TiO2 Nanoparticles and Commensal Bacteria Alter Mucus Layer Thickness and Composition in a Gastrointestinal Tract Model.

Nanoparticles (NPs) are used in food packaging and processing and have become an integral part of many commonly ingested products. There are few studies that have focused on the interaction between ingested NPs, gut function, the mucus layer, and the gut microbiota. In this work, an in vitro model of gastrointestinal (GI) tract is used to determine whether, and how, the mucus layer is affected by the presence of Gram-positive, commensal Lactobacillus rhamnosus; Gram-negative, opportunistic Escherichia coli; and/or exposure to physiologically relevant doses of pristine or digested TiO2 NPs. Caco-2/HT29-MTX-E12 cell monolayers are exposed to physiological concentrations of bacteria (expressing fluorescent proteins) and/or TiO2 nanoparticles for a period of 4 h. To determine mucus thickness and composition, cell monolayers are stained with alcian blue, periodic acid schiff, or an Alexa Fluor 488 conjugate of wheat germ agglutinin. It is found that the presence of both bacteria and nanoparticles alter the thickness and composition of the mucus layer. Changes in the distribution or pattern of mucins can be indicative of pathological conditions, and this model provides a platform for understanding how bacteria and/or NPs may interact with and alter the mucus layer.

Body-on-a-chip systems for animal-free toxicity testing.

Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing.

Effects of shear stress pattern and magnitude on mesenchymal transformation and invasion of aortic valve endothelial cells.

Understanding the role of mechanical forces on cell behavior is critical for tissue engineering, regenerative medicine, and disease initiation studies. Current hemodynamic bioreactors are largely limited to 2D substrates or the application of general flow conditions at a tissue level, which eliminates the investigation of some essential physiological and pathological responses. One example is the mesenchymal transformation of endothelial cells in response to shear stress. Endothelial to mesenchymal transformation (EndMT) is a valve morphogenic mechanism associated with aortic valve disease initiation. The aortic valve experiences oscillatory shear on the disease-susceptible fibrosa, and the role of hemodynamics on adult EndMT is unknown. The goal of this work was to develop and characterize a microfluidic bioreactor that applies physiologically relevant laminar or oscillatory shear stresses to endothelial cells and permits the quantitative analysis of 3D cell-extracellular matrix (ECM) interactions. In this study, porcine aortic valve endothelial cells were seeded onto 3D collagen I gels and exposed to different magnitudes of steady or oscillatory shear stress for 48 h. Cells elongated and aligned perpendicular to laminar, but not oscillatory shear. Low steady shear stress (2 dyne/cm(2) ) and oscillatory shear stress upregulated EndMT (ACTA2, Snail, TGFB1) and inflammation (ICAM1, NFKB1) related gene expression, EndMT-related (αSMA) protein expression, and matrix invasion when compared with static controls or cells exposed to high steady shear (10 and 20 dyne/cm(2) ). Our system enables direct testing of the role of shear stress on endothelial cell mesenchymal transformation in a dynamic, 3D environment and shows that hemodynamics regulate EndMT in adult valve endothelial cells.

Inflammatory cytokines promote mesenchymal transformation in embryonic and adult valve endothelial cells.

OBJECTIVE: Inflammatory activation of valve endothelium is an early phase of aortic valve disease pathogenesis, but subsequent mechanisms are poorly understood. Adult valve endothelial cells retain the developmental ability to undergo endothelial-to-mesenchymal transformation (EndMT), but a biological role has not been established. Here, we test whether and how inflammatory cytokines (tumor necrosis factor-α and interleukin-6) regulate EndMT in embryonic and adult valve endothelium. METHODS AND RESULTS: Using in vitro 3-dimensional collagen gel culture assays with primary cells, we determined that interleukin-6 and tumor necrosis factor-α induce EndMT and cell invasion in dose-dependent manners. Inflammatory-EndMT occurred through an Akt/nuclear factor-κB-dependent pathway in both adult and embryonic stages. In embryonic valves, inflammatory-EndMT required canonical transforming growth factor-ß signaling through activin receptor-like kinases 2 and 5 to drive EndMT. In adult valve endothelium, however, inflammatory-induced EndMT still occurred when activin receptor-like kinases 2 and 5 signaling was blocked. Inflammatory receptor gene expression was significantly upregulated in vivo during embryonic valve maturation. Endothelial-derived mesenchymal cells expressing activated nuclear factor-κB were found distal to calcific lesions in diseased human aortic valves. CONCLUSIONS: Inflammatory cytokine-induced EndMT in valve endothelium is present in both embryonic and adult stages, acting through Akt/nuclear factor-κB, but differently using transforming growth factor-ß signaling. Molecular signatures of valve EndMT may be important diagnostic and therapeutic targets in early valve disease.

