Intestinal Epithelial Cell Layer Research Articles

Direct effects of alcohol on gut-epithelial barrier: Unraveling the disruption of physical and chemical barrier of the gut-epithelial barrier that compromises the host-microbiota interface upon alcohol exposure.

The development of alcohol-associated diseases is multifactorial, mechanism of which involves metabolic alteration, dysregulated immune response, and a perturbed intestinal host-environment interface. Emerging evidence has pinpointed the critical role of the intestinal host-microbiota interaction in alcohol-induced injuries, suggesting its contribution to disease initiation and development. To maintain homeostasis in the gut, the intestinal mucosa serves as the first-line defense against exogenous factors in the gastrointestinal tract, including dietary contents and the commensal microbiota. The gut-epithelial barrier comprises a physical barrier lined with a single layer of intestinal epithelial cells and a chemical barrier with mucus trapping host regulatory factors and gut commensal bacteria. In this article, we review recent studies pertaining to the disrupted gut-epithelial barrier upon alcohol exposure and examine how alcohol and its metabolism can affect the regulatory ability of intestinal epithelium.

Host-gut microbiota derived secondary metabolite mediated regulation of Wnt/β-catenin pathway: a potential therapeutic axis in IBD and CRC.

The intestinal tract encompasses one of the largest mucosal surfaces with a well-structured layer of intestinal epithelial cells supported by a network of underlying lamina propria immune cells maintaining barrier integrity. The commensal microflora in this environment is a major contributor to such functional outcomes due to its prominent role in the production of secondary metabolites. Of the several known metabolites of gut microbial origin, such as Short Chain Fatty Acids (SCFAs), amino acid derivatives, etc., secondary bile acids (BAs) are also shown to exhibit pleiotropic effects maintaining gut homeostasis in addition to their canonical role in dietary lipid digestion. However, dysbiosis in the intestine causes an imbalance in microbial diversity, resulting in alterations in the functionally effective concentration of these secondary metabolites, including BAs. This often leads to aberrant activation of the underlying lamina propria immune cells and associated signaling pathways, causing intestinal inflammation. Sustained activation of these signaling pathways drives unregulated cell proliferation and, when coupled with genotoxic stress, promotes tumorigenesis. Here, we aimed to discuss the role of secondary metabolites along with BAs in maintaining immune-gut homeostasis and regulation of inflammation-driven tumorigenesis with emphasis on the classical Wnt/β-Catenin signaling pathway in colon cancer.

Beneficial Effect of Sustainable Magnetic Treatment of Tap Water Investigations with Cultivated Intestinal Epithelial Cells

(Electro)magnetic fields have been used as a tap water treatment since more than 60 years to prevent water pipe walls from the unwanted deposition of calcium carbonate. In the meantime a lot of research has been done on the effects of magnetic or electromagnetic treatment of water. Most of them deal with physiochemical aspects and only a few report on biological effects on prokaryotes or cultured mammalian cells. Intestinal health plays a key role in respect to systemic health and the intestinal epithelial barrier comes directly into contact with drinking water, the effect of magnetic tap water treatment on the integrity of the intestinal epithelial barrier was investigated by measurement of the transepithelial electrical resistance (TEER). Moreover, the sensitivity of the intestinal epithelial barrier against oxidative stress was investigated. Since the regeneration process of intestinal epithelial cells is essential for the reconstitution and maintenance of the barrier integrity after injury or traumatization in vivo, this aspect was also examined with water ± magnetic treatment. The animal cell line IPEC-J2 was used for the investigations. Local tap water (= initial source water) was obtained before and after magnetic treatment with water core magnets which show a very weak field strength with high magnetic gradients as a special characteristic. TEER increased by 28.5 ± 6.8% after incubation with the tap water with magnetic treatment when compared to the initial tap water (mean value ± standard deviation; p ≤ 0.01 by the two-tailed Wilcoxon-Mann-Whitney rank sum test). Measurement of TEER after oxidative stress showed no marked difference between both tap water samples ± magnetic treatment. In both experimental series there was a significant reduction in TEER by more than 90 % representing a leaky intestinal epithelial cell layer due to oxidative stress. Despite this fact the number of cells in the initial tap water sample was largely decreased whereas the cell density in the treated water samples was much higher. The magnetic treatment of the tap water improved the regeneration of the intestinal epithelial cells by 17.4 ± 7.2% (mean value ± standard deviation; p ≤ 0.05 by the two-tailed Wilcoxon-Mann-Whitney rank sum test) when compared with the initial tap water. The results of the study demonstrate that the water core magnets as used here are able to influence tap water and, subsequently, induce beneficial cellular effects on cultured intestinal epithelial cells. Thus, the tap water with magnetic treatment might be able to promote and maintain intestinal and systemic health in vivo.

