Hydrostatic pressure mediates epithelial-mesenchymal transition of cholangiocytes through RhoA/ROCK and TGF-ß/smad pathways.

Biomechanical cue within the tissue microenvironment is known to play a critical role in regulating cell behaviors and maintaining tissue homeostasis. As hydrostatic pressure often increases in biliary system under pathological states, we investigated the effect of the moderate elevation of the hydrostatic pressure on biliary epithelial cells, especially on the epithelial-mesenchymal transition (EMT). Human intrahepatic biliary epithelial cells were loaded to hydrostatic pressure using a commercial device. We found that loading the cells to 50 mmHg hydrostatic pressure induced obvious morphological changes and significantly upregulated vimentin, ZEB1, and pSmad2/3, fibronectin, and collagen 1α. All changes induced by hydrostatic pressure loading were effectively mitigated by either ROCK inhibitor (Y-27632) or ALK5 inhibitor (SB-431542). Our in vitro experimental data suggests that hydrostatic pressure loading induces EMT of cholangiocytes through RhoA/ROCK and TGF-ß/Smad pathways. Elevated hydrostatic pressure in biliary duct system under pathological states may promote the biliary epithelial cells shifting to profibrotic and mesenchymal characteristics.

Inhibition of ACSS2-mediated histone crotonylation alleviates kidney fibrosis via IL-1ß-dependent macrophage activation and tubular cell senescence.

Histone lysine crotonylation (Kcr), as a posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. In this study, we report that histone Kcr of tubular epithelial cells is abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) is found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys reveal that the hub proinflammatory cytokine IL-1ß, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppress H3K9cr-mediated IL-1ß expression, which thereby alleviate IL-1ß-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

Sounding the alarm in the lung with TL1A.

Environmental airborne antigens are central to the development of allergic asthma, but the cellular processes that trigger disease remain incompletely understood. In this report, Schmitt et al. (https://doi.org/10.1084/jem.20231236) identify TNF-like protein 1A (TL1A) as an epithelial alarmin constitutively expressed by a subset of lung epithelial cells, which is released in response to airborne microbial challenge and synergizes with IL-33 to drive allergic disease.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells.

Chronic sinusitis with nasal polyps (CRSwNP) is one of the most common chronic inflammatory diseases, and involves tissue remodeling. One of the key mechanisms of tissue remodeling is the epithelial-mesenchymal transition (EMT), which also represents one of the pathophysiological processes of CRS observed in CRSwNP tissues. To date, many transcription factors and forms of extracellular stimulation have been found to regulate the EMT process. However, it is not known whether gangliosides, which are the central molecules of plasma membranes, involved in regulating signal transmission pathways, are involved in the EMT process. Therefore, we aimed to determine the role of gangliosides in the EMT process. First, we confirmed that N-cadherin, which is a known mesenchymal marker, and ganglioside GD3 were specifically expressed in CRSwNP_NP tissues. Subsequently, we investigated whether the administration of TNF-α to human nasal epithelial cells (hNECs) resulted in the upregulation of ganglioside GD3 and its synthesizing enzyme, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialytransferase 1 (ST8Sia1), and the consequently promoted inflammatory processes. Additionally, the expression of N-cadherin, Zinc finger protein SNAI2 (SLUG), and matrix metallopeptidase 9 (MMP-9) were elevated, but that of E-cadherin, which is known to be epithelial, was reduced. Moreover, the inhibition of ganglioside GD3 expression by the siRNA or exogenous treatment of neuraminidase 3 (NEU 3) led to the suppression of inflammation and EMT. These results suggest that gangliosides may play an important role in prevention and therapy for inflammation and EMT.

Preventative Effects of Milk Fat Globule Membrane Ingredients on DSS-Induced Mucosal Injury in Intestinal Epithelial Cells.

