Abstract Background Mechanisms governing non-vesicular extracellular particle (NVEP) biogenesis and compositional diversity remain incompletely characterized. Method This study integrates multiparametric profiling to define NVEPs. NVEPs were isolated by differential ultracentrifugation and characterized using complementary biochemical, lipidomic, and imaging approaches, including protein-to-lipid ratio analysis, lipid profiling, and advanced microscopy. Result Comprehensive characterization demonstrated that NVEPs exhibit significantly elevated protein-to-lipid ratios compared to extracellular vesicles (EVs) subtypes. Lipidomic profiling revealed pronounced enrichment in cholesteryl esters and triacylglycerols, with distinct carbon chain length signatures differentiating NVEPs from small EVs. Super-resolution microscopy confirmed marker heterogeneity across populations, with NVEPs showing specific enrichment of Arf6 and CD63. Conclusion These findings provide a robust compositional framework to distinguish non-vesicular extracellular particles from canonical EVs and underscore their emerging significance as mediators of intercellular communication and potential vehicles for targeted delivery.

Abstract Several new antibiotics target multidrug-resistant pathogens, yet resistance is still evaluated mainly by drug-susceptibility, leaving consequences for bacterial pathogenicity poorly understood. Here, we develop a framework integrating resistance evolution, genomic surveillance and host–pathogen phenotyping to classify antibiotics by resistance potential and pathogenic consequences. Applying this framework to Klebsiella pneumoniae identified functionally distinct antibiotic candidates associated with elevated virulence risk. Resistance evolution rapidly increased virulence through clinically-relevant mutations, without direct selection for pathogenicity. Despite distinct genetic routes, resistance converged on cell-envelope rewiring. A single resistance mutation increased epithelial adhesion, intracellular colonization, macrophage immune-evasion, and tissue persistence in murine infection models, transforming K. pneumoniae into a more invasive and cytotoxic pathogen. Risk-profile analysis revealed partial decoupling of resistance and pathogenicity, with some low-resistance antibiotics yielding highly-virulent populations. These findings establish resistance-driven virulence as an underappreciated translational hazard and call for incorporating host–pathogen interactions into resistance surveillance and preclinical antibiotic development.

Cerium oxide (CeNPs) nanozymes represent a versatile class of biocatalytic nanomaterials capable of mimicking multiple enzyme functions, however, their practical application is often hindered by limited colloidal stability. Herein, the interaction of oppositely charged polyelectrolytes, namely heparin (HEP), chondroitin sulfate (CS), poly(acrylic acid) (PAA) and methylglycol chitosan (MGC), with CeNPs was investigated with respect to dispersion stability, peroxidase-like activity and biocompatibility. Distinct charging and aggregation regimes were quantitatively identified over a wide range of polyelectrolyte-to-particle mass ratios. At high surface coverage, adsorption of the macromolecules markedly enhanced resistance against salt-induced aggregation owing to electrosteric repulsion. Surface functionalization also improved the peroxidase-like operation of the CeNPs, with polyanion-coated particles exhibiting substantially higher enzymatic activity than the bare nanozymes. Cell viability assays confirmed excellent biocompatibility of both pristine and functionalized CeNPs, with no detectable cell death upon incubation with the nanozymes. These findings demonstrate that polyelectrolyte adsorption provides an effective strategy to simultaneously improve the colloidal stability and biocatalytic performance of CeNPs, widening their implementation in biomedical and industrial applications.

Acute pancreatitis (AP) is a common gastroenterological disorder characterized by severe abdominal pain and inflammation. Despite its high mortality rate, the pathomechanism of AP remains incompletely understood, although neurogenic inflammation - where nerve-derived mediators trigger or amplify inflammatory responses - also appears to contribute to its pathogenesis. The aim of this study was to further investigate the role of neurogenic inflammation in AP and the impact of sensory nerve desensitisation on disease severity. Sensory afferent neurons were ablated using resiniferatoxin (RTX) prior to AP induction in four distinct rat and mouse models. Additionally, TRPV1 knock-out mice were employed to assess the contribution of this ion channel to AP development. Our findings reveal that RTX-induced sensory nerve desensitisation exacerbates the severity of necrotising AP induced by L-ornithine (L-Orn) and sodium taurocholate (NaTc) in rats, as evidenced by increased tissue damage, leukocyte infiltration or serum amylase activity. Conversely, in the cerulein (Cer)-induced oedematous AP rat model, RTX treatment significantly reduced leukocyte infiltration without affecting tissue oedema. In mice, RTX administration worsened the severity of Cer-induced AP, while TRPV1 gene deletion led to a modest reduction in disease severity. These results suggest that the impact of sensory nerve disruption on AP varies depending on the model and type of AP. The study underscores the important role of sensory neurons and TRPV1 ion channels in the pathogenesis and progression of AP, highlighting the potential for targeted therapies that modulate neurogenic inflammation.

