IntroductionInterstitial cystitis (IC) is an aseptic chronic bladder inflammation of unknown etiology and poorly understood pathophysiology with symptoms resembling bacterial cystitis (BC). There is limited data about the contribution of regulatory microRNAs (miRNAs) in IC. The study aimed to identify differences in miRNA expression between mouse models of IC and BC to find potential miRNAs that would distinguish between the two types of cystitis and to evaluate the use of the mouse model of IC as a tool to study the pathogenic mechanisms of IC in humans.MethodsTwo mouse models were utilized: cyclophosphamide was used for induction of chronic aseptic cystitis, and uropathogenic E.coli for induction of acute bacterial cystitis. Potential regulatory miRNAs were selected based on publicly available human IC datasets and validated in mice. Quantitative PCR and RNA isolated from formalin-fixed, paraffin-embedded mouse bladder tissue were used. An enrichment analysis of the target mRNAs of the validated miRNAs was performed to suggest the differences in the possible mechanisms of inflammation between the IC and BC.ResultsWe observed differential expression of 20 of the 33 selected miRNAs in IC and BC compared to the control group, with 11 miRNAs showing the same trend of expression between mouse and human IC. There are 8 common reporter-assay (RA) validated targets (performed by others) of these miRNAs in mouse and human. Histopathological analysis of mouse IC and BC urinary bladders, miRNA expression analysis, and expression of their validated targets revealed significant differences between the urinary bladders of BC and IC mouse models. We identified miR-301a-3p as a possible marker of discrimination between two types of cystitis and its target gene, nuclear factor-κB (NF-κB) suppressing factor (NKRF).ConclusionOur results show that miRNA expression and its RA-validated targets, and enriched signaling pathways, differ between the two types of cystitis and might depend on the type of bladder inflammation. The mouse model of IC has some similarities with human IC, confirming that it is a useful tool to identify novel potentially discriminatory biomarkers between BC and IC, such as miR-301a-3p, and also potential therapeutic targets for IC (e.g., NKRF and NF-κB).

Background: Interstitial cystitis (IC) is a debilitating lower urinary tract condition characterised by chronic inflammation of the bladder. As the aetiology remains unknown, current treatments are symptomatic, aiming to reduce inflammation and pain. Cannabidiol (CBD), the most common cannabinoid in industrial Cannabis sativa (hemp), is one of the most important pharmacologically active cannabinoids used in medicine due to its anti-inflammatory and antioxidant effects without psychoactive properties. While other cannabinoids have shown beneficial effects in animal models of IC, the impact of CBD on the urinary bladder and overall animal well-being has not been elucidated. Methods: Using a cyclophosphamide (CYP)-induced mouse model of IC, we investigated the effects of intraperitoneally administered CBD on bladder structure, function, inflammation, and animal behaviour. A multimodal approach was applied, including light and electron microscopy, immunolabeling, qPCR, transepithelial electrical resistance (TEER) measurements, behavioural testing, and monitoring of animals. Results: CBD treatment promoted the restoration of damaged urothelial structure and improved the integrity of the blood-urine barrier. Additionally, CBD exerted an anti-inflammatory effect, reducing oedema and infiltration of inflammatory cells in the bladder wall with chronic cystitis. Finally, the increased burrowing activity of CBD-treated mice suggests a benefit of CBD on overall well-being. Conclusions: Our findings suggest that CBD has a beneficial effect on the inflamed urinary bladder and could potentially serve as an adjunct treatment for patients with IC in the future.