The goblet cells of the gastrointestinal tract (GIT) produce glycoproteins called mucins that form a protective barrier from digestive contents and external stimuli. Recent evidence suggests that the milk fat globule membrane (MFGM) and its milk phospholipid component (MPL) can benefit the GIT through improving barrier function. Our objective was to compare the effects of two digested MFGM ingredients with or without dextran sodium sulfate (DSS)-induced barrier stress on mucin proteins. Co-cultured Caco-2/HT29-MTX intestinal cells were treated with in vitro digests of 2%, 5%, and 10% (w/v) MFGM or MPL alone for 6 h or followed by challenge with 2.5% DSS (6 h). Transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran (FD4) permeability measurements were used to measure changes in barrier integrity. Mucin characterization was performed using a combination of slot blotting techniques for secreted (MUC5AC, MUC2) and transmembrane (MUC3A, MUC1) mucins, scanning electron microscopy (SEM), and periodic acid Schiff (PAS)/Alcian blue staining. Digested MFGM and MPL prevented a DSS-induced reduction in secreted mucins, which corresponded to the prevention of DSS-induced increases in FD4 permeability. SEM and PAS/Alcian blue staining showed similar visual trends for secreted mucin production. A predictive bioinformatic approach was also used to identify potential KEGG pathways involved in MFGM-mediated mucosal maintenance under colitis conditions. This preliminary in silico evidence, combined with our in vitro findings, suggests the role of MFGM in inducing repair and maintenance of the mucosal barrier.

Carboxymethylated Rhizoma alismatis polysaccharides reduces the risk of calcium oxalate stone formation by reducing cellular inflammation and oxidative stress.

This study aims to elucidate the mechanism and potential of Rhizoma alismatis polysaccharides (RAPs) in preventing oxidative damage to human renal proximal tubule epithelial cells. The experimental approach involved incubating HK-2 cells with 100 nm calcium oxalate monohydrate for 24 h to establish a cellular injury model. Protection was provided by RAPs with varying carboxyl group contents: 3.57%, 7.79%, 10.84%, and 15.33%. The safeguarding effect of RAPs was evaluated by analyzing relevant cellular biochemical indicators. Findings demonstrate that RAPs exhibit notable antioxidative properties. They effectively diminish the release of reactive oxygen species, lactate dehydrogenase, and malondialdehyde, a lipid oxidation byproduct. Moreover, RAPs enhance superoxide dismutase activity and mitochondrial membrane potential while attenuating the permeability of the mitochondrial permeability transition pore. Additionally, RAPs significantly reduce levels of inflammatory factors, including NLRP3, TNF-α, IL-6, and NO. This reduction corresponds to the inhibition of overproduced pro-inflammatory mediator nitric oxide and the caspase 3 enzyme, leading to a reduction in cellular apoptosis. RAPs also display the ability to suppress the expression of the HK-2 cell surface adhesion molecule CD44. The observed results collectively underscore the substantial anti-inflammatory and anti-apoptotic potential of all four RAPs. Moreover, their capacity to modulate the expression of cell surface adhesion molecules highlights their potential in inhibiting the formation of kidney stones. Notably, RAP3, boasting the highest carboxyl group content, emerges as the most potent agent in this regard.

MiR-2284b regulation of α-s1 casein synthesis in mammary epithelial cells of dairy goats.

The lactation character of dairy goats is the most important characteristic, and milk protein is an important index to evaluate milk quality. Casein accounts for more than 80% of the total milk protein in goat milk and is the main component of milk protein. Using GMECs (goat mammary epithelial cells) as the research object, the CHECK2 vector of the CSN1S1 gene and the overexpression vector of pcDNA 3.1 were constructed, and the mimics of miR-2284b and the interfering RNA of CSN1S1 were synthesized. Using PCR, RT-qPCR, a dual luciferase activity detection system, EdU, CCK8, cell apoptosis detection and ELISA detection, we explored the regulatory mechanism and molecular mechanism of miR-2284b regulation of αs1-casein synthesis in GMECs. miR-2284b negatively regulates proliferation and apoptosis of GMECs and αs1-casein synthesis. Two new gene sequences of CSN1S1 were discovered. CSN1S1-1/-2 promoted the proliferation of GMECs and inhibited cell apoptosis. However, it had no effect on αs1-casein synthesis. MiR-2284b negatively regulates αs1-casein synthesis in GMECs by inhibiting the CSN1S1 gene. These results all indicated that miR-2284b could regulate αs1-casein synthesis, thus playing a theoretical guiding role in the future breeding process of dairy goats and accelerating the development of dairy goat breeding.

