Epithelial Cell Culture Model Research Articles

Development and Characterization of a Human Mammary Epithelial Cell Culture Model for the Blood–Milk Barrier—A Contribution from the ConcePTION Project

It is currently impossible to perform an evidence-based risk assessment for medication use during breastfeeding. The ConcePTION project aims to provide information about the use of medicines during lactation. The study aimed to develop and characterize an in vitro model of the blood–milk barrier to determine the extent of the milk transfer of xenobiotics, relying on either on human mammary epithelial cells (hMECs) or immortalized cell lines derived from breast tissue. The hMECs were cultured and characterized for epithelial markers; further, the ability to form an epithelial barrier was investigated. Drug transporter functionality in the cultured hMECs was analyzed with specific probe substrates. The hMECs showed an epithelial morphology and the expression of epithelial markers and tight junctions. They formed a reproducible tight barrier with a transepithelial electrical resistance greater than 400 Ωcm2, unlike immortalized cell lines. Different levels of mRNA expression were detected for 81 genes of membrane transporters. Functional assays showed no evidence for the transporter-mediated secretion of medicines across the hMECs. Nevertheless, the hMEC-based in vitro model covered a 50-fold range of permeability values, differentiating between passive transcellular and paracellular-mediated transport. The cultured hMECs proved to be a promising in vitro model for biorelevance; the wide characterization of hMECs makes them useful for studying medicine partitioning in milk.

Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Establishment of Swine Primary Nasal, Tracheal, and Bronchial Epithelial Cell Culture Models for the Study of Influenza Virus Infection

We established primary porcine nasal, tracheal, and bronchial epithelial cells that recapitulate the physical and functional properties of the respiratory tract and have the ability to fully differentiate. Trans-well cultures demonstrated increased transepithelial electrical resistance over time the presence of tight junctions as demonstrated by immunohistochemistry. The nasal, tracheal, and bronchial epithelial cells developed cilia, secreted mucus, and expressed sialic acids on surface glycoproteins, the latter which are required for influenza A virus infection. Swine influenza viruses were shown to replicate efficiently in the primary epithelial cell cultures, supporting the use of these culture models to assess swine influenza and other virus infection. Primary porcine nasal, tracheal, and bronchial epithelial cell culture models enable assessment of emerging and novel influenza viruses for pandemic potential as well as mechanistic studies to understand mechanisms of infection, reassortment, and generation of novel virus. As swine are susceptible to infection with multiple viral and bacterial respiratory pathogens, these primary airway cell models may enable study of the cellular response to infection by pathogens associated with Porcine Respiratory Disease Complex.

Cytolethal Distending Toxin Modulates Cell Differentiation and Elicits Epithelial to Mesenchymal Transition.

The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus-infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture. Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis.

In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores.

Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

Temperature influences commensal-pathogen dynamics in a nasal epithelial cell co-culture model.

Chronic rhinosinusitis (CRS) is an inflammatory disease of the paranasal sinuses, and microbial dysbiosis associated with CRS is thought to be a key driver of host inflammation that contributes to disease progression. Staphylococcus aureus is a common upper respiratory tract (URT) pathobiont associated with higher carriage rates in CRS populations, where S. aureus-secreted toxins can be identified in CRS tissues. Although many genera of bacteria colonize the URT, few account for the majority of sequencing reads. These include S. aureus and several species belonging to the genus Corynebacterium, including Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum, which are observed at high relative abundance in the healthy URT. Studies have examined bacterial interactions between major microbionts of the URT and S. aureus, but few have done so in the context of a healthy versus diseased URT environment. Here, we examine the role of temperature in commensal, pathogen, and epithelial dynamics using an air-liquid interface cell culture model mimicking the nasal epithelial environment. Healthy URT temperatures change from the nares to the nasopharynx and are increased during disease. Temperatures representative of the healthy URT increase persistence and aggregate formation of commensal C. propinquum and C. pseudodiphtheriticum, reduce S. aureus growth, and lower epithelial cytotoxicity compared to higher temperatures correlating with the diseased CRS sinus. Dual-species colonization revealed species-specific interactions between Corynebacterium species and S. aureus dependent on temperature. Our findings suggest URT mucosal temperature plays a significant role in mediating polymicrobial and host-bacterial interactions that may exacerbate microbial dysbiosis in chronic URT diseases.IMPORTANCEChronic rhinosinusitis is a complex inflammatory disease with a significant healthcare burden. Although presence of S. aureus and microbial dysbiosis are considered mediators of inflammation in CRS, no studies have examined the influence of temperature on S. aureus interactions with the nasal epithelium and the dominant genus of the healthy URT, Corynebacterium. Interactions between Corynebacterium species and S. aureus have been documented in several studies, but none to date have examined how environmental changes in the URT may alter their interactions with the epithelium or each other. This study utilizes a polarized epithelial cell culture model at air-liquid interface to study the colonization and spatial dynamics of S. aureus and clinical isolates of Corynebacterium from people with CRS to characterize the role temperature has in single- and dual-species dynamics on the nasal epithelium.