Background. Acute pancreatitis (AP) is a frequent gastrointestinal emergency lacking disease‑specific therapy. Severe AP results from sustained intracellular Ca²⁺ overload in pancreatic acinar and ductal cells, which induces mitochondrial permeability transition, ATP depletion and necrotic cell death. The mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) normally extrudes Ca²⁺ to maintain mitochondrial homeostasis, but its role in AP is unknown. This study tested whether pharmacological inhibition of NCLX modifies Ca²⁺ handling and protects against toxin‑induced acinar injury. Methods. Murine pancreatic acinar cells were isolated and exposed to chenodeoxycholic acid (CDC) or ethanol + palmitic acid. NCLX expression was assessed by RT‑qPCR and immunohistochemistry. Mitochondrial Ca²⁺ and membrane potential were monitored, and cell viability, apoptosis and necrosis were quantified by fluorescent imaging and CellTiter‑Glo assays. Electron microscopy evaluated ultrastructural changes. High‑resolution respirometry determined oxidative phosphorylation and spare respiratory capacity. In vivo , mice received CGP‑37157 (an NCLX inhibitor) after initiation of cerulein‑, sodium taurocholate‑ or ethanol + palmitoleic acid–induced AP and disease severity was determined. Results. SLC8B1/NCLX mRNA and protein were detected in acinar cells, and carbachol‑evoked mitochondrial Ca²⁺ transients were reduced by CGP‑37157. In acini treated with CDC or ethanol + palmitic acid, CGP‑37157 significantly decreased the plateau phase of mitochondrial Ca²⁺ elevation and prevented mitochondrial depolarization. The inhibitor reduced apoptotic and necrotic death and restored ATP‑dependent viability by ~50 %. Electron microscopy showed that NCLX inhibition preserved endoplasmic reticulum organization and prevented mitochondrial swelling and cristolysis. CGP‑37157 maintained routine and ATP‑linked respiration, limited leak respiration and preserved reserve respiratory capacity. In all three in vivo AP models, CGP‑37157 reduced pancreatic necrosis and hyperamylasaemia and improved histological scores, although the reduction of necrosis was not significant in the sodium taurocholate model. Conclusions. These findings demonstrate that NCLX is expressed in the exocrine pancreas and that its inhibition with CGP‑37157 limits toxin‑induced mitochondrial Ca²⁺ overload, preserves mitochondrial function, reduces apoptosis and necrosis, and ameliorates pancreatic injury in several experimental AP models. The results suggest that mitochondrial Ca²⁺ efflux via NCLX contributes to acinar damage during AP, and that transient pharmacological inhibition of NCLX may be a therapeutic strategy to protect pancreatic tissue.

Az organoidok olyan háromdimenziós (3D) sejttenyészetek, amelyeket pluripotens őssejtekből, felnőttek szöveti őssejtjeiből vagy akár daganatsejtekből hozhatnak létre. Az organoidokat felépítő sejtek önszerveződésének köszönhetően mikroszkopikus méretű, szervszerű struktúrák alakulnak ki, amelyek hitelesen reprezentálják a natív gasztrointesztinális szövetek kulcsfontosságú jellemzőit. Ezek a fejlett modellek megőrzik a sejtszintű sokféleséget, a szöveti architektúrát és a funkciókat, amelyek hiányoznak a hagyományos kétdimenziós sejttenyészetekből, ezáltal áthidalják a szakadékot az in vitro kísérletek és az in vivo élettani, illetve patológiás folyamatok között, amelyek a betegek szervezetében lejátszódnak. Az összefoglaló célja, hogy a gasztroenterológusok és más klinikusok számára átfogó képet nyújtson a gasztrointesztinális organoidok gyorsan fejlődő területéről. Kitér az olyan alapvető részletekre, mint az organoidok fogalma és típusai, továbbá összefoglalja, miként hozhatók létre organoidmodellek a gasztrointesztinális traktus szöveteiből, valamint a májból és a hasnyálmirigyből. Szó esik továbbá az organoidok egyre hangsúlyosabb szerepéről a klinikai alkalmazások terén, ideértve alkalmazásukat a terápiás válaszok előrejelzésében (személyre szabott orvoslás), a gasztrointesztinális betegségek in vitro modellezésében kutatási és diagnosztikai célokra, valamint a regeneratív orvoslásban.

