Primary Human Epithelial Cells Research Articles

Tonabersat suppresses priming/activation of the NOD-like receptor protein-3 (NLRP3) inflammasome and decreases renal tubular epithelial-to-macrophage crosstalk in a model of diabetic kidney disease

BackgroundAccompanied by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, aberrant connexin 43 (Cx43) hemichannel-mediated ATP release is situated upstream of inflammasome assembly and inflammation and contributes to multiple secondary complications of diabetes and associated cardiometabolic comorbidities. Evidence suggests there may be a link between Cx43 hemichannel activity and inflammation in the diabetic kidney. The consequences of blocking tubular Cx43 hemichannel-mediated ATP release in priming/activation of the NLRP3 inflammasome in a model of diabetic kidney disease (DKD) was investigated. We examined downstream markers of inflammation and the proinflammatory and chemoattractant role of the tubular secretome on macrophage recruitment and activation.MethodsAnalysis of human transcriptomic data from the Nephroseq repository correlated gene expression to renal function in DKD. Primary human renal proximal tubule epithelial cells (RPTECs) and monocyte-derived macrophages (MDMs) were cultured in high glucose and inflammatory cytokines as a model of DKD to assess Cx43 hemichannel activity, NLRP3 inflammasome activation and epithelial-to-macrophage paracrine-mediated crosstalk. Tonabersat assessed a role for Cx43 hemichannels.ResultsTranscriptomic analysis from renal biopsies of patients with DKD showed that increased Cx43 and NLRP3 expression correlated with declining glomerular filtration rate (GFR) and increased proteinuria. In vitro, Tonabersat blocked glucose/cytokine-dependant increases in Cx43 hemichannel-mediated ATP release and reduced expression of inflammatory markers and NLRP3 inflammasome activation in RPTECs. We observed a reciprocal relationship in which NLRP3 activity exacerbated increased Cx43 expression and hemichannel-mediated ATP release, events driven by nuclear factor kappa-B (NFκB)-mediated priming and Cx43 hemichannel opening, changes blocked by Tonabersat. Conditioned media (CM) from RPTECs treated with high glucose/cytokines increased expression of inflammatory markers in MDMs, an effect reduced when macrophages were pre-treated with Tonabersat. Co-culture using conditioned media from Tonabersat-treated RPTECs dampened macrophage inflammatory marker expression and reduced macrophage migration.ConclusionUsing a model of DKD, we report for the first time that high glucose and inflammatory cytokines trigger aberrant Cx43 hemichannel activity, events that instigate NLRP3-induced inflammation in RPTECs and epithelial-to-macrophage crosstalk. Recapitulating observations previously reported in diabetic retinopathy, these data suggest that Cx43 hemichannel blockers (i.e., Tonabersat) may dampen multi-system damage observed in secondary complications of diabetes.

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells.

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

Epidemiologically relevant phthalates affect human endometrial cells in vitro through cell specific gene expression changes related to the cytoskeleton and mitochondria

Phthalates are endocrine disrupting chemicals (EDCs) found in common consumer products such as soft plastics and cosmetics. Although the knowledge regarding the adverse effects of phthalates on female fertility are accumulating, information on the hormone sensitive endometrium is still scarce. Here, we studied the effects of phthalates on endometrial cell proliferation and gene expression. Human endometrial primary epithelial and stromal cells were isolated from healthy fertile-aged women (n=3), and were compared to endometrial cell lines T-HESC and Ishikawa. Three different epidemiologically relevant phthalate mixtures were used, defined by urine samples in the Midlife Women Health Study (MWHS) cohort. Mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was used as a single phthalate control. Cells were harvested for proliferation testing and transcriptomic analyses after 24h exposure. Even though all cell models responded differently to the phthalate exposures, many overlapping differentially expressed genes (DEGs, FDR<0.1), related to cell adhesion, cytoskeleton and mitochondria were found in all cell types. The qPCR analysis confirmed that MEHHP significantly affected cell adhesion gene vinculin (VCL) and NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), important for oxidative phosphorylation. Benchmark dose modelling showed that MEHHP had significant concentration-dependent effects on cytoskeleton gene actin-beta (ACTB). In conclusion, short 24h phthalate exposures significantly altered gene expression cell-specifically in human endometrial cells, with six shared DEGs. The mixture effects were similar to those of MEHHP, suggesting MEHHP could be the main driver in the mixture. Impact of phthalate exposures on endometrial functions including receptivity should be addressed.

BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells.

Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms. In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, β-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells. In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing β-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated β-galactosidase (SA-β-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model. BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

HtrA-Dependent E-Cadherin Shedding Impairs the Epithelial Barrier Function in Primary Gastric Epithelial Cells and Gastric Organoids

Impaired E-cadherin (Cdh1) functions are closely associated with cellular dedifferentiation, infiltrative tumor growth and metastasis, particularly in gastric cancer. The class-I carcinogen Helicobacter pylori (H. pylori) colonizes gastric epithelial cells and induces Cdh1 shedding, which is primarily mediated by the secreted bacterial protease high temperature requirement A (HtrA). In this study, we used human primary epithelial cell lines derived from gastroids and mucosoids from different healthy donors to investigate HtrA-mediated Cdh1 cleavage and the subsequent impact on bacterial pathogenesis in a non-neoplastic context. We found a severe impairment of Cdh1 functions by HtrA-induced ectodomain cleavage in 2D primary cells and mucosoids. Since mucosoids exhibit an intact apico-basal polarity, we investigated bacterial transmigration across the monolayer, which was partially depolarized by HtrA, as indicated by microscopy, the analyses of the transepithelial electrical resistance (TEER) and colony forming unit (cfu) assays. Finally, we investigated CagA injection and observed efficient CagA translocation and tyrosine phosphorylation in 2D primary cells and, to a lesser extent, similar effects in mucosoids. In summary, HtrA is a crucially important factor promoting the multistep pathogenesis of H. pylori in non-transformed primary gastric epithelial cells and organoid-based epithelial models.

Construction and Identification of a Human Colorectal Adenoma Epithelial Cell Line by SV40 Large T-Antigen Transfection.

Colorectal adenoma represents the critical step in the development of colorectal cancer. The establishment of an immortalized epithelial cell line of colorectal adenoma of human origin would provide a tool for studying the mechanism of precancerous lesions, screening the efficacy of novel drugs, and constructing in vivo disease models. Currently, there is no commercially available stable supply of epithelial cells from precancerous lesions. This study aimed to establish a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection. Simian vacuolating virus 40(SV40), SV40-LT overexpressed lentivirus vector, was transfected into primary human colorectal adenomatous polyp epithelial cells. The transfected cells were screened, and the screened cells were amplified to obtain the epithelial cell line: IHCRA- CELL. The cells were identified by morphological observation, cell proliferation, Quantitative real-time PCR (qPCR), and Short Tandem Repeats (STR) experiments. Morphologically, the cells showed epithelial-like characteristics, such as polygon shape, desmosomes mitochondria, and strong positive keratin staining. There was no significant difference between the transfected cells and the primary cells. Through the STR identification experiment, no matching cell lines were found in the cell lines retrieval. We successfully established a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection, which has been patented and is now preserved in the Chinese Typical Culture Preservation Center. It was verified that the transformed cells maintained the phenotype and biological characteristics of epithelial cells. This cell line can be used to study the mechanism of precancerous lesions, screen the efficacy of novel drugs, and construct in vivo disease models.

Long-Term Maintenance of Viable Human Endometrial Epithelial Cells to Analyze Estrogen and Progestin Effects.

There are fewer investigations conducted on human primary endometrial epithelial cells (HPEECs) compared to human primary endometrial stromal cells (HPESCs). One of the main reasons is the scarcity of protocols enabling prolonged epithelial cell culture. Even though it is possible to culture HPEECs in 3D over a longer period of time, it is technically demanding. In this study, we successfully established a highly pure, stable, and long-term viable human conditionally reprogrammed endometrial epithelial cell line, designated as eCRC560. These cells stained positive for epithelial markers, estrogen and progesterone receptors, and epithelial cell-cell contacts but negative for stromal and endothelial cell markers. Estradiol (ES) reduced the abundance of ZO-1 in a time- and dose-dependent manner, in contrast to the dose-dependent increase with the progestin dienogest (DNG) when co-cultured with HPESCs. Moreover, ES significantly increased cell viability, cell migration, and invasion of the eCRC560 cells; all these effects were inhibited by pretreatment with DNG. DNG withdrawal led to a significantly disrupted monolayer of eCRC560 cells in co-culture with HPESCs, yet it markedly increased the adhesion of eCRC560 to the human mesothelial MeT-5A cells. The long-term viable eCRC560 cells are suitable for in vitro analysis of HPEECs to study the epithelial compartment of the human endometrium and endometrial pathologies.