Background/Objectives:Trefoil factor protein 3 (Tff3) is a small peptide known as an epithelial tissue-protective protein, and it is also identified as a novel participant in complex metabolic processes. In numerous mouse models of obesity, Tff3 has been found to be downregulated in the liver and its overexpression is associated with an improvement in metabolic parameters. These mouse models with metabolic phenotypes have a multigenic background, with numerous genes contributing to their phenotype. To elucidate the role of Tff3 protein in metabolic events, we developed a mouse model with Tff3 deficiency on a C57Bl6N background without other intrinsic mutations affecting metabolism. Methods: We investigated the effects of a high-fat diet (9 weeks) on the liver of Tff3 protein-deficient mice of both sexes and the corresponding wild type. We investigated the general metabolic status of the animals and analysed the expression of markers of relevant pathophysiological pathways in the liver. Results:Tff3-deficient mice had significantly lower body weight. They also had a comparable total liver fat content but it was distributed in small vesicles, indicating the protective effect of Tff3 deficiency. The results of molecular analysis showed no major gene expression changes in inflammation-, ER- and oxidative stress-, and lipid metabolism-related genes. Tff3−/−males had reduced expression of Il1α and Cxcr7 genes in the liver and no global proteome changes; Tff3-deficient females had decreased expression of Irs2 and Atf4 genes and total proteome comparison showed decreased levels of proteins related to ribosome biosynthesis and the inhibition of acetylation. Conclusions: Our results demonstrate that Tff3 deficiency reduces lipid accumulation in the liver and we set the direction for further studies aimed at uncovering the exact molecular mechanisms in other organs. Furthermore, it emphasises the need to include both sexes in future research, as the observed phenotype differs significantly depending on sex.

Nanoplastics (NPs) are released into the environment through the degradation of plastic objects, leading to human exposure. Due to their small size, concerns have been raised about the potential hazards to the respiratory tract, as ultrafine and nanoparticles are known to penetrate till the alveolar regions of the lungs, potentially impairing their functions. Thus, in the present study, we used model polystyrene nanoparticles doped with the fluorescent metal europium (PS-Eu) to enhance the understanding of NPs hazard and investigate adverse outcomes associated with exposure in human lungs using alveolar (A549) and bronchial (Calu-3) cell models grown in 2D and 3D submerged conditions or quasi air-liquid interface (ALI) conditions (3D). Briefly, after in-dept physicochemical characterization of the particles, we assessed their impact on ROS production, cell viability (AlamarBlue and lactate dehydrogenase assays) and barrier integrity (lucifer yellow assay and TEER measurement), finding no negative effects in either model. However, in alveolar cells, particles increased acidic organelle activity. Transmission electron microscopy and Raman microscopy showed, in both models, a dose- and cell-dependent particle uptake with PS-Eu accumulating in numerous and large endo-lysosomes, which, in transwells-grown A549 cells, often contained also lamellar bodies (LBs), organelles involved in surfactants storage and secretion. After extensively quantifying surfactant proteins (SP) in the pellet and supernatant fractions of treated A549 cells, we observed a significant reduction in several members of this family, including surfactant protein B, which is crucial for lamellar body formation and surface tension regulation in the lungs. In quasi-ALI Calu-3 cultures instead, PS-Eu significantly upregulated interleukin 6 (IL-6) and increased transforming growth factor beta β (TGF-β), zonula occludens 1 (ZO-1), and mucin (MUC) 5B mRNA expressions causing a moderate proinflammatory response. Our results show that PS-Eu exposure does not induce acute cytotoxicity in these models, but affects cell-specific functions like surfactant, mucin, and cytokine production. This underscores the limitations of relying solely on standard cytotoxicity tests for particle hazard assessment and highlights the importance of investigating cell function-specific signaling pathways. To support researchers in hazard assessment, we propose specific classes of biomarkers to test in in vitro lung models.

Trefoil factor family (TFF) peptides have been examined primarily in the gastrointestinal tract, where they play an important role in the epithelial regeneration. The therapeutic effects of TFFs, particularly the TFF3 protein, have been well studied in humans and in animal models of gastrointestinal injury, whereas little is known about their occurrence and function in the urinary bladder. In this study, we investigated the presence, location, and function of Tff3 in the urinary bladders of wild-type mice (Tff3WT) and compared them with Tff3 knockout mice (Tff3KO) using molecular and microscopic methods at the light and electron microscopic level. Our results show that Tff3 is expressed in the superficial cells of the urothelium, where it colocalizes with the uroplakin UP1b as one of the fundamental structural components of the apical plasma membrane, which is an important component of the blood-urine permeability barrier. Analysis of the urothelium with experimentally induced injury revealed that injury is more severe in Tff3KO mice and urothelial regeneration is attenuated compared with Tff3WT mice, suggesting that Tff3 plays a fine-tuned role in homeostasis and protection of the urothelium. This study provides the first data on the precise location and function of Tff3 in the bladder epithelium.

