Human Epithelium Research Articles

Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model.

Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.

FABP4 Is an Indispensable Factor for Regulating Cellular Metabolic Functions of the Human Retinal Choroid.

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells.

Myricitrin inhibited ferritinophagy-mediated ferroptosis in cisplatin-induced human renal tubular epithelial cell injury.

Cisplatin-induced acute kidney injury (AKI) increases the patient mortality dramatically and results in an unfavorable prognosis. A strong correlation between AKI and ferroptosis, which is a notable type of programmed cell death, was found in recent studies. Myricitrin is a natural flavonoid compound with diverse pharmacological properties. To investigate the protective effect of myricitrin against cisplatin induced human tubular epithelium (HK-2) cell injury and the underlying anti-ferroptic mechanism by this study. Firstly, a pharmacology network analysis was proposed to explore the myricitrin's effect. HK-2 cells were employed for in vitro experiments. Ferroptosis was detected by cell viability, quantification of iron, malondialdehyde, glutathione, lipid peroxidation fluorescence, and glutathione peroxidase (GPX4) expression. Ferritinophagy was detected by related protein expression (NCOA4, FTH, LC3II/I, and SQSTM1). In our study, GO enrichment presented that myricitrin might be effective in eliminating ferroptosis. The phenomenon of ferroptosis regulated by ferritinophagy was observed in cisplatin-activated HK-2 cells. Meanwhile, pretreatment with myricitrin significantly rescued HK-2 cells from cell death, reduced iron overload and lipid peroxidation biomarkers, and improved GPX4 expression. In addition, myricitrin downregulated the expression of LC3II/LC3I and NCOA4 and elevated the expression of FTH and SQTM. Furthermore, myricitrin inhibited ROS production and preserved mitochondrial function with a lower percentage of green JC-1 monomers. However, the protection could be reserved by the inducer of ferritinophagy rapamycin. Mechanically, the Hub genes analysis reveals that AKT and NF-κB are indispensable mediators in the anti-ferroptic process. In conclusion, myricitrin ameliorates cisplatin induced HK-2 cells damage by attenuating ferritinophagy mediated ferroptosis.

Structures and functions of the normal and injured human olfactory epithelium.

The olfactory epithelium (OE) is directly exposed to environmental agents entering the nasal cavity, leaving OSNs prone to injury and degeneration. The causes of olfactory dysfunction are diverse and include head trauma, neurodegenerative diseases, and aging, but the main causes are chronic rhinosinusitis (CRS) and viral infections. In CRS and viral infections, reduced airflow due to local inflammation, inflammatory cytokine production, release of degranulated proteins from eosinophils, and cell injury lead to decreased olfactory function. It is well known that injury-induced loss of mature OSNs in the adult OE causes massive regeneration of new OSNs within a few months through the proliferation and differentiation of progenitor basal cells that are subsequently incorporated into olfactory neural circuits. Although normal olfactory function returns after injury in most cases, prolonged olfactory impairment and lack of improvement in olfactory function in some cases poses a major clinical problem. Persistent inflammation or severe injury in the OE results in morphological changes in the OE and respiratory epithelium and decreases the number of mature OSNs, resulting in irreversible loss of olfactory function. In this review, we discuss the histological structure and distribution of the human OE, and the pathogenesis of olfactory dysfunction associated with CRS and viral infection.

