluronic acid permeability and tight junction modulation in human buccal TR146 cell monolayers

The oral route is considered an attractive method for drug delivery, as it avoids the hepatic and intestinal first-pass metabolism processes. Hyaluronic acid (HA) beneficial effects to the human body include anti-aging and wound healing but its effects on oral barrier integrity and mechanical function have not yet been investigated. In this study, we analyzed oral barrier integrity and the paracellular pathway of HA transportation in TR146 cell monolayers during and after permeation and using low molecular weight HA (LMW-HA, <100 kDa) and high molecular weight HA (HMW-HA, >500 kDa). Cytotoxicity assays in TR146 cells revealed that neither LMW-HA or HMW-HA altered cell viability at concentrations <0.5 % during 24 h of treatment. HA-treated TR146 cell monolayers showed enhanced oral barrier integrity and reduced apparent permeability of fluorescein. Moreover, HA significantly increased tight junction (TJ)-related genes expression, including ZO-2, marvelD3, cingulin, claudin-1, claudin-3, and claudin-4 expression. Overall, the results of the present study indicate that HA can permeate across the oral barrier and enhance oral barrier function via the upregulated expression of TJ-related genes.

Pine Pollen Polysaccharides' and Sulfated Polysaccharides' Effects on UC Mice through Modulation of Cell Tight Junctions and RIPK3-Dependent Necroptosis Pathways.

The purpose of this study is to explore the effects of pine pollen polysaccharides and sulfated polysaccharides on mice with ulcerative colitis and whether they could protect mice from inflammation by regulating the tight junctions of colonic epithelial cells and regulating the RIPK3-dependent necroptosis pathways. Pine pollen polysaccharides were prepared by water boiling and ethanol precipitation. After deproteinedization with trichloroacetic acid, the UV spectrum showed that there were no proteins. One polysaccharide component (PPM60-III) was made by gel filtration chromatography, and then sulfated polysaccharide (SPPM60-III) was derived using the chlorosulfonic acid-pyridine method. After treatment with PPM60-III and SPPM60-III, the body weight of mice with ulcerative colitis induced by dextran sodium sulfate increased, the DAI score decreased, the levels of pro-inflammatory factors and inflammation-related enzymes decreased, and the level of anti-inflammatory factors increased. In addition, after treatment, the expressions levels of tight junction proteins increased, the expressions levels of key proteins of programmed necroptosis decreased, while the level of Caspase-8 increased. The results indicated that pine pollen polysaccharides and sulfated polysaccharides have a certain therapeutic effect on UC mice, and the therapeutic effect may be achieved by regulating the tight junction of colonic epithelial cells and regulating the RIPK3-dependent necroptosis pathways.

Impact of peptide permeation enhancer on tight junctions opening cellular mechanisms

The myristoylated pentapeptide, L-R5, contains an amino acid sequence of the zeta inhibitory peptide (ZIP) portion (pseudosubstrate) of protein kinase C zeta (PKC ζ). As PKC ζ is involved in the modulation of epithelial tight junctions (TJs) through the phosphorylation of TJ proteins, L-R5 was suggested to interact with the enzyme resulting in the enhancement of paracellular permeability. This study shows that L-R5 does not bind to the enzyme but interacts directly with TJ proteins. We show here that the binding of PKC ζ to occludin and its successive phosphorylation is prevented by L-R5, which leads to TJ disruption and enhanced epithelial permeability. Although L-R5 did not show any in vitro cytotoxicity, a proteomics study revealed that L-R5 interferes with other regulatory pathways, e.g., apoptosis and immune response. We suggest that structural modification of the peptide may increase the specificity TJ protein-peptide interaction.

Abstract 374: Small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs

Abstract Although oral dosing is the most convenient route of drug administration, most anti-neoplastic drugs are administered intravenously due to poor bioavailability because of the epithelial barriers. A major organ involved in oral drug absorption is the small intestine where enterocytes are linked together by tight junctions (TJs). TJs regulate paracellular transport by creating a selectively-permeable barrier to small molecules, ions and water, thereby restricting the uptake of drugs. Transient modulation of the TJs is considered a potential strategy to improve drug delivery. Human small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. However, their geometry prevents access to the apical compartment of the epithelium (the outer, or luminal, surface being located within the organoid with the basal surface on the outside), making them unsuitable for the study of orally-administered drugs. Here we describe methods to generate transwell cell monolayers and inverted or ‘apical-out’ organoids, both derived from normal human small intestine, that allow direct access to the apical surface. We demonstrate that both formats provide an in vivo-like cell composition, comprising of all the intestinal cellular types, including stem cells, Paneth cells, goblet cells, enteroendocrine cells and enterocytes, and respond to treatment with ‘tool drug’ TJ modulators such as sodium caprate, chitosan, EGTA and various cytokines. Briefly, for the generation of transwell-grown cell monolayers, SI organoids were dissociated into single cells and plated in matrix-coated transwell inserts. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements. Treatments with TJ modulators were applied after TEER reached a plateau and effects on TJs were evaluated by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. For the generation of apical-out organoids, SI organoids were mechanically broken into fragments and seeded in low adhesion plates until the organoids switched polarity. Treatments with TJ modulators were applied and effects on TJs were evaluated by immunofluorescence. In conclusion, our models provide an innovative in vitro solution to study the effect of TJ modulators on increasing paracellular transport of orally-administered, anti-neoplastic drugs. Furthermore, as anti-neoplastic drugs can also have toxic effects on the intestinal epithelium by affecting TJs, thereby creating a leaky barrier, the models are useful to predict such effects and stratify drug candidates prior to in vivo studies. Citation Format: Valentina Ubertini, Stephanie French, James Wilson, Frida Ponthan. Small intestinal organoids to study the effects of tight junction modulators that may improve the absorption of orally-administered, anti-neoplastic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 374.

Structure–activity relationship of a peptide permeation enhancer

ABSTRACT The pentapeptide L-R5 has previously been shown to transiently increase the permeability of nasal epithelial cell layers in vitro, allowing paracellular transport of molecules of up to 4 kDa. Protein kinase C zeta (PKC ζ), a member of a family of serine/threonine kinases was shown to be involved in tight junction modulation induced by L-R5. We show here that the ability of L-R5 to modulate tight junctions is comparable to other permeability enhancers such as bilobalide, latrunculin A or C10. Interaction of the peptide with the target protein occurs via electrostatic interaction, with the presence of positive charges being essential for its functionality. L-R5 is myristoylated to allow quick cell entry and onset of activity. While no epithelial cytotoxicity was detected, the hydrophobic myristoyl rest was shown to cause haemolysis. Taken together, these data show that a structural optimization of L-R5 may be possible, both from a toxicological and an efficacy point of view.

Structure characterization of soybean peptides and their protective activity against intestinal inflammation

Soybean peptides serve as functional foods with impressive health benefits. The structure characteristics of peptides are highly related to the health benefits. The structure–activity relationship and mechanism underlined are important scientific questions in this field. To answer these questions, soybean peptides were produced by combinatory enzymatic hydrolysis in this work. Fifty-two peptide sequences were identified by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The anti-inflammatory activities of these peptides were investigated by using a lipopolysaccharide (LPS)-induced inflammation cell model. Soybean peptides could significantly promote cell proliferation. Additionally, soybean peptides could alleviate LPS-induced inflammation by reducing the production and expression of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Moreover, soybean peptides could promote the mRNA expression of proteins related to inflammation inhibition (IL-10) and tight junction modulation. The structure–activity relationship was addressed. The results documented the potential of soybean peptides as functional foods.