Mouse tumor models are an important tool in cancer research, and the orthotopic cancer cell transplantation model is the most widely used among them. Methods for establishing tumor models may differ in many ways, including the selection of cancer cell lines and the type of urinary bladder pretreatment. Here, we describe our mouse orthotopic bladder tumor model using a labeled MB49 urothelial cancer cell line and chemical pretreatment with the cationic polypeptide poly-L-lysine to traumatize the bladder epithelium. Double labeling of MB49 cancer cells by their transduction with GFP and internalization of metal nanoparticles allows the study of their implantation process from the first hours to several days after intravesical injection, as well as the analysis of developed tumors after 3 weeks. Thus, our model provides a comprehensive analysis of the early and late stages of tumor development in the bladder at the light and electron microscopic level.

Animal models are invaluable in the research of the pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic aseptic urinary bladder disease of unknown etiology that primarily affects women. Here, a mouse model of IC/BPS was induced with multiple low-dose cyclophosphamide (CYP) applications and thoroughly characterized by RNA sequencing, qPCR, Western blot, and immunolabeling to elucidate key inflammatory processes and sex-dependent differences in the bladder inflammatory response. CYP treatment resulted in the upregulation of inflammatory transcripts such as Ccl8, Eda2r, and Vegfd, which are predominantly involved in innate immunity pathways, recapitulating the crucial findings in the bladder transcriptome of IC/BPS patients. The JAK/STAT signaling pathway was analyzed in detail, and the JAK3/STAT3 interaction was found to be most activated in cells of the bladder urothelium and lamina propria. Sex-based data analysis revealed that cell proliferation was more pronounced in male bladders, while innate immunity and tissue remodeling processes were the most distinctive responses of female bladders to CYP treatment. These processes were also reflected in prominent histological changes in the bladder. The study provides an invaluable reference dataset for preclinical research on IC/BPS and an insight into the sex-specific mechanisms involved in the development of IC/BPS pathology, which may explain the more frequent occurrence of this disease in women.

The urothelium is a vital permeability barrier that prevents the uncontrolled flow of urinary components into and out of the bladder interstitium. Our study addressed the question of possible sex-specific variations in the urothelium of healthy mice and their impact on chronic bladder inflammation. We found that healthy female bladders have a less robust barrier function than male bladders, as indicated by significant differences in transepithelial electrical resistance (TEER) values. These differences could be attributed to detected higher claudin 2 mRNA expression and a less pronounced glycocalyx in females than in males. In addition, TEER measurements showed delayed barrier recovery in chronically inflamed female bladders. We found subtle differences in the expressions of genes involved in the regulation of the actin cytoskeleton between the sexes, as well as pronounced urothelial hyperplasia in females compensating for attenuated barrier function. The identified genetic variations in glycosylation pathways may also contribute to this divergence. Our findings add to the growing body of literature on the intricate sex-specific nuances of urothelial permeability function and their implications for chronic bladder inflammation. Understanding these differences could lead to tailored diagnostic and therapeutic approaches in the treatment of bladder disorders in the future.

The aim of our study was to evaluate the experimental conditions under which the cationic polymer poly-L-lysine induces effective and safe desquamation of the urinary bladder epithelium, also called the urothelium. Induced desquamation of the urothelium is a frequently used approach in studies of urothelium regeneration and also to establish animal disease models. Chitosan has previously been shown to induce large-scale desquamation of urothelial cells. We have investigated the desquamation ability of another positively charged polymer poly-L-lysine. Its effect on the functional and structural integrity of the urothelium was studied in ex vivo and in vivo experiments. The results revealed that the effect of poly-L-lysine on the structure and function of the urothelium was mainly influenced by the concentration of the polymer, while the treatment time and the molecular weight of poly-L-lysine had little effect. Fifteen minutes exposure of the urothelium to 0.01% poly-L-lysine was found to be efficient and safe in ex vivo and in vivo experiments and resulted in intense cell desquamation, which was limited to the superficial cell layer only. Functional and structural regeneration of the urothelium occurred ex vivo within 2 to 3 hours after the induced injury by poly-L-lysine. Therefore, poly-L-lysine is a promising inducer of urothelial cells desquamation that has the potential to be used in the clinic for the treatment of urologic disorders.