Early Atf4 activity drives airway club and goblet cell differentiation.

Activating transcription factor 4 (Atf4), which is modulated by the protein kinase RNA-like ER kinase (PERK), is a stress-induced transcription factor responsible for controlling the expression of a wide range of adaptive genes, enabling cells to withstand stressful conditions. However, the impact of the Atf4 signaling pathway on airway regeneration remains poorly understood. In this study, we used mouse airway epithelial cell culture models to investigate the role of PERK/Atf4 in respiratory tract differentiation. Through pharmacological inhibition and silencing of ATF4, we uncovered the crucial involvement of PERK/Atf4 in the differentiation of basal stem cells, leading to a reduction in the number of secretory cells. ChIP-seq analysis revealed direct binding of ATF4 to regulatory elements of genes associated with osteoblast differentiation and secretory cell function. Our findings provide valuable insights into the role of ATF4 in airway epithelial differentiation and its potential involvement in innate immune responses and cellular adaptation to stress.

Quercetin improves and protects Calu-3 airway epithelial barrier function

Introduction: In light of the impact of airway barrier leaks in COVID-19 and the significance of vitamin D in COVID-19 outcomes, including airway barrier protection, we investigated whether the very common dietary flavonoid quercetin could also be efficacious in supporting airway barrier function.Methods: To address this question, we utilized the widely used airway epithelial cell culture model, Calu-3.Results: We observed that treating Calu-3 cell layers with quercetin increased transepithelial electrical resistance while simultaneously reducing transepithelial leaks of 14C-D-mannitol (Jm) and 14C-inulin. The effects of quercetin were concentration-dependent and exhibited a biphasic time course. These effects of quercetin occurred with changes in tight junctional protein composition as well as a partial inhibition of cell replication that resulted in decreased linear junctional density. Both of these effects potentially contribute to improved barrier function. Quercetin was equally effective in reducing the barrier compromise caused by the pro-inflammatory cytokine TNF-α, an action that seemed to derive, in part, from reducing the elevation of ERK 1/2 caused by TNF-α.Discussion: Quercetin improved Calu-3 barrier function and reduced TNF-α-induced barrier compromise, mediated in part by changes in the tight junctional complex.

An oral commensal attenuates Pseudomonas aeruginosa-induced airway inflammation and modulates nitrite flux in respiratory epithelium.

Polymicrobial airway infections in persons with cystic fibrosis (pwCF) can positively or negatively impact the course of disease. A major CF pathogen, Pseudomonas aeruginosa, often establishes a chronic infection leading to lung deterioration. Interestingly, the presence of certain oral commensal streptococci is correlated with improved outcomes for pwCF. We previously reported that hydrogen peroxide production by these commensals combined with nitrite generates reactive nitrogen intermediates (RNI), which inhibit P. aeruginosa in vitro. In this study, we utilized a rat co-infection lung model to assess whether oral commensal-generated RNI can restrict the pathogenesis of a CF isolate of P. aeruginosa. We report that the oral commensal Streptococcus parasanguinis and nitrite reduce P. aeruginosa-induced host inflammation in wild-type rats. To better recapitulate CF-specific airway conditions, we used a bronchial epithelial cell culture model to gain a better understanding of how S. parasanguinis and nitrite may influence P. aeruginosa burden during a CF infection. Hence, we co-infected wild-type and cystic fibrosis transconductance regulator (CFTR) channel-deficient bronchial epithelial cells with P. aeruginosa and S. parasanguinis with or without nitrite. Strikingly, S. parasanguinis reduced the bacterial burden of P. aeruginosa without nitrite, promoted epithelial cell viability, and stimulated nitrite production in the wild-type and CFTR-deficient epithelial cells, where nitrite induction was more apparent in the CFTR mutant cells. Taken together, our study demonstrates that the commensal S. parasanguinis may provide protection against P. aeruginosa-induced inflammation and cell death, as well as modulate nitrite flux in airway epithelial cells. IMPORTANCE Respiratory infections are a leading cause of morbidity and mortality in people with cystic fibrosis (CF). These infections are polymicrobial in nature with overt pathogens and other colonizing microbes present. Microbiome data have indicated that the presence of oral commensal bacteria in the lungs is correlated with improved outcomes. We hypothesize that one oral commensal, Streptococcus parasanguinis, inhibits CF pathogens and modulates the host immune response. One major CF pathogen is Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterium with intrinsic drug resistance and an arsenal of virulence factors. We have previously shown that S. parasanguinis inhibits P. aeruginosa in vitro in a nitrite-dependent manner through the production of reactive nitrogen intermediates. In this study, we demonstrate that while this mechanism is evident in a cell culture model of the CF airway, an alternative mechanism by which S. parasanguinis may improve outcomes for people with CF is through immunomodulation.