Understanding inborn errors of immunity: A lens into the pathophysiology of monogenic inflammatory bowel disease.

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract, including Crohn’s disease, ulcerative colitis and inflammatory bowel disease-undefined (IBD-U). IBD are understood to be multifactorial, involving genetic, immune, microbial and environmental factors. Advances in next generation sequencing facilitated the growing identification of over 80 monogenic causes of IBD, many of which overlap with Inborn errors of immunity (IEI); Approximately a third of currently identified IEI result in gastrointestinal manifestations, many of which are inflammatory in nature, such as IBD. Indeed, the gastrointestinal tract represents an opportune system to study IEI as it consists of the largest mass of lymphoid tissue in the body and employs a thin layer of intestinal epithelial cells as the critical barrier between the intestinal lumen and the host. In this mini-review, a selection of pertinent IEI resulting in monogenic IBD is described involving disorders in the intestinal epithelial barrier, phagocytosis, T and B cell defects, as well as those impairing central and peripheral tolerance. The contribution of disrupted gut-microbiota-host interactions in disturbing intestinal homeostasis among patients with intestinal disease is also discussed. The molecular mechanisms driving pathogenesis are reviewed along with the personalized therapeutic interventions and investigational avenues this growing knowledge has enabled.

Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate

Intestinal epithelial cells and the intestinal microbiota are in a mutualistic relationship that is dependent on communication. This communication is multifaceted, but one aspect is communication through compounds produced by the microbiota such as the short-chain fatty acids (SCFAs) butyrate, propionate and acetate. Studying the effects of SCFAs and especially butyrate in intestinal epithelial cell lines like Caco-2 cells has been proven problematic. In contrast to the in vivo intestinal epithelium, Caco-2 cells do not use butyrate as an energy source, leading to a build-up of butyrate. Therefore, we used human induced pluripotent stem cell derived intestinal epithelial cells, grown as a cell layer, to study the effects of butyrate, propionate and acetate on whole genome gene expression in the cells. For this, cells were exposed to concentrations of 1 and 10 mM of the individual short-chain fatty acids for 24 h. Unique gene expression profiles were observed for each of the SCFAs in a concentration-dependent manner. Evaluation on both an individual gene level and pathway level showed that butyrate induced the biggest effects followed by propionate and then acetate. Several known effects of SCFAs on intestinal cells were confirmed, such as effects on metabolism and immune responses. The changes in metabolic pathways in the intestinal epithelial cell layers in this study demonstrate that there is a switch in energy homeostasis, this is likely associated with the use of SCFAs as an energy source by the induced pluripotent stem cell derived intestinal epithelial cells similar to in vivo intestinal tissues where butyrate is an important energy source.

Postnatal intestinal mucosa and gut microbial composition develop hand in hand: A mouse study

BackgroundDuring the early postnatal life, gut microbiota development experiences dynamic changes in their structural and functional composition. The postnatal period is the critical window to develop a host defense mechanism. The maturation of intestinal mucosal barrier integrity is one of the essential defense mechanisms to prevent the entry of pathogens. However, the co-development of intestinal microbial colonization, formation of barrier integrity, and intestinal epithelial cell layer is not entirely understood. MethodsWe studied the gut microbial composition and diversity using 16S rRNA marker gene-based sequencing in mice to understand postnatal age-dependent association kinetics between gut microbial and intestinal development. Next, we assessed the intestinal development by in vivo gut permeability assay, mRNA gene expression of different tight junction proteins and intestinal epithelial cell markers, goblet cells population, villus length, and cecal IgA quantification. ResultsOur results showed a significant shift in gut microbial structural and functional composition from postnatal day 14 onwards with early life Proteobacteria abundance. Relative abundance of Verrucomicrobia was maximum at postnatal day 14 and showed a gradual decrease over time. We also observed an age-dependent biphasic pattern in barrier integrity improvement and differentiation of intestinal epithelial cells (IECs). A significant improvement in barrier integrity between days 1 and 7 showed the host factor contribution, while that beyond day 14 revealed an association with changes in microbiota composition. Our temporal correlation analysis associated Bacteroidetes phylum with the mucosal barrier formation during postnatal development. ConclusionsThe present study revealed the importance and interplay of host factors and the microbiome in gut development and intestinal mucosal homeostasis.