Multiscale computational modeling of aortic valve calcification.

Calcific aortic valve disease (CAVD) is a common cardiovascular disease that affects millions of people worldwide. The disease is characterized by the formation of calcium nodules on the aortic valve leaflets, which can lead to stenosis and heart failure if left untreated. The pathogenesis of CAVD is still not well understood, but involves several signaling pathways, including the transforming growth factor beta (TGF ß ) pathway. In this study, we developed a multiscale computational model for TGF ß -stimulated CAVD. The model framework comprises cellular behavior dynamics, subcellular signaling pathways, and tissue-level diffusion fields of pertinent chemical species, where information is shared among different scales. Processes such as endothelial to mesenchymal transition (EndMT), fibrosis, and calcification are incorporated. The results indicate that the majority of myofibroblasts and osteoblast-like cells ultimately die due to lack of nutrients as they become trapped in areas with higher levels of fibrosis or calcification, and they subsequently act as sources for calcium nodules, which contribute to a polydispersed nodule size distribution. Additionally, fibrosis and calcification processes occur more frequently in regions closer to the endothelial layer where the cell activity is higher. Our results provide insights into the mechanisms of CAVD and TGF ß signaling and could aid in the development of novel therapeutic approaches for CAVD and other related diseases such as cancer. More broadly, this type of modeling framework can pave the way for unraveling the complexity of biological systems by incorporating several signaling pathways in subcellular models to simulate tissue remodeling in diseases involving cellular mechanobiology.

A glomerulus and proximal tubule microphysiological system simulating renal filtration, reabsorption, secretion, and toxicity.

Microphysiological systems (MPS) are powerful predictive tools for assessing drug-induced kidney injuries. Previous MPS have examined single regions of the nephron, but lack simultaneous filtration, reabsorption, and secretion functionality. Here, we developed a partially open MPS that structurally and functionally recapitulated the glomerular filtration barrier, proximal tubular reabsorption, and secretion for seven days. The system introduced a recirculation circuit and an open filtrate output as a source of functional testing. As a proof-of-concept, a tri-culture of immortalized podocytes, umbilical vein endothelial cells, and proximal tubule (PCT) cells were housed in a single MPS: T-junction, glomerulus housing unit, and PCT chip. The MPS successfully retained blood serum protein, reabsorbed glucose, secreted creatinine, and expressed cell-type specific proteins (VE-cadherin, nephrin, and ZO-1). To simulate drug-induced kidney injuries, the system was perfused with cisplatin and adriamycin, and then tested using serum albumin filtration, glucose clearance, and lactate dehydrogenase release. The glomerulus and PCT MPS demonstrated a complex, dynamic microenvironment and recreated some in vivo-like functions in basal and drug-induced conditions, offering a novel prototype for preclinical testing.

Food-Grade Metal Oxide Nanoparticles Exposure Alters Intestinal Microbial Populations, Brush Border Membrane Functionality and Morphology, In Vivo (Gallus gallus).

Among food additive metal oxide nanoparticles (NP), titanium dioxide (TiO2) and silicon dioxide (SiO2) are commonly used as food coloring or anti-caking agents, while zinc oxide (ZnO) and iron oxide (Fe2O3) are added as antimicrobials and coloring agents, respectively, and can be used as micronutrient supplements. To elucidate potential perturbations associated with NP consumption on gastrointestinal health and development, this in vivo study utilized the Gallus gallus (broiler chicken) intraamniotic administration to assess the effects of physiologically relevant concentrations of food-grade metal oxide NP on brush border membrane (BBM) functionality, intestinal morphology and intestinal microbial populations in vivo. Six groups with 1 mL injection of the following treatments were utilized: non-injected, 18 MΩ DI H2O; 1.4 × 10-6 mg TiO2 NP/mL, 2.0 × 10-5 mg SiO2 NP/mL, 9.7 × 10-6 mg ZnO NP/mL, and 3.8 × 10-4 mg Fe2O3 NP/mL (n = 10 per group). Upon hatch, blood, cecum, and duodenum were collected to assess mineral (iron and zinc) metabolism, BBM functional, and pro-inflammatory-related protein gene expression, BBM morphometric analysis, and the relative abundance of intestinal microflora. Food additive NP altered mineral transporter, BBM functionality, and pro-inflammatory cytokine gene expression, affected intestinal BBM development and led to compositional shifts in intestinal bacterial populations. Our results suggest that food-grade TiO2 and SiO2 NP have the potential to negatively affect intestinal functionality; food-grade ZnO NP exposure effects were associated with supporting intestinal development or compensatory mechanisms due to intestinal damage, and food-grade Fe2O3 NP was found to be a possible option for iron fortification, though with potential alterations in intestinal functionality and health.