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells.

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity-the condensates in oral epithelium disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4-7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic "macromolecular crowding") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm-the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid-liquid phase separation (LLPS) in cells of the oral mucosa.

HISTOLOGICAL AND BIOCHEMICAL STUDY OF THE EFFECT OF FEXOFENADINE ON SALIVARY GLAND IN RATS.

Fexofenadine is a newly introduced oral non-sedating agent used for allergic diseases. We sought to investigate the effects of the use of fexofenadine on the salivary gland of adult male albino rats. 30 adult male albino rats were classified randomly into 3 groups, as follows: Group A (control group) which consisted of 10 healthy rats. Group B (treated group) which consisted of 10 rats received FEX 5mg/kg/day, and Group C (treated group) which consisted of 10 rats received FEX 10mg/kg/day. Blood samples were obtained to assess serum levels of Thioredoxin reductase (TRX) and malondialdehyde (MDA). Salivary glands were removed and prepared for histological examination. This study showed that significantly (p<0.05) higher TRX and MDA levels were observed in group B and group C, compared to group A. The histological examination for salivary tissues revealed degenerative changes in serous cells of acini were present with deep pyknotic nuclei. Vacuolar cytoplasmic degeneration is also seen in other certain cells. Blood congestion was present in the intralobular blood vessels, particularly around the striated ducts. The glandular secretion duct contained mucus and serous secretion and the wall of the duct was surrounded by many WBCs with macrophage. Fexofenadine hydrochloride use induces remarkable histopathological changes with dose-dependent response and remarkably linked to elevation of oxidative stress markers.

The mechanical influence of densification on epithelial architecture.

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayered epithelial tissue-whether protective, secretory, absorptive, or filtrative-relies on the side-by-side arrangement of its component cells. The mechanical parameters that determine the shape of epithelial cells in the apical-basal plane are not well-understood. Epithelial tissue architecture in culture is intimately connected to cell density, and cultured layers transition between architectures as they proliferate. This prompted us to ask to what extent epithelial architecture emerges from two mechanical considerations: A) the constraints of densification and B) cell-cell adhesion, a hallmark feature of epithelial cells. To address these questions, we developed a novel polyline cell-based computational model and used it to make theoretical predictions about epithelial architecture upon changes to density and cell-cell adhesion. We tested these predictions using cultured cell experiments. Our results show that the appearance of extended lateral cell-cell borders in culture arises as a consequence of crowding-independent of cell-cell adhesion. However, cadherin-mediated cell-cell adhesion is associated with a novel architectural transition. Our results suggest that this transition represents the initial appearance of a distinctive epithelial architecture. Together our work reveals the distinct mechanical roles of densification and adhesion to epithelial layer formation and provides a novel theoretical framework to understand the less well-studied apical-basal plane of epithelial tissues.

Kaempferol attenuates inflammation in lipopolysaccharide-induced gallbladder epithelial cells by inhibiting the MAPK/NF-κB signaling pathway.

