Per Artursson

The human ileal epithelium forms a critical interface governing interactions with luminal microorganisms and nanomaterials, yet physiologically relevant in vitro models with accessible apical surfaces remain limited. Here, human apical-out ileoids derived from healthy ileal biopsies are established as a platform that recapitulates the architecture and complexity of the native ileal epithelium. Assessment of donor- and passage-dependent variability identifies a representative ileoid line and a robust culture window. Integrated proteomic, imaging, ultrastructural, and functional analyses demonstrate preservation of key epithelial features, including brush-border organization, barrier integrity, absorptive capacity, and multiple endocytic pathways. Intact ileoids internalize nanoparticles of distinct surface charge, including empty lipid nanoparticles compositionally matched to approved mRNA vaccine formulations, establishing that uptake can be quantified across an unperturbed apical surface. This uptake is restrained by a retained mucin-rich glycocalyx. Cleavage of surface-attached mucins selectively enhances uptake of negatively charged nanoparticles, leaving near-neutral and positively charged nanoparticles unchanged, and increasing entry of Salmonella bacteria. These findings reveal the glycocalyx as a charge-selective regulator of epithelial uptake. In conclusion, our study provides a foundation for nanoparticle-epithelium investigations using human apical-out ileoids, and a versatile, translationally relevant platform for nanomedicine delivery, host-microbe interactions, and epithelial transport.

This work explores the applicability of new approach methodologies (NAMs) using a read-across approach to predict systemic toxicity with liver as the critical target organ, and derive a point of departurefor the target compound (TC) difenoconazole. Eight conazoles were used as source compounds (SCs), alongside metabolites hydroxyitraconazole and 1,2,4-triazole. The case study was structured according to the Alternative Safety Profiling Algorithm (ASPA) workflow developed within the RISK-HUNT3R project. Hierarchical clustering using chemical fingerprints identified similarity of different SCs to the TC, supporting their inclusion in the read-across analysis. A broad screening panel of NAMs, including nuclear receptor reporter assays, ToxProfiler, Cell Painting PLUS, transcriptomics in primary human hepatocytes and H295R steroidogenesis assays, was used to characterize biological activity of the TC and SCs and to substantiate the structure-based read-across. In a first prediction round, the nearest neighbour quantitative read-across approach (based on allometrically-scaled human lowest-observed-adverse-effect levels (LOAELs) of the SC) was used to predict 6.57 mg/kg/day as the LOAEL of the TC. This closely aligned with the in vivo-based value of 4.88 mg/kg/day. In a second approach, data from NAMs were used to predict human equivalent dose (HED), and the respective LOAELs of SC and TC, based on physiologically based kinetic modelling with quantitative in vitro to in vivo extrapolation. The HED derived from NAMs was approximately ten-fold lower than the LOAELs from traditional approaches. This case study illustrates suitable (ASPA-guided) approaches as well as the toxicological applicability of a NAM-based read-across assessment to obtain safe/conservative estimates of toxicity. Graphical Abstract A stepwise procedure was used in this work for assessment of systemic toxicity of the target compound, difenoconazole, using a read-across approach

Background and Purpose: Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of end‐stage renal disease with limited treatment options. Drug repurposing offers a promising strategy to find effective treatments. Experimental Approach: We identified birinapant, bardoxolone methyl and salicylic acid as repurposing candidates for ADPKD and applied thermal proteome profiling to identify disease‐relevant targets. Key Results: Our results uncovered shared effects between the three drugs, including a thermal shift in ADP/ATP translocases ( SLC25A4/5/6 ) caused by reduced ATP production. As expected, salicylic acid displayed a polypharmacological profile but also engaged enzymes along the N‐linked glycosylation pathway and inhibited PTK2 (FAK), an activator of the proliferative PI3K/Akt/mTOR pathway. Orthogonal assays and computational modelling confirmed these findings and located the binding site (499–504) of salicylic acid within PTK2 's ATP‐binding pocket. Engagement of several targets was achieved at clinically relevant concentrations. Conclusions and Implications: We have identified novel targets of three repurposing candidates that contribute to the cyst growth reducing effects in preclinical ADPKD models and identify salicylic acid, the metabolite of aspirin, as the repurposing candidate with the most promising mechanisms of action.

Gut pathogens such as Salmonella enterica serovar Typhimurium target intestinal epithelial cells for adhesion and type-3-secretion-system-dependent invasion, while also invading multiple other cell types as the infection progresses. Mechanistic studies have uncovered virulence factors involved in this process, but host cell determinants affecting Salmonella cell surface targeting remain less deeply explored. Furthermore, cell surface protein expression and glycosylation patterns differ dramatically between epithelial and blood-derived cell types, and even across maturation states of the same cell type. Here, we explored bottom-up reconstruction of the host cell surface, using simplistic suspension-growing K562 cells, to determine the contribution of individual cell surface constituents during Salmonella targeting. Combined with flow cytometry and a stringently tunable gene expression system, this model enabled high-throughput analysis and combined genetic manipulations in both pathogen and host cells. Transcriptomic and proteomic data along with lectin characterization revealed minimal K562 surface glycosylation at baseline. Chemical manipulations substantiated the role of cell membrane cholesterol in promoting Salmonella targeting, whereas ectopic expression of glycoproteins such as transmembrane mucins introduced a size-dependent steric barrier towards invading bacteria. Strikingly, even established glycoprotein receptors for Salmonella adhesins hampered rather than promoted invasion, suggesting that adhesins are required to overcome cellular glycocalyces in vivo, while a bare host cell membrane would be the pathogens preferred interaction surface.