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

The FAM13A Long Isoform Regulates Cilia Movement and Co-ordination in Airway Mucociliary Transport.

SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

High-magnitude cyclic stretch induces protein sialylation and RNA de-sialylation at the alveolar epithelial membrane

Sialylated glycans such as sialoglycoproteins, sialoglycolipids and sialoglycoRNA are ubiquitous in the glycocalyx of cells where they play a key role in barrier function, mechanosensation, infection, immune cell traffcking, and inflammation. These functions are particularly relevant at the blood-gas barrier of the lung. The alveolar epithelial glycocalyx is furthermore unique in that it is constantly exposed to cyclic stretch because of normal breathing. Yet, the abundance, regulation and function of sialylated glycans in the alveolar epithelial glycocalyx remains unknown. Here, we focused on the effects of mechanical stretch on the abundance and de novo formation of sialoglyco-proteins and -RNA as major constituents of the alveolar epithelial sialome. Human primary alveolar epithelial cells (hPAEpiC) were exposed to high magnitude cyclic stretch (18% CS) at 0.25 Hz for 24-48 hours to mimic biomechanical forces at the alveolar epithelium during high tidal volume ventilation. Total sialic acids in sialoglycoproteins were labeled with biotin by periodate oxidation and aldehyde ligation (pAL), and total sialic acids in sialoglycoRNA were quantified by 1, 2-diamino-4,5-methylene dioxybenzene ( DMB) assay. De novo formed sialic acids were labeled with biotin using N-azidoacetyl-mannosamine (Ac4ManNAz). Biotinylated siaolglyco-proteins and -RNAs were detected with streptavidin by Western and Northern blots, respectively, and quantified densitometrically. Stretch-dependent changes in the genes regulating sialic acid biosynthesis were assessed by RNA sequencing. High magnitude cyclic stretch increased the sialylation of proteins of different sizes (35-190 kDa) in the hPAEpiC glycocalyx. MGE showed this increase to be attributable to an increased de novo synthesis of sialic acids. On the other hand, high magnitude cyclic stretch reduced the sialylation of hPAEpiC sialoglycoRNA with no alteration in de novo synthesis, suggesting shedding, release or loss of sialoglycoRNA. RNA seq of hPAEpiC revealed that cyclic stretch upregulated the gene GNE (which encodes the enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), the master regulator of sialic acid biosynthesis. Here, we demonstrate cyclic stretch-induced sialylation of proteins and de-sialylation of RNA in the alveolar epithelial glycocalyx. Given that sialic acids promote the adhesion of both infectious pathogens and immune cells, these effects may contribute to the pathogenesis of ventilator-associated pneumonia and ventilator-induced lung injury. This work is supported by the German Research Foundation, CRC 1449 subproject B01 to MO &amp; WMK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AJAP1 Regulates Human Epithelial Cell Behaviors Required for Epicardium and EPDC Formation During Cardiac Development

Introduction: The epicardium forms the outermost layer of the heart. It is composed of a single layer of specialized epithelial cells that make important cellular contributions to cardiovascular development. During embryonic development, the epicardium is a source of multipotent progenitor cells, growth factors and extracellular matrix (ECM) components. The epicardium gives rise to multipotent epicardial-derived cells (EPDCs) which undergo epithelial-to-mesenchymal transition and differentiate into non-myocyte cardiac lineages. It is also a source of paracrine signals essential for myocardial growth and coronary vessel formation that become dormant after gestation. Adult epicardial cells can be reactivated in response to myocardial injury or ischemia to express a fetal gene program for regenerative cardiac repair. The drive to harness epicardial cells for cardiac regeneration is tethered to its roles in cardiovascular development. Thus, it is necessary to identify molecules that regulate epicardial cell behaviors required for epicardium formation and EPDC differentiation. Using our Tbx5-deficient mouse model of defective epicardium formation ( Tbx5epi−/−), we identified a transcript that is novel to the heart. Adherens junction associated protein 1 is a cell adhesion molecule that is encoded by the AJAP1 transcript which was downregulated in Tbx5epi−/− mice with impaired epicardium formation. Our preliminary studies revealed that AJAP is expressed in epithelial cells of the epicardium during heart development. Silencing of AJAP1 in primary cultured human epithelial cells reduces their ability to migrate. A role for AJAP1 in the epicardium during cardiac development remains to be identified. Study Objective: This study was designed to investigate the impact of AJAP1 on epithelial cell functions that are required for formation of the epicardium and differentiation of EPDCs. Methods: Studies were conducted in a primary human epithelial cell line, HMEpiC, which served as an in vitro model system to examine the contributions of AJAP1 to epithelial cell behaviors. HMEpiCs were transfected with small interfering RNAs (siRNAs) targeting either human AJAP1 to knock down its endogenous expression or a non-targeting negative control siRNA. AJAP1 knockdown was confirmed by quantitative PCR and immunofluorescence. We conducted assays on control and AJAP1 knockdown (KD) cells to examine the effects on epithelial cell behaviors contributing to epicardium formation and EPDC differentiation. This included analysis of cell proliferation, apoptosis, and adhesion to extracellular matrices. Results: Endogenous AJAP1 mRNA expression was reduced significantly by siRNA-mediated knockdown in human epithelial cells. We observed changes in cell morphology that correlated with a significant increase in apoptosis in AJAP1 KD cells compared to negative control cells. Proliferation of HMEpiCs was unchanged by AJAP1 knockdown. Also, we observed a significant increase in the adhesion of AJAP1 KD cells to certain extracellular matrices versus control cells. Conclusion: These findings provide evidence for the AJAP1 cell adhesion molecule to regulate epithelial cell behaviors that are crucial to formation of the epicardium and differentiation of EPDCs during cardiac development. PCOM Center for Chronic Disorders of Aging; PCOM Division of Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-inflammatory effect of curcuminoids and their analogs in hyperosmotic human corneal limbus epithelial cells