Endoplasmic reticulum/plasma membrane (ER/PM) junctions are a major site of cellular signal transduction including in epithelia; however, whether their lipid membrane environment affects junctional ion transporters function remains unclear. Here, we show that epithelial secretion is governed by phosphatidylserine (PtdSer) levels in ER/PM nanodomains, specified by the antagonistic action of the lipid transfer proteins E-Syt3 and ORP5, which transduce cAMP signals to the chloride channel CFTR and activate the sodium-bicarbonate cotransporter NBCe1-B by IRBIT. Lipid transfer by E-Syt3, along with restricted plasma membrane localization by the E-Syt3 C2C domain, are essential for E-Syt3 function, as removal of PtdSer from junctions by E-Syt3 dissociated the cAMP signaling pathway complex, preventing CFTR activation, and prevented NBCe1-B activation by IRBIT. CFTR and NBCe1-B PtdSer sensor domains responded to PtdSer reduction by E-Syt3; which was reversed by exogenous PtdSer or by PtdSer supplied by ORP5. In mice, E-Syt3 depletion improved chloride flux and fluid secretion in salivary glands and isolated pancreatic ducts. These findings provide a framework for understanding the role of junctional lipids in the assembly of functional ion protein complexes and cellular communication at epithelial signaling hubs.

Abstract Background Inflammatory Bowel Diseases (IBD) are chronic immune-mediated diseases of the gastrointestinal tract. Fibrosis is frequent in IBD driven by inflammatory cytokines. During fibrosis, extracellular matrix proteins accumulate in the tissue, causing loss of epithelial function and stenosis, leading to malabsorption and intestinal motility problems. This can ultimately lead to serious complications, strictures, fistulas, or ileus. Anti-inflammatory drugs cannot target fibrosis, only surgical intervention can be used. No relevant primary in vitro cell culture model is available for intestinal fibrosis modelling. Therefore, we aimed to establish and optimize human primary fibroblast cell culture from intestinal biopsies of control and IBD patients. We characterized and compared the fibrosis phenotype and gene expression as well. Methods Fibroblast cell cultures were generated from colonic biopsy samples by enzymatic digestion. In a suitable medium, the cells attach to the bottom of the vessel and proliferate, at 90% confluence the cultures were passaged. The phenotype was confirmed by immunofluorescent staining. Target proteins: COL1A1 (Collagen type 1 alpha 1), TGF-ß1 (Transforming growth factor beta), PAI-1 (Plasminogen activator inhibitor type 1), α-SMA (Alpha smooth muscle actin), and PHH3 (Phosphohistone H3). For gene expression analysis RNA was isolated and qRT-PCR technique was used for the following target genes: COL1A1, ACTA2 (Actin alpha 2), TGF-ß1, FN1 (Fibronectin 1), PAI-1, H3C4 (H3 clustered histone 4). We evaluated the collected data and performed statistical analysis. Results We can efficiently create and maintain human colon primary fibroblast cell cultures from colonic biopsies of healthy and CD origins. Cell morphology differences were observed from control and diseased patient samples. At the staining, the fluorescent intensity of fibrotic markers (COL1A1, TGF-ß1, PAI1) and the mitosis marker PHH3 were significantly higher in the CD samples, indicating that the cells maintained fibrotic phenotype, and the diseased fibroblasts had a higher proliferation rate. The myofibroblast marker, α-SMA didn’t show a significant difference. Interestingly, significant differences were not noted between the two groups in the qRT-PCR measurements. Conclusion In conclusion, we can generate primary human intestinal fibroblast cell cultures and maintain them to passage number 3. Fibrotic protein markers and morphology distinguish the diseased samples from the healthy subjects, therefore it could be useable for in vitro fibrosis modelling. However, the gene expression analysis doesn’t discriminate between the two groups at passage number 3. In the future, we would like to observe the fibrotic properties in earlier passage numbers.