Pumped and pumpless microphysiological systems to study (nano)therapeutics.

Fluidic microphysiological systems (MPS) are microfluidic cell culture devices that are designed to mimic the biochemical and biophysical in vivo microenvironments of human tissues better than conventional petri dishes or well-plates. MPS-grown tissue cultures can be used for probing new drugs for their potential primary and secondary toxicities as well as their efficacy. The systems can also be used for assessing the effects of environmental nanoparticles and nanotheranostics, including their rate of uptake, biodistribution, elimination, and toxicity. Pumpless MPS are a group of MPS that often utilize gravity to recirculate cell culture medium through their microfluidic networks, providing some advantages, but also presenting some challenges. They can be operated with near-physiological amounts of blood surrogate (i.e., cell culture medium) that can recirculate in bidirectional or unidirectional flow patterns depending on the device configuration. Here we discuss recent advances in the design and use of both pumped and pumpless MPS with a focus on where pumpless devices can contribute to realizing the potential future role of MPS in evaluating nanomaterials. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

An In Vitro Small Intestine Model Incorporating a Food Matrix and Bacterial Mock Community for Intestinal Function Testing.

Consumed food travels through the gastrointestinal tract to reach the small intestine, where it interacts with the microbiota, forming a complex relationship with the dietary components. Here we present a complex in vitro cell culture model of the small intestine that includes human cells, digestion, a simulated meal, and a microbiota represented by a bacterial community consisting of E. coli, L. rhamnosus, S. salivarius, B. bifidum, and E. faecalis. This model was used to determine the effects of food-grade titanium dioxide nanoparticles (TiO2 NPs), a common food additive, on epithelial permeability, intestinal alkaline phosphatase activity, and nutrient transport across the epithelium. Physiologically relevant concentrations of TiO2 had no effect on intestinal permeability but caused an increase in triglyceride transport as part of the food model, which was reversed in the presence of bacteria. Individual bacterial species had no effect on glucose transport, but the bacterial community increased glucose transport, suggesting a change in bacterial behavior when in a community. Bacterial entrapment within the mucus layer was reduced with TiO2 exposure, which may be due to decreased mucus layer thickness. The combination of human cells, a synthetic meal, and a bacterial mock community provides an opportunity to understand the implications of nutritional changes on small intestinal function, including the microbiota.

Incorporation of Targeting Biomolecule Improves Interpolymer Complex-Superparamagnetic Iron Oxide Nanoparticles Attachment to and Activation of T2 MR Signals in M2 Macrophages.