Porous membranes are frequently used as supports of cell monolayers in functional studies of epithelial and endothelial barriers. However, commonly used conventional polymer-based membranes such as those made of polycarbonate (transwells) do not mimic the structural and biochemical properties of native basement membranes, which may limit cellular differentiation and function. Here, we use a nanofibrillar membrane that mimics the microenvironment of the basement membrane and is made of recombinant spider silk functionalized with the integrin-binding RGD motif of fibronectin and coated with human kidney-specific laminin-521 (FN-silk). The FN-silk membranes were evaluated as culture substrates for renal epithelial cells (RPTEC/TERT1), a cell type notoriously difficult to differentiate in culture. FN-silk membrane structure, cellular morphology, mRNA expression, barrier properties and transporter activity were assessed using scanning and transmission electron microscopy, RNA-sequencing, permeability and transport assays. Both FN-silk and conventional membranes supported barrier integrity and tight junction expression. In contrast to cultures on conventional membranes, the RPTEC monolayers on FN-silk exhibited a differentiated morphology, low expression of cell death markers, and, directional anion and cation transport. Further, the commonly used conventional membranes released endocrine-disrupting bisphenols that activated estrogen-mediated signaling. In summary, these findings indicate that FN-silk membranes with kidney-specific basement membrane laminin 521 reduces cellular stress, supports and maintains cellular differentiation and preserves important cellular functions. Our work establishes FN-silk membranes as a next-generation biomaterial that enables the differentiation of renal epithelial monolayers into a physiologically relevant in vitro model.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. Its progressively expanding, fluid‐filled renal cysts eventually lead to end‐stage renal disease. Despite the relatively high prevalence, treatment options are currently limited to a single drug approved by the FDA and EMA. Here, we investigated human ADPKD patient‐derived three‐dimensional cyst cultures (3DCC) as an in vitro model for ADPKD and drug repurposing research. First, we analyzed the proteomes of 3DCC derived from healthy and diseased tissues. We then compared the protein expression profiles with those of reference tissues, mainly from the same patients. We quantified 290 proteins affecting drug disposition and proposed target proteins for drug treatment. Lastly, we investigated the functional response of the quantified target proteins after exposure to repurposing candidates in the 3DCC. Proteomic profiling of human 3DCC reflected previously reported pathophysiological alterations, including aberrant protein expression in inflammation and metabolic reprogramming. While the 3DCCs largely recapitulated the disease phenotype in vitro, drug transporter expression was reduced compared to in vivo conditions. Target proteins for proposed repurposing candidates showed similar expression in vitro and in tissues. Exposure to these repurposing candidates inhibited cyst swelling in vitro, supporting the suitability of the 3DCC for ADPKD drug screening. In summary, our results provide new insights into the ADPKD proteome and offer a starting point for further research to improve treatment options for affected individuals.

Intestinal enteroids are stem cell-based “mini-guts” that mimic many aspects of the corresponding epithelial barrier in vivo . Here, we established and characterized differentiated apical-out (AO) and basal-out (BO) jejunal enteroids in suspension and followed their differentiation by quantitative global proteomics and different microscopic techniques. The barrier integrity and function and subcellular location of nutrient and clinically important drug transporters were investigated in the matured enteroids using live-cell microscopy. The presystemic metabolism of two drugs by CYP3A4 was determined and the results were used to predict the pharmacokinetics after oral administration by a PBPK population model. The differentiated AO enteroids displayed a protein profile that overlapped both qualitatively and quantitatively with that of freshly isolated jejunal enterocytes and tissue. They exhibited a morphology that recapitulates the mature villus enterocyte in vivo , formed an intact barrier with a well-developed glycocalyx and are impermeable to the hydrophilic low molecular weight compound lucifer yellow and transported a medium chain fatty acid derivative by FATP4 into lipid deposits. The clinically important ABC-transporters Pgp and BCRP were expressed at near in vivo levels, had the correct subcellular localization and effluxed their substrates. Terfenadine and midazolam were metabolized by CYP3A4 and the results were used to predict the clinical pharmacokinetics of the drugs after oral administration with good accuracy. We conclude that suspended 3D AO enteroids provide a physiologically relevant model for studies of intestinal function that offers convenient access to the apical surface and is easy to dispense in multi-wells formats for large scale experimentation.