In vitro and in vivo studies of ocular topically administered NLC for the treatment of uveal melanoma

Uveal melanoma is one of the most common and aggressive intraocular malignancies, and, due to its great capability of metastasize, it constitutes the most incident intraocular tumor in adults. However, to date there is no effective treatment since achieving the inner ocular tissues still constitutes one of the greatest challenges in actual medicine, because of the complex structure and barriers. Uncoated and PEGylated nanostructured lipid carriers were developed to achieve physico-chemical properties (mean particle size, homogeneity, zeta potential, pH and osmolality) compatible for the ophthalmic administration of (S)-(–)-MRJF22, a new custom-synthetized prodrug for the potential treatment of uveal melanoma. The colloidal physical stability was investigated at different temperatures by Turbiscan® Ageing Station. Morphology analysis and mucoadhesive studies highlighted the presence of small particles suitable to be topically administered on the ocular surface. In vitro release studies performed using Franz diffusion cells demonstrated that the systems were able to provide a slow and prolonged prodrug release. In vitro cytotoxicity test on Human Corneal Epithelium and Human Uveal Melanoma cell lines and Hen’s egg-chorioallantoic membrane test showed a dose-dependent cytotoxic effect of the free prodrug on corneal cells, whose cytocompatibility improved when encapsulated into nanoparticles, as also confirmed by in vivo studies on New Zealand albino rabbits. Antiangiogenic capability and preventive anti-inflammatory properties were also investigated on embryonated eggs and rabbits, respectively. Furthermore, preliminary in vivo biodistribution images of fluorescent nanoparticles after topical instillation in rabbits’ eyes, suggested their ability to reach the posterior segment of the eye, as a promising strategy for the treatment of choroidal uveal melanoma.

Preclinical development of ATR04-484, an auxotrophic strain of Staphylococcus epidermidis, for the topical treatment of epidermal growth factor receptor (EGFR) inhibitor-induced dermal toxicity.

e24074 Background: Agents targeting the EGFR-mediated signaling pathway are commonly used for the treatment of advanced lung, pancreatic, colorectal, and head and neck cancers. Significant dermal toxicities, which occur in up to 90% of patients treated with EGFR inhibitors (EGFRis) and other therapies inhibiting downstream signaling pathways, may be disruptive to a patient’s quality of life and adherence to therapy. Inhibition of the EGFR pathway may suppress host defenses and lead to opportunistic pathogenic colonization or infection. EGFRi-induced dermal toxicity is associated with elevated levels of Staphylococcus aureus and IL-36γ. ATR04-484 is a topical ointment containing S. epidermidis strain SE484, isolated from a healthy human volunteer that was selected for its ability to inhibit S. aureus and IL-36γ . Methods: Reconstructed human epidermis (RHE) and ex vivo pig skin were used to measure the effect of ATR04-484 on S. aureus in therapeutic and prophylactic settings with S. aureus added prior to or after ATR04-484, respectively. To evaluate the anti-inflammatory effects of ATR04-484, IL-36γ levels were measured on untreated RHE and RHE treated with erlotinib alone or in combination with ATR04-484. 104 colony-forming units (CFU) of methicillin-resistant (MRSA) or methicillin-sensitive (MSSA) S. aureus were used. Toxicology was assessed by measuring cell viability in three in vitro toxicology assays: the eye irritant potential was tested on 3D reconstituted human cornea-like epithelium (RhCE), the skin irritant potential of the strain in the ointment was assessed on 3D RHE, and the skin corrosive potential was evaluated on RHE. Results: ATR04-484 comparably inhibited growth of both MRSA and MSSA in therapeutic and prophylactic settings. In a therapeutic setting, ATR04-484 inhibited MRSA growth by 1 log (90%) compared to untreated MRSA in both RHE and pig skin. In a prophylactic setting, ATR04-484 inhibited MRSA growth by approximately 5 logs on RHE and 2 logs on pig skin. Additionally, application of ATR04-484 reduced IL-36γ to a level comparable to untreated RHE. The effect was dose-dependent; application of 109CFU/cm2 of ATR04-484 showed more potent IL-36γ reduction compared to 108 CFU/cm2. The three toxicology assays indicate that ATR04-484 does not cause irritation or corrosion. Conclusions: In these preclinical studies, ATR-04-484 showed a promising preclinical profile for the treatment of EGFRi-induced dermal toxicity by significantly reducing S. aureus growth, completely ameliorating IL-36γ levels, and showing no safety concerns.