Kaempferol (KPR), a flavonoid compound found in various plants and foods, has garnered attention for its anti-inflammatory, antioxidant, and anticancer properties. In preliminary studies, KPR can modulate several signaling pathways involved in inflammation, making it a candidate for treating cholecystitis. This study aimed to explore the effects and mechanisms of KPR on lipopolysaccharide (LPS)-induced human gallbladder epithelial cells (HGBECs). To assess the impact of KPR on HGBECs, the HGBECs were divided into control, KPR, LPS, LPS + KPR, and LPS + UDCA groups. Cell viability and cytotoxicity were evaluated by MTT assay and lactate dehydrogenase (LDH) assay, respectively, and concentrations of KPR (10-200 µM) were tested. LPS-induced inflammatory responses in HGBECs were to create an in vitro model of cholecystitis. The key inflammatory markers (IL-1ß, IL-6, and TNF-α) levels were quantified using ELISA, The modulation of the MAPK/NF-κB signaling pathway was measured by western blot using specific antibodies against pathway components (p-IκBα, IκBα, p-p65, p65, p-JNK, JNK, p-ERK, ERK, p-p38, and p38). The cell viability and LDH levels in HGBECs were not significantly affected by 50 µM KPR, thus it was selected as the optimal KPR intervention concentration. KPR increased the viability of LPS-induced HGBECs. Additionally, KPR inhibited the inflammatory factors level (IL-1ß, IL-6, and TNF-α) and protein expression (iNOS and COX-2) in LPS-induced HGBECs. Furthermore, KPR reversed LPS-induced elevation of p-IκBα/IκBα, p-p65/p65, p-JNK/JNK, p-ERK/ERK, and p-p38/p38 ratios. KPR attenuates the LPS-induced inflammatory response in HGBECs, possibly by inhibiting MAPK/NF-κB signaling.

Transcriptional patterns of human retinal pigment epithelial cells under protracted high glucose.

BACKGROUND: The retinal pigment epithelium (RPE) is essential for retinal homeostasis. Comprehensively exploring the transcriptional patterns of diabetic human RPE promotes the understanding of diabetic retinopathy (DR). METHODS AND RESULTS: A total of 4125 differentially expressed genes (DEGs) were screened out from the human primary RPE cells subjected to prolonged high glucose (HG). The subsequent bioinformatics analysis is divided into 3 steps. In Step 1, 21 genes were revealed by intersecting the enriched genes from the KEGG, WIKI, and Reactome databases. In Step 2, WGCNA was applied and intersected with the DEGs. Further intersection based on the enrichments with the GO biological processes, GO cellular components, and GO molecular functions databases screened out 12 candidate genes. In Step 3, 13 genes were found to be simultaneously up-regulated in the DEGs and a GEO dataset involving human diabetic retinal tissues. VEGFA and ERN1 were the 2 starred genes finally screened out by overlapping the 3 Steps. CONCLUSION: In this study, multiple genes were identified as crucial in the pathological process of RPE under protracted HG, providing potential candidates for future researches on DR. The current study highlights the importance of RPE in DR pathogenesis.

[Clinicopathological and molecular genetic features of Crohn's disease].

Objective: To investigate the clinicopathological and molecular genetic characteristics of Crohn's disease (CD). Methods: A retrospective analysis was conducted on 52 CD patients who underwent surgical resection at the First Affiliated Hospital of Nanjing Medical University between January 2014 and June 2023. Clinical presentations and histopathological features were assessed. Whole-genome sequencing was performed on 17 of the samples, followed by sequencing and pathway enrichment analyses. Immunohistochemistry was used to assess the expression of frequently mutated genes. Results: Among the 52 patients, 34 were males and 18 were females, male-to-female ratio was 1.9∶1.0, with a median age of 45 years at surgery and 35 years at diagnosis. According to the Montreal classification, A3 (51.9%,27/52), B2 (61.5%, 32/52), and L3 (50.0%,26/52) subtypes were the most predominant. Abdominal pain and diarrhea were the common symptoms. Histopathological features seen in all 52 patients included transmural inflammation, disruption of cryptal architecture, lymphoplasmacytic infiltration, varying degrees of submucosal fibrosis and thickening, increased enteric nerve fibers and neuronal proliferation. Mucosal defects, fissure ulcers, abscesses, pseudopolyps, and adenomatous proliferation were also observed in 51 (98.1%), 38 (73.1%), 28 (53.8%), 45 (86.5%), and 28 (53.8%) cases, respectively. Thirty-one (59.6%) cases had non-caseating granulomas, and 3 (5.8%) cases had intestinal mucosal glandular epithelial dysplasia. Molecular analysis showed that 12/17 CD patients exhibited mutations in at least one mucin family gene (MUC2, MUC3A, MUC4, MUC6, MUC12, MUC17), and MUC4 was the most frequently mutated in 7/17 of cases. Immunohistochemical stains showed reduced MUC4 expression in epithelial cells, with increased MUC4 expression in the epithelial surface, particularly around areas of inflammatory cell aggregation; and minimal expression in the lower half of the epithelium. Conclusions: CD exhibits diverse clinical and pathological features, necessitating a comprehensive multidimensional analysis for diagnosis. Mutations and expression alterations in mucin family genes, particularly MUC4, may play crucial roles in the pathogenesis of CD.