BackgroundTo assess the efficacy of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin [BDC]) and their analogs (tetrahydrocurcumin [THC], tetrahydrodemethoxycurcumin [THDC], tetrahydrobisdemethoxycurcumin) in reducing inflammatory cytokines and their toxicity to primary human corneal limbal epithelial cells, these cells were cultured and exposed to these compounds.MethodsThe PrestoBlue assay assessed cell viability after treatment. Anti-inflammatory effects on hyperosmotic cells were determined using real-time polymerase chain reaction and significance was gauged using one-way analysis of variance and Tukey’s tests, considering p-values < 0.05 as significant.ResultsCurcuminoids and their analogs, at 1, 10, and 100 µM, exhibited no effect on cell viability compared to controls. However, cyclosporin A 1:500 significantly reduced cell viability more than most curcuminoid treatments, except 100 µM curcumin and BDC. All tested curcuminoids and analogs at these concentrations significantly decreased mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-17 A, matrix metallopeptidase-9, and intercellular adhesion molecule-1 after 90 mM NaCl stimulation compared to untreated cells. Furthermore, proinflammatory cytokine levels from hyperosmotic cells treated with 1, 10, and 100 µM curcumin, 100 µM BDC, 100 µM THC, 1 and 100 µM THDC mirrored those treated with cyclosporin A 1:500.ConclusionThe anti-inflammatory efficiency of 1 and 10 µM curcumin, 100 µM THC, 1 and 100 µM THDC was comparable to that of cyclosporin A 1:500 while maintaining cell viability.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

In vitro co-culture of Clostridium scindens with primary human colonic epithelium protects the epithelium against Staphylococcus aureus.

A complex and dynamic network of interactions exists between human gastrointestinal epithelium and intestinal microbiota. Therefore, comprehending intestinal microbe-epithelial cell interactions is critical for the understanding and treatment of intestinal diseases. Primary human colonic epithelial cells derived from a healthy human donor were co-cultured with Clostridium scindens (C. scindens), a probiotic obligate anaerobe; Staphylococcus aureus (S. aureus), a facultative anaerobe and intestinal pathogen; or both bacterial species in tandem. The co-culture hanging basket platform used for these experiments possessed walls of controlled oxygen (O2) permeability to support the formation of an O2 gradient across the intestinal epithelium using cellular O2 consumption, resulting in an anaerobic luminal and aerobic basal compartment. Both the colonic epithelial cells and C. scindens remained viable over 48h during co-culture. In contrast, co-culture with S. aureus elicited significant damage to colonic epithelial cells within 24h. To explore the influence of the intestinal pathogen on the epithelium in the presence of the probiotic bacteria, colonic epithelial cells were inoculated sequentially with the two bacterial species. Under these conditions, C. scindens was capable of repressing the production of S. aureus enterotoxin. Surprisingly, although C. scindens converted cholic acid to secondary bile acids in the luminal medium, the growth of S. aureus was not significantly inhibited. Nevertheless, this combination of probiotic and pathogenic bacteria was found to benefit the survival of the colonic epithelial cells compared with co-culture of the epithelial cells with S. aureus alone. This platform thus provides an easy-to-use and low-cost tool to study the interaction between intestinal bacteria and colonic cells in vitro to better understand the interplay of intestinal microbiota with human colonic epithelium.