Development and characterisation of a novel complex triple cell culture model of the human alveolar epithelial barrier

Owing to increased pressure from ethical groups and the public to avoid unnecessary animal testing, the need for new, responsive and biologically relevant in vitro models has surged. Models of the human alveolar epithelium are of particular interest since thorough investigations into air pollution and the effects of inhaled nanoparticles and e-cigarettes are needed. The lung is a crucial organ of interest due to potential exposures to endogenous material during occupational and ambient settings. Here, an in vitro model of the alveolar barrier has been created in preparation for use in the quasi-air liquid interface (qALI) and (aerosol) air–liquid interface (ALI) exposures. The model consists of an alveolar type 1-like cell line (TT1), an alveolar type 2-like cell line (NCI-H441) and a model of (alveolar) macrophages (dTHP-1). The model formulates a complex, multi-cellular system, cultured at the air–liquid interface, that mimics the apical layer of the alveolar epithelial region in the human lung. Characterisation data has shown that both TT1 and NCI-H441 epithelial cells are able to be cultured together in addition to dTHP-1 cells through imaging (morphology), pro-inflammatory response and viability measurements. This dataset also demonstrates evidence of a reasonable barrier created by the cell culture in comparison to negative controls. Furthermore, it shows that while maintaining a low baseline of (pro)-inflammatory mediator expression during normal conditions, the model is highly responsive to inflammatory stimuli. This model is proposed to be suitable for use in toxicology testing of inhaled exogenous agents.

Multi-omics profiling of mouse polycystic kidney disease progression at a single cell resolution.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

Comparative analysis of the effect of drugs lowering intraocular pressure on a primary culture of human corneal epithelium and A549 immortalized cell line

Background. Glaucoma as one of the most common eye diseases can be a comorbid condition of an epithelial corneal defect of various etiologies. Maintaining an optimal level of ophthalmotonus includes the prescription of antiglaucoma drugs, including benzalkonium chloride-preserved drugs.The aim of the study. To compare the effect of antiglaucoma drugs, as well as benzalkonium chloride (BC), on a primary culture of human corneal epithelium and A549 immortalized cell line.Methods. The effect of brimonidine, dorzolamide and timolol (1/100, 1/50, 1/20 and 1/10 dilutions; exposure 24 hours) on a monolayer of a human corneal epithelial primary culture and A549 immortalized cell line was assessed by structural changes in cells (phase-contrast microscopy) and MTT assay data. The cytotoxic effect of BC was studied in concentrations corresponding to its content in these dilutions of the antiglaucoma drug. Using a model of a linear defect in the monolayer of a corneal epithelial primary culture and A549 immortalized cell line, the effects of brimonidine, dorzolamide and timolol (1/100 and 1/20 dilutions; exposure 48 hours) were assessed by cell migration activity.Results. Among drugs (BC-free), dorzolamide (1/50, 1/20 and 1/10 dilutions) causes minor structural changes in human corneal epithelium and A549 immortalized cell line; timolol (1/100, 1/50, 1/20 and 1/10 dilutions) causes minor structural changes in A549 immortalized cell line. Structural changes in both types of cells, a decrease in their metabolic and migration activity occur under the influence of dorzolamide, brimonidine and timolol (BC-preserved) in 1/100, 1/50, 1/20 and 1/10 dilutions. BC at the studied concentrations exhibits a similar effect.Conclusion. The cytotoxic effect of antiglaucoma drugs is caused by the presence of BC in their composition. Despite similar morphofunctional changes in cells, A549 immortalized cell line is more resistant to the effects of drugs than the human corneal epithelial primary culture. When using it as a cellular model, it is advisable to change the experimental conditions (duration of exposure and concentration of the studied drug).

Enhancing Corneal Drug Penetration Using Penetratin for Ophthalmic Suspensions.