Exosomes Derived from Human Palatal Mesenchymal Cells Mediate Intercellular Communication During Palatal Fusion by Promoting Oral Epithelial Cell Migration.

Purpose: Exosomes are important "messengers" in cell-cell interactions, but their potential effects on palatal fusion are still unknown. This study aimed to explore the role and mechanism of exosomes derived from palatal mesenchymal cells in epithelial-mesenchymal communication during palatogenesis. Methods: The expression of exosome marker CD63 and CD81 in palatal cells during palatogenesis was detected by immunofluorescence staining. After being purified from the supernatant of human embryonic palatal mesenchymal (HEPM) cells, exosomes (HEPM-EXO) were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot. HEPM-EXO were co-cultured with human immortalized oral epithelial cells (HIOEC). The effects of HEPM-EXO on the cell proliferation, migration, apoptosis and epithelial-mesenchymal transition (EMT) of HIOEC were evaluated. The proteins encapsulated in HEPM-EXO were analyzed by proteomic analysis. Results: The extensive expression of CD63 and CD81 in palatal epithelial and mesenchymal cells were continuously detected during E12.5~E14.5, suggesting that exosomes were involved in the process of palatal fusion. The expression of CD63 was also observed in the acellular basement membrane between the palatal epithelium and the mesenchyme in vivo, and HEPM-EXO could be internalized by HIOEC in vitro, suggesting that exosomes are potent to diffuse through the cellular tissue boundary to mediate palatal cell-cell communication. Exposure of HEPM-EXO to HIOEC substantially inhibited the proliferation and stimulated the migration of HIOEC, but had no significant effect on cell apoptosis and EMT. Proteomic analysis revealed the basic characteristics of the proteins in HEPM-EXO and that exosomal THBS1 may potentially regulate the cell behaviors of HIOEC, which needs further verification. Gene ontology (GO) analysis uncovered that the proteins highly expressed in HEPM-EXO are closely related to wound healing, implying a promising therapeutic opportunity of HEPM-EXO in tissue injury treatment with future studies. Conclusion: HEPM-EXO mediated cell-cell communication by regulating cell proliferation and migration of oral epithelial cells during palatogenesis.

Insights from bioinformatics analysis reveal that lipopolysaccharide induces activation of chemokine-related signaling pathways in human nasal epithelial cells.

Lipopolysaccharide (LPS) is known to elicit a robust immune response. This study aimed to investigate the impact of LPS on the transcriptome of human nasal epithelial cells (HNEpC). HNEpC were cultured and stimulated with LPS (1 µg/mL) or an equivalent amount of normal culture medium. Subsequently, total RNA was extracted, purified, and sequenced using next-generation RNA sequencing technology. Differentially expressed genes (DEGs) were identified and subjected to functional enrichment analysis. A protein-protein interaction (PPI) network of DEGs was constructed, followed by Ingenuity Pathway Analysis (IPA) to identify molecular pathways influenced by LPS exposure on HNEpC. Validation of key genes was performed using quantitative real-time PCR (qRT-PCR). A total of 97 DEGs, comprising 48 up-regulated genes and 49 down-regulated genes, were identified. Results from functional enrichment analysis, PPI, and IPA indicated that DEGs were predominantly enriched in chemokine-related signaling pathways. Subsequent qRT-PCR validation demonstrated significant upregulation of key genes in these pathways in LPS-treated HNEpC compared to control cells. In conclusion, LPS intervention profoundly altered the transcriptome of HNEpC, potentially exacerbating inflammatory responses through the activation of chemokine-related signaling pathways.