Preparation and characterization of citreorosein nanoparticles with improved bioavailability and cytotoxic potential

The present study was designed to improve the aqueous solubility and dissolution rate of citreorosein, a poorly water soluble drug, by producing its nanoparticles (NPs) using an “antisolvent precipitation with a syringe pump” (APSP) method. Various process parameters including flow rate, stirring speed, temperature, drug concentration and solvent-antisolvent ratio were investigated and optimized to obtain the smallest particle size of citreorosein. The prepared NPs were subjected to different analytical techniques such as SEM, FTIR, XRD and DLS. The NPs were tested for different dissolution parameters including difference (f1) and similarity factors (f2), dissolution efficiency (DE) and mean dissolution time (MDT). The antioxidant potential of citreorosein and its NPs were evaluated using DPPH, ABTS, and FRAP models, while their cytotoxicity was tested on various cancer cell lines such as breast (MDA-MB-231), lung (A549) and liver (HepG2), using the MTT assay. The specificity of the test compound was investigated against two normal human primary epithelial cells including renal (HRPTEpiC) and alveolar (HPAEpiC) cells. DLS analysis revealed that the prepared NPs were less than 200 nm in size while SEM and XRD confirmed their rod shape and amorphous nature, respectively. Citreorosein-NPs exhibited enhanced solubility and dissolution rate in all medias as compared to pure citreorosein. The NPs displayed significant antioxidant effects against ABTS, DPPH and FRAP models, with IC50 values of22.6, 21.45 and 27.30 µg/ml, respectively. Moreover, it showed significant cytotoxic activity against MDA-MB-231, A549 and HepG2 cells, with IC50 values of 3.45, 4.5 and 6.23 µg/ml, respectively. Furthermore, the NPs demonstrated high selectivity index values for the aforementioned cell lines as compared to reference drugs. This study demonstrated that APSP method successfully produced citreorosein-NPs and hence, showed better aqueous solubility, dissolution rate, antioxidant and cytotoxic activities than the pure compound.

Transcriptome exploration of ferroptosis-related genes in TGFβ- induced lens epithelial to mesenchymal transition during posterior capsular opacification development

BackgroundPosterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFβ) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO.MethodsRNA sequencing (RNA-seq) was performed on both TGF-β2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR).ResultsOur analysis revealed 1253 DEGs between TGF-β2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq.ConclusionsOur RNA-Seq data identified 38 ferroptosis-related genes in TGF-β2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-β2 stimulation.Graphical Our study initially observed ferroptosis related genes in primary human lens epithelial cells stimulated by TGF-β2. These findings may improve the understanding of the molecular mechanisms of PCO and provide a new direction for exploring the potential mechanisms of PCO

Identification and characterization of endogenous retroviruses upon SARS-CoV-2 infection.

Endogenous retroviruses (ERVs) derived from the long terminal repeat (LTR) family of transposons constitute a significant portion of the mammalian genome, with origins tracing back to ancient viral infections. Despite comprising approximately 8% of the human genome, the specific role of ERVs in the pathogenesis of COVID-19 remains unclear. In this study, we conducted a genome-wide identification of ERVs in human peripheral blood mononuclear cells (hPBMCs) and primary lung epithelial cells from monkeys and mice, both infected and uninfected with SARS-CoV-2. We identified 405, 283, and 206 significantly up-regulated transposable elements (TEs) in hPBMCs, monkeys, and mice, respectively. This included 254, 119, 68, and 28 ERVs found in hPBMCs from severe and mild COVID-19 patients, monkeys, and transgenic mice expressing the human ACE2 receptor (hACE2) and infected with SARS-CoV-2. Furthermore, analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT) revealed certain parental genomic sequences of these up-regulated ERVs in COVID-19 patients may be involved in various biological processes, including histone modification and viral replication. Of particular interest, we identified 210 ERVs specifically up-regulated in the severe COVID-19 group. The genes associated with these differentially expressed ERVs were enriched in processes such as immune response activation and histone modification. HERV1_I-int: ERV1:LTR and LTR7Y: ERV1:LTR were highlighted as potential biomarkers for evaluating the severity of COVID-19. Additionally, validation of our findings using RT-qPCR in Bone Marrow-Derived Macrophages (BMDMs) from mice infected by HSV-1 and VSV provided further support to our results. This study offers insights into the expression patterns and potential roles of ERVs following viral infection, providing a valuable resource for future studies on ERVs and their interaction with SARS-CoV-2.