Eye drops, including solutions and suspensions, are essential dosage forms to treat ophthalmic diseases, with poorly water-soluble drugs typically formulated as ophthalmic suspensions. In addition to low bioavailability, suspensions exhibit limited efficacy, safety, and usability due to the presence of drug particles. Improving bioavailability can reduce the drug concentrations and the risk of problems associated with suspended drug particles. However, practical penetration enhancers capable of improving bioavailability remain elusive. Herein, we focused on penetratin (PNT), a cell-penetrating peptide (CPP) that promotes active cellular transport related to macromolecule uptake, such as micropinocytosis. According to the in vitro corneal uptake study using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24, PNT enhanced the uptake of Fluoresbrite® YG carboxylate polystyrene microspheres without covalent binding. In an ex vivo porcine eye model, the addition of 10 µM PNT to rebamipide ophthalmic suspension markedly improved the corneal uptake of rebamipide; however, the addition of 100 µM PNT was ineffective due to potentially increased particle size by aggregation. This article provides basic information on the application of PNT as a penetration enhancer in ophthalmic suspensions, including the in vitro and ex vivo studies mentioned above, as well as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay and storage stability at different pH values.

Unraveling Divergent Transcriptomic Profiles: A Comparative Single-Cell RNA Sequencing Study of Epithelium, Gingiva, and Periodontal Ligament Tissues.

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions.

Faecalibacterium prausnitzii A2-165 metabolizes host- and media-derived chemicals and induces transcriptional changes in colonic epithelium in GuMI human gut microphysiological system.

Aim: Recently, a GuMI gut microphysiological system has been established to coculture oxygen-intolerant Faecalibacterium prausnitzii (F. prausnitzii) A2-165 with organoids-derived primary human colonic epithelium. This study aims to test if this GuMI system applies to different donors with different healthy states and uses metabolomics to reveal the role of gut microbes in modulating host- and diet-derived molecules in the gut lumen. Methods: Organoids-derived colonic monolayers were generated from an uninflamed region of diverticulitis, ulcerative colitis, and Crohn's disease patients and then integrated into the GuMI system to coculture with F. prausnitzii A2-165 for 2 to 4 days. Apical media was collected for metabolomic analysis. Targeted metabolomics was performed to profile 169 polar chemicals under three conditions: conventional static culture without bacteria, GuMI without bacteria, and GuMI with F. prausnitzii. The barrier function of monolayers was measured using transepithelial resistance. Results: GuMI successfully cocultured patient-derived monolayers and F. prausnitzii for up to 4 days, with active bacterial growth. Introducing flow and oxygen gradient significantly increases the barrier function, while exposure to F. prausnitzii slightly increases the barrier function. Targeted metabolomics screened 169 compounds and detected 76 metabolites, of which 70 significantly differed between at least two conditions. F. prausnitzii significantly modulates the levels of nucleosides, nucleobases, and amino acids on the apical side. Further analysis suggests that F. prausnitzii changes the mRNA level of 260 transcription factor genes in colonic epithelial cells. Conclusion: The GuMI physiomimetic system can maintain the coculture of F. prausnitzii and colonic epithelium from different donors. Together with metabolomics, we identified the modulation of F. prausnitzii in extracellular chemicals and colonic epithelial cell transcription in coculture with human colonic epithelium, which may reflect its function in gut lumen in vivo.

Gold Nanoparticles: Tunable Characteristics and Potential for Nasal Drug Delivery.