Introduction: Inflammatory diseases are the leading cause of death in the world, accounting for 3 out of 5 deaths. Despite the abundance of diagnostic tools for detection, most screening and diagnostic methods are indirect and insufficient as they are unable to reliably discriminate between high-risk or low-risk stages of inflammatory diseases. Previously, we showed that the selective activation of interpolymer complexed superparamagnetic iron oxide nanoparticles (IPC-SPIOs) under oxidative conditions can be detected by a change in T2 magnetic resonance (MR) contrast. In this work, IPC-SPIOs were further modified by incorporating mannose as a targeting biomolecule to enhance nanoparticle delivery to M2 macrophages at inflammatory sites. Methods: Uncoated SPIOs were synthesized via coprecipitation from a mixture of FeCl2 and FeCl3, PEGylated by adsorbing PEG 300 kDa (40 mg/mL in water) to SPIOs (3 mg/mL in water) over 24 hours, and complexed by mixing 0.25 mg/mL aqueous poly(gallol) with 2 mg/mL PEG-SPIOs and adding 1 M of phosphate buffer in a 9:9:2 ratio. Mannose-PEG attachment was accomplished conducting a second complexation of mannose-PEG to IPC-SPIOs. M2 macrophages were treated with 150, 100, and 75 µg/mL of IPC-SPIOs and mannose-IPC-SPIOs to investigate activation of T2 MRI signals. Results and Discussion: Surface modification resulted in a slight reduction in ROS scavenging capacity; however, nanoparticle uptake by M2 macrophages increased by over 50%. The higher uptake did not cause a reduction in cellular viability. In fact, mannose-IPC-SPIOs induced significant T2 MR contrast in M2 macrophages compared to IPC-SPIOs and nanoparticles exposed to M1 macrophages. M2 macrophages activated over 30% of mannose-IPC-SPIOs after 6 hours of exposure compared to M1 macrophages and untargeted M2 macrophages. These findings demonstrated that mannose-IPC-SPIOs specifically targeted M2 macrophages and scavenged cellular ROS to activate T2 MR signal, which can be used to detect inflammation.

Shear and endothelial induced late-stage calcific aortic valve disease-on-a-chip develops calcium phosphate mineralizations.

Calcific aortic valve disease (CAVD) is an active pathobiological process leading to severe aortic stenosis, where the only treatment is valve replacement. Late-stage CAVD is characterized by calcification, disorganization of collagen, and deposition of glycosaminoglycans, such as chondroitin sulfate (CS), in the fibrosa. We developed a three-dimensional microfluidic device of the aortic valve fibrosa to study the effects of shear stress (1 or 20 dyne per cm2), CS (1 or 20 mg mL-1), and endothelial cell presence on calcification. CAVD chips consisted of a collagen I hydrogel, where porcine aortic valve interstitial cells were embedded within and porcine aortic valve endothelial cells were seeded on top of the matrix for up to 21 days. Here, we show that this CAVD-on-a-chip is the first to develop human-like calcified nodules varying in calcium phosphate mineralization maturity resulting from high shear and endothelial cells, specifically di- and octa-calcium phosphates. Long-term co-culture microfluidic studies confirmed cell viability and calcium phosphate formations throughout 21 days. Given that CAVD has no targeted therapies, the creation of a physiologically relevant test-bed of the aortic valve could lead to advances in preclinical studies.

Upcycling Compact Discs for Flexible and Stretchable Bioelectronic Applications.

Electronic waste is a global issue brought about by the short lifespan of electronics. Viable methods to relieve the inundated disposal system by repurposing the enormous amount of electronic waste remain elusive. Inspired by the need for sustainable solutions, this study resulted in a multifaceted approach to upcycling compact discs. The once-ubiquitous plates can be transformed into stretchable and flexible biosensors. Our experiments and advanced prototypes show that effective, innovative biosensors can be developed at a low-cost. An affordable craft-based mechanical cutter allows pre-determined patterns to be scored on the recycled metal, an essential first step for producing stretchable, wearable electronics. The active metal harvested from the compact discs was inert, cytocompatible, and capable of vital biopotential measurements. Additional studies examined the material's resistive emittance, temperature sensing, real-time metabolite monitoring performance, and moisture-triggered transience. This sustainable approach for upcycling electronic waste provides an advantageous research-based waste stream that does not require cutting-edge microfabrication facilities, expensive materials, and high-caliber engineering skills.

The mechanistic effects of human digestion on magnesium oxide nanoparticles: implications for probiotics Lacticaseibacillus rhamnosus GG and Bifidobacterium bifidum VPI 1124.

The effects of nanoparticles (NPs) on the human gut microbiota are of high interest due to the link between the gut homeostasis and overall human health. The human intake of metal oxide NPs has increased due to its use in the food industry as food additives. Specifically, magnesium oxide nanoparticles (MgO-NPs) have been described as antimicrobial and antibiofilm. Therefore, in this work we investigated the effects of the food additive MgO-NPs, on the probiotic and commensal Gram-positive Lactobacillus rhamnosus GG and Bifidobacterium bifidum VPI 1124. The physicochemical characterization showed that food additive MgO is formed by nanoparticles (MgO-NPs) and after a simulated digestion, MgO-NPs partially dissociate into Mg2+. Moreover, nanoparticulate structures containing magnesium were found embedded in organic material. Exposures to MgO-NPs for 4 and 24 hours increased the bacterial viability of both L. rhamnosus and B. bifidum when in biofilms but not when as planktonic cells. High doses of MgO-NPs significantly stimulated the biofilm development of L. rhamnosus, but not B. bifidum. It is likely that the effects are primarily due to the presence of ionic Mg2+. Evidence from the NPs characterization indicate that interactions bacteria/NPs are unfavorable as both structures are negatively charged, which would create repulsive forces.