Epigenetic responses to rhinovirus exposure in airway epithelial cells are correlated with key transcriptional pathways in chronic rhinosinusitis.

Viruses may drive immune mechanisms responsible for chronic rhinosinusitis with nasal polyposis (CRSwNP), but little is known about the underlying molecular mechanisms. To identify epigenetic and transcriptional responses to a common upper respiratory pathogen, rhinovirus (RV), that are specific to patients with CRSwNP using a primary sinonasal epithelial cell culture model. Airway epithelial cells were collected at surgery from patients with CRSwNP (cases) and from controls without sinus disease, cultured, and then exposed to RV or vehicle for 48 h. Differential gene expression and DNA methylation (DNAm) between cases and controls in response to RV were determined using linear mixed models. Weighted gene co-expression analysis (WGCNA) was used to identify (a) co-regulated gene expression and DNAm signatures, and (b) genes, pathways, and regulatory mechanisms specific to CRSwNP. We identified 5585 differential transcriptional and 261 DNAm responses (FDR <0.10) to RV between CRSwNP cases and controls. These differential responses formed three co-expression/co-methylation modules that were related to CRSwNP and three that were related to RV (Bonferroni corrected p < .01). Most (95%) of the differentially methylated CpGs (DMCs) were in modules related to CRSwNP, whereas the differentially expressed genes (DEGs) were more equally distributed between the CRSwNP- and RV-related modules. Genes in the CRSwNP-related modules were enriched in known CRS and/or viral response immune pathways. RV activates specific epigenetic programs and correlated transcriptional networks in the sinonasal epithelium of individuals with CRSwNP. These novel observations suggest epigenetic signatures specific to patients with CRSwNP modulate response to viral pathogens at the mucosal environmental interface. Determining how viral response pathways are involved in epithelial inflammation in CRSwNP could lead to therapeutic targets for this burdensome airway disorder.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air-liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA.

Calcitriol modifies tight junctions, improves barrier function, and reduces TNF-α-induced barrier leak in the human lung-derived epithelial cell culture model, 16HBE 14o.

Using the 16HBE 14o- human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics - increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14 C-D-mannitol (Jm ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-α-induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF-α-induced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF- α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF- α - induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF- α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials.

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease.

Autosomal dominant polycystic liver and kidney disease is a spectrum of hereditary diseases, which display disturbed function of primary cilia leading to cyst formation. In autosomal dominant polycystic kidney disease a genetic cause can be determined in almost all cases. However, in isolated polycystic liver disease (PLD) about half of all cases remain genetically unsolved, suggesting more, so far unidentified genes to be implicated in this disease. Customized next-generation sequencing was used to identify the underlying pathogenesis in two related patients with PLD. A variant identified in SEC61A1 was further analysed in immortalized patients' urine sediment cells and in an epithelial cell model. In both patients, a heterozygous missense change (c.706C>T/p.Arg236Cys) was found in SEC61A1, which encodes for a subunit of the translocation machinery of protein biosynthesis at the endoplasmic reticulum (ER). While kidney disease is absent in the proposita, her mother displays an atypical polycystic kidney phenotype with severe renal failure. In immortalized urine sediment cells, mutant SEC61A1 is expressed at reduced levels, resulting in decreased levels of polycystin-2 (PC2). In an epithelial cell culture model, we found the proteasomal degradation of mutant SEC61A1 to be increased, whereas its localization to the ER is not affected. Our data expand the allelic and clinical spectrum for SEC61A1, adding PLD as a new and the major phenotypic trait in the family described. We further demonstrate that mutant SEC61A1 results in enhanced proteasomal degradation and impaired biosynthesis of PC2.

Resveratrol Supplementation Attenuates Excessive Inflammation and Helps Restore Impaired Restitution in an Intestinal Epithelial Cell Culture Model

Resveratrol Supplementation Attenuates Excessive Inflammation and Helps Restore Impaired Restitution in an Intestinal Epithelial Cell Culture Model

Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease.

The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.