Understanding the tissue changes and molecular mechanisms of preclinical models is essential for creating an optimal experimental design for credible translation into clinics. In our study, a chlorhexidine (CHX)-induced mouse model of peritoneal fibrosis was used to analyze histological and molecular/cellular alterations induced by 1 and 3 weeks of intraperitoneal CHX application. CHX treatment for 1 week already caused injury, degradation, and loss of mesothelial cells, resulting in local inflammation, with the most severe structural changes occurring in the peritoneum around the ventral parts of the abdominal wall. The local inflammatory response in the abdominal wall showed no prominent differences between 1 and 3 weeks. We observed an increase in polymorphonuclear cells in the blood but no evidence of systemic inflammation as measured by serum levels of serum amyloid A and interleukin-6. CHX-induced fibrosis in the abdominal wall was more pronounced after 3 weeks, but the gene expression of fibrotic markers did not change over time. Complement system molecules were strongly expressed in the abdominal wall of CHX-treated mice. To conclude, both histological and molecular changes were already present in week 1, allowing examination at the onset of fibrosis. This is crucial information for refining further experiments and limiting the amount of unnecessary animal suffering.

Pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS) remains poorly understood, as well as its effective diagnosis and therapy. Studying changes in tissue glycosylation patterns under pathological conditions is a promising way of discovering novel biomarkers and therapeutic targets. The glycobiology of IC/BPS is largely understudied, therefore we compared glycosylation patterns of normal human urothelium with the urothelium of IC/BPS patients using a selection of 10 plant-based lectins with different monosaccharide preferences. We also compared lectin binding to human urothelium with the two most cited experimental models of IC/BPS, specifically, TNFα-treated human urothelial cell line RT4 and cyclophosphamide-induced chronic cystitis in C57BL6/J mice. Furthermore, binding of four of the selected lectins (ConA, DSL, Jacalin and WGA) was evaluated qualitatively by means of fluorescence microscopy, and quantitatively by fluorescence intensity (F.I.) measurements. Our results reveal a significant reduction in F.I. of Jacalin, as well as a prominent change in the WGA labeling pattern in the urothelium of IC/BPS patients, suggesting their potential use as promising additional biomarkers for histopathological diagnosis of IC/BPS. We have also shown that urothelial glycosylation patterns between selected experimental models and patients with IC/BPS are similar enough to offer an adequate platform for preclinical study of IC/BPS glycobiology.

Urothelial cells of the urinary bladder play a critical role in the development and progression of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic and debilitating inflammatory disease. Given the lack of data on the exact phenotype and function of urothelial cells in an inflammatory setting (as in IC/BPS), we performed the first in-depth characterization of these cells using RNA sequencing, qPCR, ELISA, Western blot, and immunofluorescence. After TNFα stimulation, urothelial cells in the in vitro model of IC/BPS showed marked upregulation of several proinflammatory mediators, such as SAA, C3, IFNGR1, IL1α, IL1β, IL8, IL23A, IL32, CXCL1, CXCL5, CXCL10, CXCL11, TNFAIPR, TNFRSF1B, and BIRC3, involved in processes and pathways of innate immunity, including granulocyte migration and chemotaxis, inflammatory response, and complement activation, as well as TLR-, NOD-like receptor- and NFkB-signaling pathways, suggesting their active role in shaping the local immune response of the bladder. Our study demonstrates that the TNFα-stimulated urothelial cells recapitulate key observations found in the bladders of patients with IC/BPS, underpinning their utility as a suitable in vitro model for understanding IC/BPS mechanisms and confirming the role of TNFα signaling as an important component of the associated pathology. The present study also identifies novel upregulated gene targets of TNFα in urothelial cells, including genes encoding the acute phase protein SAA, complement component C3, and the cytokine receptor IFNGR1, which could be exploited as therapeutic targets of IC/BPS. Altogether, our study provides a reference database of the phenotype of urothelial cells in an inflammatory environment that will not only increase our knowledge of their role in IC/BPS, but also advance our understanding of how urothelial cells shape tissue immunity in the bladder.