Emissions from plastic incineration induce inflammation, oxidative stress, and impaired bioenergetics in primary human respiratory epithelial cells.

Inhalation exposure to plastic incineration emissions (PIEs) is a problem of increasing human relevance, as plastic production and waste creation have drastically increased since mainstream integration during the 20th century. We investigated the effects of PIEs on human nasal epithelial cells (HNECs) to understand if such exposures cause damage and dysfunction to respiratory epithelia. Primary HNECs from male and female donors were cultured at air-liquid interface (ALI), and 16HBE cells were cultured on coverslips. Smoke condensates were generated from incineration of plastic at flaming (640°C) and smoldering (500°C) temperatures, and cells were subsequently exposed to these materials at 5-50 μg/cm2 concentrations. HNECs were assessed for mitochondrial dysfunction and 16HBE cells for glutathione oxidation in real-time analyses. HNEC culture supernatants and total RNA were collected at 4-h postexposure for cytokine and gene expression analysis, and results show that PIEs can acutely induce inflammation, oxidative stress, and mitochondrial dysfunction in HNECs, and that incineration temperature modifies biological responses. Specifically, condensates from flaming and smoldering PIEs significantly increased HNEC secretion of cytokines IL-8, IL-1β, and IL-13, as well as expression of xenobiotic metabolism pathways and genes such as CYP1A1 and CYP1B1 at 5 and 20 μg/cm2 concentrations. Only 50 μg/cm2 flaming PIEs significantly increased glutathione oxidation in 16HBEs, and decreased respiration and ATP production in HNEC mitochondria. Impact Statement: Our data reveal the impact of incineration temperatures on biological outcomes associated with PIE exposures, emphasizing the importance of temperature as a factor when evaluating respiratory disease associated with PIEs exposure.

Abstract 5899: Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy

Abstract Recent research into prostate cancer treatment has unveiled a promising contender known as Up284. In laboratory experiments, this compound has exhibited a remarkable preference for targeting prostate cancer cells as opposed to normal cells. Specifically, in vitro cell viability assays using MTT have shown that prostate cancer cells display high sensitivity to Up284 at low nanomolar IC50 values, while primary human prostate epithelial cells and mouse keratinocytes exhibit minimal sensitivity. This establishes a notable therapeutic window between cancerous and non-cancerous cells, with Up284 significantly inhibiting colony formation in PC3 cells. Additional functional assays have confirmed Up284's potential, including the 4UbFL Reporter Gene assay, which has revealed robust, dose-dependent proteasome inhibition. This effect is supported by the accumulation of polyubiquitinated proteins in various prostate cancer cell lines following Up284 treatment, suggesting a distinct mechanism of toxicity compared to bortezomib. Furthermore, Up284 has been found to induce apoptosis in PC3 cells, surpassing the efficacy of bortezomib. Competitive binding assays have shown that Up284 exhibits a stronger binding affinity to the proteasomal receptor RPN13 than the prototype inhibitor RA190B. This is further supported by a Cellular Thermo Stabilization Assay (CETSA), which indicates that Up284 stabilizes RPN13 in cells. A preliminary in vivo study utilizing the bone metastatic prostate cancer genetically engineered mouse model(BMPC GEMM) model for metastatic prostate adenocarcinoma has demonstrated that Up284 leads to significant tumor regression and increased levels of poly-ubiquitinated proteins in residual tumor tissues. Moreover, when Up284 is conjugated with an E3 ligase ligand, it successfully degrades RPN13, resulting in the death of PC3 cells. Up284 has also shown favorable pharmacokinetics/pharmacodynamics (PK/PD) profiles and safety in animal models. It is well-tolerated in mice and holds promise for oral administration, demonstrating a proteasome-dependent reporter protein stabilization effect superior to ixazomib. Importantly, Up284 has induced tumor regression and improved survival rates in various cancer models, including ovarian and breast cancers. In conclusion, Up284 emerges as a lead candidate for targeted prostate cancer therapy due to its high specificity, substantial therapeutic index, and potent anti-cancer activity both in vitro and in vivo. Further development and clinical investigation of Up284 are warranted to fully explore its therapeutic potential. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung Chang. Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5899.

Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression. Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3027.