Chondroitin Sulfate Promotes Interstitial Cell Activation and Calcification in an In Vitro Model of the Aortic Valve.

PURPOSE: Calcific aortic valve disease (CAVD), has been characterized as a cascade of cellular changes leading to leaflet thickening and valvular calcification. In diseased aortic valves, glycosaminoglycans (GAGs) normally found in the valve spongiosa migrate to the collagen I-rich fibrosa layer near calcified nodules. Current treatments for CAVD are limited to valve replacement or drugs tailored to other cardiovascular diseases. METHODS: Porcine aortic valve interstitial cells and porcine aortic valve endothelial cells were seeded into collagen I hydrogels of varying initial stiffness or initial stiffness-matched collagen I hydrogels containing the glycosaminoglycans chondroitin sulfate (CS), hyaluronic acid (HA), or dermatan sulfate (DS). Assays were performed after 2 weeks in culture to determine cell gene expression, protein expression, protein secretion, and calcification. Multiple regression analyses were performed to determine the importance of initial hydrogel stiffness, GAGs, and the presence of endothelial cells on calcification, both with and without osteogenic medium. RESULTS: High initial stiffness hydrogels and osteogenic medium promoted calcification, while for DS or HA the presence of endothelial cells prevented calcification. CS was found to increase the expression of pro-calcific genes, increase activated myofibroblast protein expression, induce the secretion of collagen I by activated interstitial cells, and increase calcified nodule formation. CONCLUSION: This study demonstrates a more complete model of aortic valve disease, including endothelial cells, interstitial cells, and a stiff and disease-like ECM. In vitro models of both healthy and diseased valves can be useful for understanding the mechanisms of CAVD pathogenesis and provide a model for testing novel therapeutics.

Glycosaminoglycans affect endothelial to mesenchymal transformation, proliferation, and calcification in a 3D model of aortic valve disease.

Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells' loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.

Cardiac developmental toxicity.

Congenital heart disease (CHD) is a highly prevalent problem with mostly unknown origins. Many cases of CHD likely involve an environmental exposure coupled with genetic susceptibility, but practical and ethical considerations make nongenetic causes of CHD difficult to assess in humans. The development of the heart is highly conserved across all vertebrate species, making animal models an excellent option for screening potential cardiac teratogens. This review will discuss exposures known to cause cardiac defects, stages of heart development that are most sensitive to teratogen exposure, benefits and limitations of animal models of cardiac development, and future considerations for cardiac developmental toxicity research.

Modelling Renal Filtration and Reabsorption Processes in a Human Glomerulus and Proximal Tubule Microphysiological System.

Kidney microphysiological systems (MPS) serve as potentially valuable preclinical instruments in probing mechanisms of renal clearance and osmoregulation. Current kidney MPS models target regions of the nephron, such as the glomerulus and proximal tubule (PCT), but fail to incorporate multiple filtration and absorption interfaces. Here, we describe a novel, partially open glomerulus and PCT microdevice that integrates filtration and absorption in a single MPS. The system equalizes pressure on each side of the PCT that operates with one side "closed" by recirculating into the bloodstream, and the other "opened" by exiting as primary filtrate. This design precisely controls the internal fluid dynamics and prevents loss of all fluid to the open side. Through this feature, an in vitro human glomerulus and proximal tubule MPS was constructed to filter human serum albumin and reabsorb glucose for seven days of operation. For proof-of-concept experiments, three human-derived cell types-conditionally immortalized human podocytes (CIHP-1), human umbilical vein endothelial cells (HUVECs), and human proximal tubule cells (HK-2)-were adapted into a common serum-free medium prior to being seeded into the three-component MPS (T-junction splitter, glomerular housing unit, and parallel proximal tubule barrier model). This system was optimized geometrically (tubing length, tubing internal diameter, and inlet flow rate) using in silico computational modeling. The prototype tri-culture MPS successfully filtered blood serum protein and generated albumin filtration in a physiologically realistic manner, while the device cultured only with proximal tubule cells did not. This glomerulus and proximal convoluted tubule MPS is a potential prototype for the human kidney used in both human-relevant testing and examining pharmacokinetic interactions.