Therapy with mesenchymal stem cells (MSCs) is promising in many diseases. Evaluation of their efficacy depends on adequate follow-up of MSCs after transplantation. Several studies have shown that MSCs can be labeled and subsequently visualized with magnetic nanoparticles (NPs). We investigated the homing of MSCs labeled with magnetic cobalt ferrite NPs in experimentally induced acute kidney injury in mice. To explore the homing of MSCs after systemic infusion into mice, we developed a pre-infusion strategy for optimal tracing and identification of MSCs with polyacrylic acid-coated cobalt ferrite (CoFe2O4) NPs by light and transmission electron microscopy (TEM) in various organs of mice with cisplatin-induced acute kidney injury and control mice. By correlative microscopy, we detected MSCs labeled with NPs in the lungs, spleen, kidney, and intestine of cisplatin-treated mice and in the lungs and spleen of control mice. Our results confirm that labeling MSCs with metal NPs did not affect the ultrastructure of MSCs and their ability to settle in various organs. This study demonstrates the usefulness of cobalt ferrite NPs in ex vivo visualization of MSCs and offers correlative microscopy as a useful method in routine histopathology laboratories for tracing MSCs in paraffin-embedded tissue.

Superparamagnetic iron oxide nanoparticles (SPIONs) have great potential for use in medicine, but they may cause side effects due to oxidative stress. In our study, we investigated the effects of silica-coated SPIONs on endothelial cells and whether oleic acid (OA) can protect the cells from their harmful effects. We used viability assays, flow cytometry, infrared spectroscopy, fluorescence microscopy, and transmission electron microscopy. Our results show that silica-coated SPIONs are internalized by endothelial cells, where they increase the amount of reactive oxygen species (ROS) and cause cell death. Exposure to silica-coated SPIONs induced accumulation of lipid droplets (LD) that was not dependent on diacylglycerol acyltransferase (DGAT)-mediated LD biogenesis, suggesting that silica-coated SPIONs suppress LD degradation. Addition of exogenous OA promoted LD biogenesis and reduced SPION-dependent increases in oxidative stress and cell death. However, exogenous OA protected cells from SPION-induced cell damage even in the presence of DGAT inhibitors, implying that LDs are not required for the protective effect of exogenous OA. The molecular phenotype of the cells determined by Fourier transform infrared spectroscopy confirmed the destructive effect of silica-coated SPIONs and the ameliorative role of OA in the case of oxidative stress. Thus, exogenous OA protects endothelial cells from SPION-induced oxidative stress and cell death independent of its incorporation into triglycerides.

BACKGROUND:There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue.METHODS:We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions.RESULTS:Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time.CONCLUSIONS:Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a multifactorial, chronic bladder disorder with limited therapeutic options currently available. The present review provides an extensive overview of therapeutic approaches used in in vitro, ex vivo, and in vivo experimental models of IC/BPS. Publications were identified by electronic search of three online databases. Data were extracted for study design, type of treatment, main findings, and outcome, as well as for methodological quality and the reporting of measures to avoid bias. A total of 100 full-text articles were included. The majority of identified articles evaluated therapeutic agents currently recommended to treat IC/BPS by the American Urological Association guidelines (21%) and therapeutic agents currently approved to treat other diseases (11%). More recently published articles assessed therapeutic approaches using stem cells (11%) and plant-derived agents (10%), while novel potential drug targets identified were proteinase-activated (6%) and purinergic (4%) receptors, transient receptor potential channels (3%), microRNAs (2%), and activation of the cannabinoid system (7%). Our results show that the reported methodological quality of animal studies could be substantially improved, and measures to avoid bias should be more consistently reported in order to increase the value of preclinical research in IC/BPS for potential translation to a clinical setting.