Modulation of Milk and Lipid Synthesis and Secretion in a3-Dimensional Mouse Mammary Epithelial Cell Culture Model: Effects of Palmitate and Orlistat.

Human milk synthesis is impacted by maternal diet, serum composition, and substrate uptake and synthesis by mammary epithelial cells (MECs). The milk of obese/high-fat-diet women has an increased fat content, which promote excess infant weight gain and the risk of childhood/adult obesity. Yet, the knowledge of milk synthesis regulation is limited, and there are no established approaches to modulate human milk composition. We established a 3-dimensional mouse MEC primary culture that recreates the milk production pathway and tested the effects of the major saturated fatty acid in human milk (palmitate) and a lipoprotein lipase inhibitor (orlistat) on triglyceride production. Positive immunostaining confirmed the presence of milk protein and intracellular lipid including milk globules in the cytoplasm and extracellular space. The treatment with palmitate activated "milk" production by MECs (β-casein) and the lipid pathway (as evident by increased protein and mRNA expression). Consistent with these cellular changes, there was increased secretion of milk protein and triglyceride in MEC "milk". The treatment with orlistat suppressed milk triglyceride production. Palmitate increased milk and lipid synthesis, partly via lipoprotein lipase activation. These findings demonstrate the ability to examine MEC pathways of milk production via both protein and mRNA and to modulate select pathways regulating milk composition in MEC culture.

Isolation and Characterization of a Phapecoctavirus Infecting Multidrug-Resistant Acinetobacter baumannii in A549 Alveolar Epithelial Cells.

Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is an emerging pathogen in the ESKAPE group. The global burden of antimicrobial resistance has led to renewed interest in alternative antimicrobial treatment strategies, including phage therapy. This study isolated and characterized a phage vB_AbaM_ ABPW7 (vABPW7) specific to MDR A. baumannii. Morphological analysis showed that phage vABPW7 belongs to the Myoviridae family. Genome analysis showed that the phage DNA genome consists of 148,647 bp and that the phage is a member of the Phapecoctavirus genus of the order Caudovirales. A short latent period and a large burst size indicated that phage vABPW7 was a lytic phage that could potentially be used in phage therapy. Phage vABPW7 is a high-stability phage that has high lytic activity. Phage vABPW7 could effectively reduce biofilm formation and remove preformed biofilm. The utility of phage vABPW7 was investigated in a human A549 alveolar epithelial cell culture model. Phage vABPW7 was not cytotoxic to A549 cells, and the phage could significantly reduce planktonic MDR A. baumannii and MDR A. baumannii adhesion on A549 cells without cytotoxicity. This study suggests that phage vABPW7 has the potential to be developed further as a new antimicrobial agent against MDR A. baumannii.

Higher Concentrations of Folic Acid Cause Oxidative Stress, Acute Cytotoxicity, and Long-Term Fibrogenic Changes in Kidney Epithelial Cells.

Kidney fibrosis is a common step during chronic kidney disease (CKD), and its incidence has been increasing worldwide. Aberrant recovery after repeated acute kidney injury leads to fibrosis. The mechanism of fibrogenic changes in the kidney is not fully understood. Folic acid-induced kidney fibrosis in mice is an established in vivo model to study kidney fibrosis, but the mechanism is poorly understood. Moreover, the effect of higher concentrations of folic acid on kidney epithelial cells in vitro has not yet been studied. Oxidative stress is a common property of nephrotoxicants. Therefore, this study evaluated the role of folic acid-induced oxidative stress in fibrogenic changes by using the in vitro renal proximal tubular epithelial cell culture model. To obtain comprehensive and robust data, three different cell lines derived from human and mouse kidney epithelium were treated with higher concentrations of folic acid for both acute and long-term durations, and the effects were determined at the cellular and molecular levels. The result of cell viability by the MTT assay and the measurement of reactive oxygen species (ROS) levels by the DCF assay revealed that folic acid caused cytotoxicity and increased levels of ROS in acute exposure. The cotreatment with antioxidant N-acetyl cysteine (NAC) protected the cytotoxic effect, suggesting the role of folic acid-induced oxidative stress in cytotoxicity. In contrast, the long-term exposure to folic acid caused increased growth, DNA damage, and changes in the expression of marker genes for EMT, fibrosis, oxidative stress, and oxidative DNA damage. Some of these changes, particularly the acute effects, were abrogated by cotreatment with antioxidant NAC. In summary, the novel findings of this study suggest that higher concentrations of folic acid-induced oxidative stress act as the driver of cytotoxicity as an acute effect and of fibrotic changes as a long-term effect in kidney epithelial cells.