Cystic fibrosis (CF) is a life-shortening monogenic disease caused by mutations in the CFTR gene, but the functional expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−/HCO3− channel is determined by more than its genetic sequence. Beyond the well-known folding defect of the common F508del mutation, CFTR activity is dynamically modulated by a network of intracellular signaling pathways that control the channel’s gating, trafficking to, and retention at the apical membrane. Foremost is the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, which drives CFTR opening via phosphorylation of its regulatory (R) domain and coordination by scaffolding proteins (e.g., A-kinase anchoring proteins (AKAPs) and Na+/H+ exchanger regulatory factor 1 (NHERF1)). Equally important, Ca2+-dependent signaling cascades provide complementary fine-tuning: Ca2+-bound calmodulin can directly bind and increase the CFTR open probability, Ca2+-activated kinases such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the tyrosine kinase Pyk2 (with Src) can phosphorylate CFTR through noncanonical routes, and signaling intermediates such as IP3 receptor binding protein released with IP3(IRBIT) connect Ca2+ release to CFTR activation. These cAMP- and Ca2+-driven pathways intersect in specialized subcellular nanodomains, enabling precise spatiotemporal regulation of CFTR function. Clinically, although new CFTR modulator drugs have greatly improved outcomes, their effectiveness is limited by mutation-specific responses and incomplete restoration of channel activity. Understanding how cAMP–Ca2+ crosstalk governs CFTR in context can reveal novel therapeutic strategies targeting the channel’s regulatory microenvironment. This review highlights how compartmentalized cAMP and Ca2+ signals orchestrate CFTR function and discusses emerging approaches to harness this insight for better therapies across CF-affected organs.

Abstract Background Over recent decades, significant progress has been made in the pharmacological management of Inflammatory Bowel Diseases (IBD). However, the sustained efficacy of existing anti-inflammatory therapies remains suboptimal, highlighting the urgent need for novel treatments. Recent studies have implicated the coagulation pathway, particularly Plasminogen Activator Inhibitor 1 (PAI-1), as a crucial link between epithelium and inflammation in IBD. Genetic deletion of PAI-1 has been shown to reduce experimental colitis severity in mice. Based on these findings, we aimed to develop and evaluate novel PAI-1 inhibitors for IBD treatment. Methods Using the A3SMO® platform, we synthesized LDN-071, an amino acid-based PAI-1 inhibitor, and assessed its inhibitory potential and cytotoxicity in vitro. Acute and chronic colitis models were induced in mice using DSS, with 10 mg/bwkg LDN-071 administered orally. Body weight, spleen weight, and colon length were monitored, while colon histology was analysed. Cytokine expression was measured via ELISA and qRT-PCR, and collagen deposition was quantified using a hydroxyproline assay. Human colon organoid (HCO) and fibroblast (FB) cultures from IBD patients were used to evaluate LDN-071’s effects. Toxicity was assessed in vivo in mice. Results LDN-071 effectively inhibited PAI-1 in vitro without cytotoxicity (1µM–1mM). In DSS-treated mice, 10 mg/bwkg LDN-071 prevented body weight loss, bloody diarrhea, colon shortening, and spleen enlargement. Histological analysis showed that PAI-1 inhibition significantly reduced severe inflammation, ulceration, crypt structure loss, and goblet cell depletion in acute and chronic colitis models. Additionally, PAI-1 inhibition reduced mucosal TNF-α, IL-1β, and IL-6 expression during acute inflammation. In chronic colitis, LDN-071 decreased inflammation, epithelial erosion, and fibrosis while significantly lowering collagen deposition. Importantly, no toxicity was observed at 100 mg/bwkg LDN-071 for seven days. In vitro, LDN-071 reduced pro-inflammatory cytokine expression in HCOs from IBD patients and significantly decreased fibrosis-related gene expression in patient-derived FB cultures. Conclusion Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1, significantly ameliorated the severity of acute and chronic colitis in mice. However, the LDN-071 reduced the expression of inflammation- and fibrosis-related genes and proteins in vitro human models. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