Critical Considerations for the Design of Multi-Organ Microphysiological Systems (MPS).

Identification and approval of new drugs for use in patients requires extensive preclinical studies and clinical trials. Preclinical studies rely on in vitro experiments and animal models of human diseases. The transferability of drug toxicity and efficacy estimates to humans from animal models is being called into question. Subsequent clinical studies often reveal lower than expected efficacy and higher drug toxicity in humans than that seen in animal models. Microphysiological systems (MPS), sometimes called organ or human-on-chip models, present a potential alternative to animal-based models used for drug toxicity screening. This review discusses multi-organ MPS that can be used to model diseases and test the efficacy and safety of drug candidates. The translation of an in vivo environment to an in vitro system requires physiologically relevant organ scaling, vascular dimensions, and appropriate flow rates. Even small changes in those parameters can alter the outcome of experiments conducted with MPS. With many MPS devices being developed, we have outlined some established standards for designing MPS devices and described techniques to validate the devices. A physiologically realistic mimic of the human body can help determine the dose response and toxicity effects of a new drug candidate with higher predictive power.

Bacteria Remediate the Effects of Food Additives on Intestinal Function in an in vitro Model of the Gastrointestinal Tract.

As the site of nutrient absorption, the small intestine is continuously exposed to preservatives and additives present in consumed food. While the effects of diet on the lower gastrointestinal tract are widely studied, the effects of food additives on the small intestinal epithelium and microbiota are less clearly understood. The goal of this work was to develop and establish a physiologically relevant model of the upper gastrointestinal tract to study the complex interactions between food additives, individual bacterial species, and intestinal function. To achieve this, an in vitro model incorporating simulated digestion, human intestinal epithelial cells, and the commensal, Gram-positive Lactobacillus rhamnosus, or the opportunistic, Gram-negative Escherichia coli was developed. This model was used to assess intestinal permeability and alkaline phosphatase activity following exposure to high glucose (HG), salt, emulsifier (TWEEN 20), food (milk chocolate candies) or chemical grade titanium dioxide nanoparticles (TiO2-NP), and food (whole wheat bread) or chemical grade gluten. It was found that HG increased intestinal permeability, the presence of bacteria remediated the negative effects of HG on intestinal permeability, and a decrease in permeability and IAP activity was observed with increasing concentration of TWEEN 20 both in the presence and absence of bacteria. While L. rhamnosus influenced the activity of intestinal alkaline phosphatase and tight junction protein distribution, E. coli produced indole to reinstate intestinal permeability. The source of TiO2 and gluten led to altered impacts on permeability and IAP activity. The growth of E. coli and L. rhamnosus was found to depend on the type of food additive used. Overall, the presence of bacteria in the in vitro model influenced the effects of food additives on intestinal function, suggesting a complex association between diet and upper GI microbiota. This model provides a method to study small intestinal function and host-microbe interactions in vitro in both healthy and diseased conditions.

Intra-amniotic administration (Gallus gallus) of TiO2, SiO2, and ZnO nanoparticles affect brush border membrane functionality and alters gut microflora populations.

Metal oxide nanoparticles (NP) are increasingly used in the food and agriculture industries, making human consumption nearly unavoidable. The goal of this study was to use the Gallus gallus (broiler chicken) intra-amniotic administration of physiologically relevant concentrations of TiO2, SiO2, and ZnO to better understand the effects of NP exposure on gut health and function. Immediately after hatch, blood, cecum, and small intestine were collected for assessment of iron (Fe)-metabolism, zinc (Zn)-metabolism, brush border membrane (BBM) functional, and pro-inflammatory related proteins gene expression; blood Fe and Zn levels; cecum weight; and the relative abundance of intestinal microflora. NP type, dose, and the presence or absence of minerals was shown to result in altered mineral transporter, BBM functional, and pro-inflammatory gene expression. Metal oxide NP also altered the abundance of intestinal bacterial populations. Overall, the data suggest that the in vivo results align with in vitro studies, and that NP have the potential to negatively affect intestinal functionality and health.