Non-muscle-invasive bladder cancer is the most common form of bladder cancer. The main problem in managing bladder tumors is the high recurrence after the transurethral resection of bladder tumors (TURBT). Our study aimed to examine the fate of intravesically applied cancer cells as the implantation of cancer cells after TURBT is thought to be a cause of tumor recurrence. We established an orthotopic mouse bladder tumor model with MB49-GFP cancer cells and traced them during the first three days to define their location and contacts with normal urothelial cells. Data were obtained by Western blot, immunolabeling, and light and electron microscopy. We showed that within the first two hours, applied cancer cells adhered to the traumatized epithelium by cell projections containing α3β1 integrin on their tips. Cancer cells then migrated through the epithelium and on day 3, they reached the basal lamina or even penetrated it. In established bladder tumors, E-cadherin and desmoplakin 1/2 were shown as feasible immunohistochemical markers of tumor margins based on the immunolabeling of various junctional proteins. Altogether, these results for the first time illustrate cancer cell implantation in vivo mimicking cellular events of tumor recurrence in bladder cancer patients.

Background: Mesenchymal stromal cell (MSC) - based therapies are emerging as promising treatment of various autoimmune diseases, however the utility of different MSC tissue sources remains elusive. We aimed to characterize MSC from different origins, namely bone marrow (BM), adipose tissue (AT) and umbilical cord (UC) and determine their functional effects on normal human lung fibroblasts (NHLF). Methods: BM-, AT- or UC-MSC were isolated each from 3 different healthy donors. The gene expression and protein secretion were analyzed at basal level, along with TNF-alpha, IL-1 beta- and SAA- stimulated cells using real-time PCR and Luminex technology. Effect of conditioned medium (CM) from different MSC sources on migration was determined with wound scratch assay, while mitotic and apoptotic rates were studied using immunofluorescence microscopy. Results: BM-MSC expressed highest basal mRNA levels of SDF1 and VCAM-1, while other genes were similarly expressed between MSC origins. TNF alpha priming of AT-MSC gained a prominent increase in IDO1 and CCL5 gene expression, with 928-fold and 4396-fold changes, respectively. Among all tissue sources, basal UC-MSC released highest protein levels of most measured analytes, including IL-6, IL-8, MCP-1, ICAM1, HGF, MMP1 and CH3L1. BM- and AT-MSC derived CM enhanced wound closure in NHLF, while an opposite effect was observed with UC-MSC derived CM. Our data also suggests that MSC-CM could contribute to decreased mitotic potential and increased apoptotic rate in lung fibroblasts. Conclusions: Our study highlights origin-specific MSC profile differences and emphasizes a heterogenic response of different MSC to inflammatory stimuli. (C) 2020 Elsevier Masson SAS. All rights reserved.

The aging of organisms leads to a decreased ability of tissue to regenerate after injury. The regeneration of the bladder urothelium after induced desquamation with biopolymer chitosan has been studied in young mice but not in old mice. Chitosan is a suitable inducer of urothelial desquamation because it is known to be non-toxic. We used chitosan for desquamation of urothelial cells in order to compare the dynamics of urothelial regeneration after injury between young and old mice. Our aim was to determine whether the urothelial function and structure of old mice is restored as fast as in young mice, and to evaluate the inflammatory response due to chitosan treatment. We discovered that the urothelial function restored comparably fast in both age groups and that the urothelium of young and old mice recovered within 5 days after injury, although the onset of proliferation and differentiation appeared later in old mice. Acute inflammation markers showed some differences in the inflammatory response in young versus old mice, but in both age groups, chitosan caused short-term acute inflammation. In conclusion, the restoration of urothelial function is not impaired in old mice, but the regeneration of the urothelial structure in old mice slightly lags behind the regeneration in young mice.