Abstract Background Inflammatory Bowel Diseases (IBD) require lifelong treatment and patient monitoring. Current predictors of relapse and therapeutic success have limitations, mostly invasive, time-consuming, and expensive. The faecal biomarker can help in disease activity and therapeutic response monitoring by simple and non-invasive way. Faecal Calprotectin (FC) is the gold-standard, but it has many disadvantages. We previously described that the correlation between the faecal Plasminogen activator inhibitor type 1 (PAI-1) (FP) level and the endoscopic activity and therapeutic response promote that it could be used as a novel non-invasive faecal biomarker in IBD diagnosis.1 To observe the exact faecal biomarker potential, we aimed to define the FP level of IBD patients and subjects with other gastrointestinal (GI) diseases and compare it with the FC. Methods Faecal samples were collected from 84 patients with IBD (CD-active: 12, CD-inactive: 10, UC-active: 9, UC-inactive: 10) and other GI diseases [colorectal cancer (CRC): 10, diverticulosis [DIV]: 9, irritable bowel syndrome [IBS]: 5, Polyp: 10) and 9 healthy subjects. ELISA method was applied to define FP level and it was validated for stool samples by us. In our previous study, 0.68 ng/g PAI-1 level was defined as the cut-off value. For FC observation diagnostic FC ELISA kit (Orgentec) was used. FC cut-off value was 50 mg/g. ROC analysis was applied to define Youden-index, specificity, and sensitivity of the measured values. Results FP level was significantly higher in patients with active IBD compared to inactive IBD and healthy subjects. No significant differences were observed between CD-active and UC-active groups. In addition, FP concentrations were significantly increased in active IBD patients compared to inactive IBD, CRC, DIV, IBS, Polyp, and healthy subjects. However, FC showed the same pattern as FP, except at the inactive IBD and DIV. In these groups, there were no significant differences compared to the active IBD subjects. ROC analysis defined high specificity (88.9%) and moderate sensitivity (47.6%) values (TP: 10, FP: 7, FN: 11, TN: 56, PPV: 0.588, NPV: 0.836) at the FP measurements. Nevertheless, at FC levels 34.9% specificity and 95.2% sensitivity were measured (TP: 20, FP: 41, FN: 1, TN: 22, PPV: 0.328, NPV: 0.957). Calculated Youden-index was higher at the FP (0.37) compared to the FC (0.3). Conclusion Our results suggest that the FP level selectively increased in the active IBD and discriminated more specifically from the other GI diseases compared to the FC. However, the sensitivity was lower, but it could be improved by the increasing patient numbers. Thus, the FP level could be useful in the diagnosis of IBD independently or combined with FC. References 1.Jójárt B, Resál T, Kata D, Molnár T, Bacsur P, Szabó V, Varga Á, Szántó KJ, Pallagi P, Földesi I, Molnár T, Maléth J, Farkas K. Plasminogen Activator Inhibitor 1 Is a Novel Faecal Biomarker for Monitoring Disease Activity and Therapeutic Response in Inflammatory Bowel Diseases. J Crohns Colitis. 2024 Mar 1;18(3):392-405. doi: 10.1093/ecco-jcc/jjad160. PMID: 37751311; PMCID: PMC10906952.

Cystic fibrosis transmembrane conductance regulator (CFTR) determines epithelial ion secretion, which is fundamental in various organs. The synergy between cyclic AMP (cAMP) and Ca2+ signaling fine-tunes CFTR-mediated secretion; however, the organization of such signaling complexes and their physiological impact remained largely unknown. Here, we identified an apical membrane signaling complex consisting of secretory pathway Ca2+-ATPase (SPCA2), stromal interaction molecule 1 (STIM1)/calcium release-activated calcium channel protein 1 (ORAI1), adenylyl cyclases, and CFTR. In this complex, SPCA2 facilitates constitutive, store-independent but STIM1-dependent ORAI1-mediated Ca2+ influx by activating ORAI1 and promoting STIM1/ORAI1 interaction, which is essential for basal CFTR function. Analysis by super-resolution dSTORM revealed constitutive organization of the proteins in a nanodomain on the apical membrane, which translates local Ca2+ increases to cAMP elevation and CFTR activation in unstimulated cells required for ion secretion. The same system operates in the pancreas, airways, and liver. Our findings reveal an essential, self-directing regulatory mechanism of CFTR-mediated ion secretion in secretory epithelial cells, independent from neurohormonal stimuli.

Alterations to intestinal microbiota are assumed to occur in the pathogenesis of inflammatory bowel disease (IBD). This study aims to analyze the association of fecal microbiota composition, body composition, and lipid characteristics in patients with Crohn’s disease (CD). In our cross-sectional study, patients with CD were enrolled and blood and fecal samples were collected. Clinical and endoscopic disease activity and body composition were assessed and laboratory tests were made. Fecal bacterial composition was analyzed using the shotgun method. Microbiota alterations based on obesity, lipid parameters, and disease characteristics were analyzed. In this study, 27 patients with CD were analyzed, of which 37.0% were obese based on visceral fat area (VFA). Beta diversities were higher in non-obese patients (p < 0.001), but relative abundances did not differ. C. innocuum had a higher abundance at a high cholesterol level than Bacillota (p = 0.001, p = 0.0034). Adlercreutzia, B. longum, and Blautia alterations were correlated with triglyceride levels. Higher Clostridia (p = 0.009) and B. schinkii (p = 0.032) and lower Lactobacillus (p = 0.035) were connected to high VFA. Disease activity was coupled with dysbiotic elements. Microbiota alterations in obesity highlight the importance of gut microbiota in diseases with a similar inflammatory background and project therapeutic options.