Bacterial translocation in colorectal cancer patients

Bacterial translocation is the penetration of intestinal bacteria through the intestinal mucosa into usually sterile tissues and internal organs. Commensal bacteria, despite their presence in the intestine in extremely large numbers, rarely cause local or systemic inflammation, because the unicellular epithelial layer of the intestinal mucosa prevents the migration of these bacteria from the intestine. For years, researchers have wondered how a single layer of intestinal epithelial cells can prevent microorganisms from entering the systemic circulation. Today, the phenomenon of bacterial translocation is considered as one of the main mechanisms of endotoxemia and systemic inflammatory response syndrome in various pathologies, including colorectal cancer and acute bowel obstruction. This narrative review is devoted to the search for factors promoting to bacterial translocation from the intestine in colorectal cancer and acute malignant bowel obstruction.

Dynamic Imaging of IEL-IEC Co-Cultures Allows for Quantification of CD103-Dependent T Cell Migration.

Intraepithelial lymphocytes (IEL) are widely distributed within the small intestinal epithelial cell (IEC) layer and represent one of the largest T cell pools of the body. While implicated in the pathogenesis of intestinal inflammation, detailed insight especially into the cellular cross-talk between IELs and IECs is largely missing in part due to lacking methodologies to monitor this interaction. To overcome this shortcoming, we employed and validated a murine IEL-IEC (organoids) ex vivo co-culture model system. Using livecell imaging we established a protocol to visualize and quantify the spatio-temporal migratory behavior of IELs within organoids over time. Applying this methodology, we found that IELs lacking CD103 (i.e., integrin alpha E, ITGAE) surface expression usually functioning as a retention receptor for IELs through binding to E-cadherin (CD324) expressing IECs displayed aberrant mobility and migration patterns. Specifically, CD103 deficiency affected the ability of IELs to migrate and reduced their speed during crawling within organoids. In summary, we report a new technology to monitor and quantitatively assess especially migratory characteristics of IELs communicating with IEC ex vivo. This approach is hence readily applicable to study the effects of targeted therapeutic interventions on IEL-IEC cross-talk.

Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers

The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (STm) within minutes elicited focal contractions with inward movements of up to ∼1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.

Endoplasmic Reticulum Stress and Intestinal Inflammation: A Perilous Union.

The intestinal tract encompasses the largest mucosal surface fortified with a fine layer of intestinal epithelial cells along with highly sophisticated network of the lamina propria immune cells that are indispensable to sustain gut homeostasis. However, it can be challenging to uphold homeostasis when these cells in the intestine are perpetually exposed to insults of both endogenous and exogenous origin. The complex networking and dynamic microenvironment in the intestine demand highly functional cells ultimately burdening the endoplasmic reticulum (ER) leading to ER stress. Unresolved ER stress is one of the primary contributors to the pathogenesis of inflammatory bowel diseases (IBD). Studies also suggest that ER stress can be the primary cause of inflammation and/or the consequence of inflammation. Therefore, understanding the patterns of expression of ER stress regulators and deciphering the intricate interplay between ER stress and inflammatory pathways in intestinal epithelial cells in association with lamina propria immune cells contribute toward the development of novel therapies to tackle IBD. This review provides imperative insights into the molecular markers involved in the pathogenesis of IBD by potentiating ER stress and inflammation and briefly describes the potential pharmacological intervention strategies to mitigate ER stress and IBD. In addition, genetic mutations in the biomarkers contributing to abnormalities in the ER stress signaling pathways further emphasizes the relevance of biomarkers in potential treatment for IBD.

Glutamate metabolism in a human intestinal epithelial cell layer model.

Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-13C] or [15N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the 13C was incorporated into alanine, proline, ornithine, and glutamine, and the 15N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8-85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with 13C, 15N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.