PALB2-mutated human mammary cells display a broad spectrum of morphological and functional abnormalities induced by increased TGFß signaling.

Heterozygous mutations in any of three major genes, BRCA1, BRCA2 and PALB2, are associated with high-risk hereditary breast cancer susceptibility frequently seen as familial disease clustering. PALB2 is a key interaction partner and regulator of several vital cellular activities of BRCA1 and BRCA2, and is thus required for DNA damage repair and alleviation of replicative and oxidative stress. Little is however known about how PALB2-deficiency affects cell function beyond that, especially in the three-dimensional setting, and also about its role during early steps of malignancy development. To answer these questions, we have generated biologically relevant MCF10A mammary epithelial cell lines with mutations that are comparable to certain clinically important PALB2 defects. We show in a non-cancerous background how both mono- and biallelically PALB2-mutated cells exhibit gross spontaneous DNA damage and mitotic aberrations. Furthermore, PALB2-deficiency disturbs three-dimensional spheroid morphology, increases the migrational capacity and invasiveness of the cells, and broadly alters their transcriptome profiles. TGFß signaling and KRT14 expression are enhanced in PALB2-mutated cells and their inhibition and knock down, respectively, lead to partial restoration of cell functions. KRT14-positive cells are also more abundant with DNA damage than KRT14-negative cells. The obtained results indicate comprehensive cellular changes upon PALB2 mutations, even in the presence of half dosage of wild type PALB2 and demonstrate how PALB2 mutations may predispose their carriers to malignancy.

Reduction of elective lymph node volume in radiotherapy of early anal squamous cell cancer: a comparative study between two Swedish university hospitals.

BACKGROUND: Anal squamous cell cancer (ASCC) in early stages (T1-2N0M0) is treated with chemoradiotherapy with a 3-year overall survival (OS) exceeding 90%. In Swedish guidelines, it has been optional to include the external iliac and presacral lymph node (LN) stations in radiotherapy (RT) treatment fields in early ASCC. Two Swedish hospitals treating ASCC (SU: Sahlgrenska University Hospital; UU: Uppsala University Hospital) have chosen different approaches since 2010. MATERIAL AND METHODS: This study included consecutive patients with early ASCC (T1-2N0M0) treated between 2010 and 2017 at both sites (SU n = 70; UU n = 46). Data were retrieved from medical records and RT charts. RESULTS: At SU, the external iliac and presacral LN stations were included in elective LN irradiation in 96.8% (n = 60) and 95.2% (n = 59) patients compared to 2.4% (n = 1) and 29.3% (n = 12) at UU. The mean elective LN volume was 2,313 cc (interquartile range [IQR] 1,951-2,627) in the SU cohort compared to 1,317 cc (IQR 1,192-1,528) in the UU cohort, p < 0.0001. No case of regional LN recurrence was seen in either cohort. Disease specific survival (DSS) at 5 years was 95.7% (confidence interval [CI] 90.1-100.0) in the SU cohort and 97.8% (CI 93.2-100.0) in the UU cohort (p 0.55). OS at 5 years was 84.5% (CI 76.1-93.0) in the SU cohort and 82.6% (CI 69.6-89.1) in the UU cohort (p 0.8). INTERPRETATION: We found no differences in regional recurrence, DSS or OS between the cohorts treated with different elective LN volumes. In this population-based study, reduction of RT volume in early ASCC did not lead to inferior outcome.