A general procedure to prepare gold nanourchins (GNUs) via a seed-mediated method was followed using dopamine hydrochloride as a reducing agent and silver nitrate salt (AgNO3) as a shape-directing agent. The novelty of this study comes from the successful incorporation of the prepared gold urchins as an aqueous suspension in a nasal pressurized metered dose inhaler (pMDI) formulation and the investigation of their potential for olfactory targeting for direct nose-to-brain drug delivery (NTBDD). The developed pMDI formulation was composed of 0.025% w/w GNUs, 2% w/w Milli-Q water, and 2% w/w EtOH, with the balance of the formulation being HFA134a propellant. Particle integrity and aerosolization performance were examined using an aerosol exposure system, whereas the nasal deposition profile was tested in a sectioned anatomical replica of human nasal airways. The compatibility of the gold dispersion with the nasal epithelial cell line RPMI 2650 was also investigated in this study. Colloidal gold was found to be stable following six-month storage at 4 °C and during the lyophilization process utilizing a pectin matrix for complete re-dispersibility in water. The GNUs were intact and discrete following atomization via a pMDI, and 13% of the delivered particles were detected beyond the nasal valve, the narrowest region in the nasal cavity, out of which 5.6% was recovered from the olfactory region. Moreover, the formulation was found to be compatible with the human nasal epithelium cell line RPMI 2650 and excellent cell viability was observed. The formulated GNU-HFA-based pMDI is a promising approach for intranasal drug delivery, including deposition in the olfactory region, which could be employed for NTBDD applications.

Influence of Vitamin D Receptor Signalling and Vitamin D on Colonic Epithelial Cell Fate Decisions in Ulcerative Colitis.

Epidemiological studies have shown that subnormal levels of vitamin D (25[OH]D) are associated with a more aggravated clinical course of ulcerative colitis [UC]. Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor [VDR] signalling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25[OH]2D3/VDR) signalling in human organoids could influence the maintenance of the colonic epithelium. Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterise the functional and transcriptional effects of 1,25[OH]2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9. Our results suggest that 1,25[OH]2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25[OH]2D3/VDR signalling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC. We identified an important role of vitamin D signalling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signalling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC.

IN VITRO PERMEABILITY STUDY OF NASAL MEDICINAL PRODUCT FOR SYSTEMIC ACTION

For systemic medicinal products bioavailability is a fundamental characteristic that determines their efficacy and onset of action. Biopharmaceutical parameters of nasal preparations (physicochemical properties of the active substance such as membrane permeability, solubility, lipophilicity, pKa, polymorphic state, type of finished dosage form, pH, osmolarity, composition and characteristics of excipients), affecting bioavailability, are variable and controllable within pharmaceutical development. Besides the use of components that affect enzymatic activity, mucoadhesion and effective con-tact time, mucociliary clearance and viscosity of mucous secretion, changing the rate and degree of absorption of the active substance is possible by modulation of tight junctions and paracellular transport. These processes can be particularly determined by the osmolarity of the dosage form and the use of permeation enhancers. Taking into account the portfolio of excipients available to scientists used in a wide range of concentrations, and, in addi-tion, the requirements of the target product profile in terms of safety and pharmacokinetic parameters, as well as regulatory recommendations for jus-tification of the composition within pharmaceutical development, with particular emphasis on the selection of functional components and the concen-trations used, it is important to have relevant and reliable screening tools when formulating a drug product. Due to the absence of standard bioavailabil-ity assessment methods for nasal preparations, literature review was conducted and methodology of in vitro permeability study for of nasal medicinal products for systemic action with the use of artificial membranes as well as cell line RPMI 2650 of human nasal epithelium is developed. The use of such sufficiently homogeneous barrier films allows to minimize the variability of test conditions. Permeation study of developed compositions of an-timigraine medicinal product is conducted. The results obtained provide evidence of their qualitative compliance and allow to recommend laboratory permeability study on cellulose membranes for the selection and justification of the composition of pharmaceutical compositions. Bioavailability is a fundamental characteristic of pharmaceutical products for systemic action, which defines efficacy and speed of action. Permeability through biological membranes is defined not only by the properties of active component but also by the particulars of formulation. Due to the absence of standard bioavailability assessment methods for nasal preparations, literature review was conducted and methodology of in vitro permeability study for of nasal medicinal products for systemic action with the use of artificial membranes as well as cell line RPMI 2650 is developed. Permeation study of developed compositions of antimigraine medicinal product is conducted. The results obtained provide evidence of their qualitative compliance and allow to recommend laboratory permeability study on cellulose membranes as a screening tool in pharmaceutical development.

ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification.

Dynamic changes in mechanical microenvironments, such as cell crowding, regulate lineage fates as well as cell proliferation. Although regulatory mechanisms for contact inhibition of proliferation have been extensively studied, it remains unclear how cell crowding induces lineage specification. Here we found that a well-known oncogene, ETS variant transcription factor 4 (ETV4), serves as a molecular transducer that links mechanical microenvironments and gene expression. In a growing epithelium of human embryonic stem cells, cell crowding dynamics is translated into ETV4 expression, serving as a pre-pattern for future lineage fates. A switch-like ETV4 inactivation by cell crowding derepresses the potential for neuroectoderm differentiation in human embryonic stem cell epithelia. Mechanistically, cell crowding inactivates the integrin-actomyosin pathway and blocks the endocytosis of fibroblast growth factor receptors (FGFRs). The disrupted FGFR endocytosis induces a marked decrease in ETV4 protein stability through ERK inactivation. Mathematical modelling demonstrates that the dynamics of cell density in a growing human embryonic stem cell epithelium precisely determines the spatiotemporal ETV4 expression pattern and, consequently, the timing and geometry of lineage development. Our findings suggest that cell crowding dynamics in a stem cell epithelium drives spatiotemporal lineage specification using ETV4 as a key mechanical transducer.

Intranasal PAMAM-G3 scavenges cell-free DNA attenuating the allergic airway inflammation

Allergic airway inflammation (AAI), including allergic rhinitis (AR) and allergic asthma, is driven by epithelial barrier dysfunction and type 2 inflammation. However, the underlying mechanism remains uncertain and available treatments are constrained. Consequently, we aim to explore the role of cell-free DNA (cfDNA) in AAI and assess the potential alleviating effects of cationic polymers (CPs) through cfDNA elimination. Levels of cfDNA were evaluated in AR patients, allergen-stimulated human bronchial epithelium (BEAS-2B cells) and primary human nasal epithelium from both AR and healthy control (HC), and AAI murine model. Polyamidoamine dendrimers-generation 3 (PAMAM-G3), a classic type of cationic polymers, were applied to investigate whether the clearance of cfDNA could ameliorate airway epithelial dysfunction and inhibit AAI. The levels of cfDNA in the plasma and nasal secretion from AR were higher than those from HC (P < 0.05). Additionally, cfDNA levels in the exhaled breath condensate (EBC) were positively correlated with Interleukin (IL)-5 levels in EBC (R = 0.4191, P = 0.0001). Plasma cfDNA levels negatively correlated with the duration of allergen immunotherapy treatment (R = −0.4297, P = 0.006). Allergen stimulated cfDNA secretion in vitro (P < 0.001) and in vivo (P < 0.0001), which could be effectively scavenged with PAMAM-G3. The application of PAMAM-G3 inhibited epithelial barrier dysfunction in vitro and attenuated the development of AAI in vivo. This study elucidates that cfDNA, a promising biomarker for monitoring disease severity, aggravates AAI and the application of intranasal PAMAM-G3 could potentially be a novel therapeutic intervention for AAI.Allergen stimulates the secretion of cell-free DNA (cfDNA) in both human and mouse airway. Intranasal polyamidoamine dendrimers-generation 3 (PAMAM-G3) scavenges cfDNA and alleviates allergic airway inflammation.