Abstract 3364: Preclinical evaluation of BiTE®immune therapy targeting MUC17 or CLDN18.2 for gastric cancer

Abstract Advanced gastric cancer remains a disease of high unmet medical need. In the United States, most patients present with symptomatic, incurable disease and prognosis is poor, with a 5-year survival rate of less than 10%. BiTE® (bispecific T cell engager) immune therapy activates a patient's own T cells to kill tumor cells and has the potential to overcome common mechanisms of therapy resistance. We generated fully human, half-life extended (HLE) BiTE® molecules against the tumor antigens MUC17 and CLDN18.2 for the treatment of gastric cancer. The mucin MUC17 is a protein normally found in the mucosal layer of intestinal epithelial cells that is delocalized and expressed in 45% of gastric tumors. The claudin CLDN18.2 is a protein normally found in the cellular tight junctions of gastric mucosa and intestinal epithelium that is delocalized and expressed in >60% of gastric tumors. AMG 199 (MUC17 HLE BiTE®) and AMG 910 (CLDN18.2 HLE BiTE®) show potent cytotoxic activity against gastric cancer cell lines that express MUC17 or CLDN18.2, respectively, in vitro, and promote significant tumor growth inhibition against established gastric tumor xenograft models in vivo. In preclinical non-human primate (NHP) toxicology studies, both molecules show evidence for BiTE® target engagement, including T cell activation and proliferation, but demonstrate different effects on target-expressing tissue. Weekly administration of AMG 199 is well tolerated in NHP with minimal findings in MUC17-expressing normal tissues. In contrast, treatment with AMG 910 led to direct cell killing of CLDN18.2-expressing gastric mucosal cells in NHP, a finding which was fully reversible once treatment was stopped. AMG 199 and AMG 910 may offer the potential to improve outcomes in advanced gastric patients worldwide. Citation Format: Julie M. Bailis, Petra Lutterbuese, Oliver Thomas, Kathrin Locher, John Harrold, Michael Boyle, Joachim Wahl, Shyun Li, Alexander Sternjak, Anja Henn, Christoph Dahlhoff, Virginie Naegele, Benno Rattel, Tobias Raum, Angela Coxon. Preclinical evaluation of BiTE®immune therapy targeting MUC17 or CLDN18.2 for gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3364.

Zwitterionic micelles efficiently deliver oral insulin without opening tight junctions.

Oral delivery of protein drugs is considered a life-changing solution for patients who require regular needle injections. However, clinical translation of oral protein formulations has been hampered by inefficient penetration of drugs through the intestinal mucus and epithelial cell layer, leading to low absorption and bioavailability, and safety concerns owing to tight junction openings. Here we report a zwitterionic micelle platform featuring a virus-mimetic zwitterionic surface, a betaine side chain and an ultralow critical micelle concentration, enabling drug penetration through the mucus and efficient transporter-mediated epithelial absorption without the need for tight junction opening. This micelle platform was used to fabricate a prototype oral insulin formulation by encapsulating a freeze-dried powder of zwitterionic micelle insulin into an enteric-coated capsule. The biocompatible oral insulin formulation shows a high oral bioavailability of >40%, offers the possibility to fine tune insulin acting profiles and provides long-term safety, enabling the oral delivery of protein drugs.

Microfluidic chip for culturing intestinal epithelial cell layers: Characterization and comparison of drug transport between dynamic and static models

Dynamic flow in vitro models are currently widely explored for their applicability in drug development research. The application of gut-on-chip models in toxicology is lagging behind. Here we report the application of a gut-on-chip model for biokinetic studies and compare the observed biokinetics of reference compounds with those obtained using a conventional static in vitro model. Intestinal epithelial Caco-2 cells were cultured on a porous membrane assembled between two glass flow chambers for the dynamic model, or on a porous membrane in a Transwell model. Confocal microscopy, lucifer yellow translocation, and alkaline phosphatase activity evaluation revealed that cells cultured in the gut-on-chip model formed tight, differentiated, polarized monolayers like in the static cultures. In the dynamic gut-on-chip model the transport of the high permeability compounds antipyrine, ketoprofen and digoxin was lower (i.e. 4.2-, 2.7- and 1.9-fold respectively) compared to the transport in the static Transwell model. The transport of the low permeability compound, amoxicillin, was similar in both the dynamic and static in vitro model. The obtained transport values of the compounds are in line with the compound Biopharmaceuticals Classification System. It is concluded that the gut-on-chip provides an adequate model for transport studies of chemicals.

Epithelial Toll-like receptors and their role in gut homeostasis and disease.

The human gastrointestinal tract is colonized by trillions of microorganisms that interact with the host to maintain structural and functional homeostasis. Acting as the interface between the site of the highest microbial burden in the human body and the richest immune compartment, a single layer of intestinal epithelial cells specializes in nutrient absorption, stratifies microorganisms to limit colonization of tissues and shapes the responses of the subepithelial immune cells. In this Review, we focus on the expression, regulation and functions of Toll-like receptors (TLRs) in the different intestinal epithelial lineages to analyse how epithelial recognition of bacteria participates in establishing homeostasis in the gut. In particular, we elaborate on the involvement of epithelial TLR signalling in controlling crypt dynamics, enhancing epithelial barrier integrity and promoting immune tolerance towards the gut microbiota. Furthermore, we comment on the regulatory mechanisms that fine-tune TLR-driven immune responses towards pathogens and revisit the role of TLRs in epithelial repair after injury. Finally, we discuss how dysregulation of epithelial TLRs can lead to the generation of dysbiosis, thereby increasing susceptibility to colitis and tumorigenesis.

The High Permeability of Nanocarriers Crossing the Enterocyte Layer by Regulation of the Surface Zonal Pattern.

The intestinal epithelium is a major barrier that limits the absorption of oral drugs. The integrity of the epithelial tissue is a very important factor for preventing intestinal diseases. However, destabilization of the epithelium can promote the transportation of nanocarriers and increase the absorption of oral drugs. In our research, three different gold nanoparticles (GNPs) of the same size but with differing negative surface charge were designed and constructed as a model to determine the surface properties crucial for promoting absorptivity and bioavailability of the nanocarriers. The higher the ratio of surface carboxyl groups on GNPs, the higher capacity to induce transepithelial electrical resistance change and cell monolayer tight junction opening with higher permeability. The half carboxyl and half methyl surfaced GNPs displayed unique zonal surface patterns exhibited the greater ability to pass through intestinal epithelial cell layer but had a relatively small influence on tight junction distribution.

Materials and Microenvironments for Engineering the Intestinal Epithelium.

The barrier functions of the gastrointestinal tract rely in large part on a single layer of columnar intestinal epithelial cells. These epithelial cells are mediators of intestinal homeostasis, regulating and communicating biochemical signals between underlying stromal cells and luminal cues. The development of representative in vitro models to recapitulate the gastrointestinal epithelium is crucial to understanding cell-cell interactions during intestinal homeostasis and dysfunction. Ideally, models would contain microbiota/immune cells, polarized intestinal architecture, multilayered cellular complexity, extracellular matrix, biochemical cues, and mechanical deformation. This review focuses on historical and state of the art biomaterials and substrates used in the field to establish static and fluidic models of the intestinal epithelium. A discussion of conventional adenocarcinoma colon cancer cell lines, primary intestinal epithelial cells derived from organoids, and stromal support cells such as enteric neurons, myofibroblasts, and immune cells, as well as the importance of increasing cellular complexity and future outlook is included.

Intestinal Epithelial Cells Respond to Chronic Inflammation and Dysbiosis by Synthesizing H2O2.

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS, however, has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here, we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2, a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-γ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings, we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation, as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2, chronic inflammation caused IECs to release higher levels of H2O2. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively, these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa.

Effects of Iron Oxide Nano-Particles on the Intestinal Tissue of Common Carp, Cyprinus Carpio

Background: The application of nano-particles (NPs) in various industries is growing. Since their toxicity is not clearly understood, they can cause adverse effects on the environment. The aim of this study was to evaluate the histopathological effects of iron oxide nano-particles on the small intestine of common carp, Cyprinus carpio. Methods: Four experimental treatments were designed (15 fish/treatment). Treatment 1 was the controls while Treatments 2, 3 and 4 were experimental. The experimental groups were exposed to 50, 75 and the 100 mg/L of iron oxide NPs, respectively. On days 14, 21 and 28, the fish were randomly picked from each tank, samples of the small intestine were dissected, and were examined for both the accumulation of the iron NPs and the tissue histopathologies. Results: The highest concentration of iron accumulation was detected for Treatment 3 on day 21, compared to all other treatment groups (p<0.05). However, iron accumulation in the tissue declined unexpectedly after day 21 despite the continued treatments at 100 mg/L of the iron NPs. The histopathological examinations revealed that the treatment beyond 21 days caused damages to the intestinal epithelial cells, including enterocytes, villi and the goblet cells. Conclusion: This study demonstrated that the effect of iron oxide NPs on the small intestinal tissue was dependant on the dose and duration of exposure. We conclude that the iron accumulation in the small intestine declined despite increasing the iron oxide NPs concentration and the exposure duration secondary to damages caused to the intestinal epithelial cell layer.

Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.