Cellular landscape of the esophageal epithelium in systemic sclerosis

Abstract Systemic sclerosis (SSc) is a rare autoimmune disease characterized by vasculopathy and progressive fibrosis of the skin and internal organs. Individuals with SSc often suffer from chronic acid reflux and dysphagia due to loss of esophageal motility, but this pathogenesis is poorly understood. Recently, studies have suggested that esophageal epithelial cells (EECs) may play a central role in the pathogenesis of SSc esophageal dysmotility. In this study, we performed a thorough transcriptomic investigation of the SSc esophageal epithelium in humans to determine whether distinct changes in EECs contribute to esophageal impairment in SSc. We performed single-cell RNA sequencing of paired proximal and distal esophageal mucosa biopsies from 10 individuals with SSc, 4 comparator individuals with gastroesophageal reflux disease (GERD), and 6 healthy controls (HCs), yielding 230,720 EECs across 40 samples. SSc and GERD samples had significantly fewer terminally differentiated, superficial cells than HCs, and differential gene expression between conditions was primarily limited to superficial EECs. Gene dysregulation in SSc was highly correlated with GERD, but was relatively greater in the proximal region, including unique dysregulation of immune mediators that correlated with esophageal dysmotility. This work sheds light on the cellular roots of esophageal dysfunction in SSc and serves as an atlas to guide future efforts to identify actionable targets in the esophagus in SSc.

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

TRPM3-mediated inhibition of active Na-K transport by the ocular lens epithelium

Purpose: The TRPM3 channel is selectively expressed in the lens epithelium, a monolayer at the anterior lens surface, but undetectable in fiber cells that make up the lens bulk. A point mutation in TRPM3 gene (substitution of isoleucine by methionine at codon 65; I65M) causes early onset autosomal dominant cataract in humans and in mice. From studies on transgenic mice, it is apparent the I65M TRPM3 mutation disrupts lens Na-K homeostasis. However, the normal role of lens TRPM3 in the lens is currently unknown. Here, we explore the lens response to TRPM3 activation. Methods: Studies were carried out using intact mouse lenses, cultured mouse lens epithelial cells and a human lens epithelium cell line (HLEB3). Active Na-K transport was studied by using rubidium (Rb) as a tracer for potassium entry. Rb was detected by atomic absorption spectrophotometry. Intracellular calcium was measured in nonconfluent single cells by Fura-2 ratiometric imaging. Results: Rb uptake was measured over a period of 10 min in intact mouse lenses exposed to a TRPM3 agonist CIM0216 (10 μM). CIM0216 reduced the rate of Rb uptake by ~20% (Ctrl 6.4±0.4 vs CIM 4.5±0.4 mmoles/kg lens dry weight/10 min, n=7, p&lt;.01). The concentration dependence of the response to CIM0216 (1 to 50 μM) was examined in primary cultured mouse lens epithelium and Rb uptake was reduced by CIM0216 concentrations &gt; 10 μM. The TRPM3 agonist reduced Rb uptake to a similar extent (~20%) in mouse lens epithelium, HLEB3 cells and HLEB3 cells that had been stably transfected with the I65M mutant TRPM3. However, the mutant cells responded to CIM0216 at a 10-fold lower concentration than WT mouse and HLEB3 cells. The Rb uptake response to CIM0216 was significantly inhibited by a TRPM3 antagonist, primidone (50 μM), and was abolished when cells were bathed in calcium-free solution. To study calcium entry, Fura2-loaded mouse lens epithelial cells were exposed to the TRPM3 agonist pregnenolone (100 μM). Pregnenolone caused an immediate increase in cytoplasmic calcium that was inhibited by primidone (Peak calcium concentration Ctrl 201±37 vs primidone 137±8 nM, n=5, p&lt;.001). In cultures lens epithelium, agents that increase cellular cAMP, including IBMX, forskolin and 8p-CPT-cAMP, all caused a marked reduction of Rb uptake. Conclusions: TRPM3 activation causes a significant reduction in the rate of active Na-K transport and the response is associated with calcium entry. Because most lens Rb uptake is ouabain-sensitive, the response points to Na,K-ATPase inhibition. Studies are under way to test whether the Na-K transport response to TRPM3 activation involves cAMP signaling. This work was supported by NIH grants R01EY029171 and R01EY009532. 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.