Epithelial Cell Markers Research Articles

Aging alters the expression of trophic factors and tight junction proteins in the mouse choroid plexus

BackgroundThe choroid plexus (CP) is an understudied tissue in the central nervous system and is primarily implicated in cerebrospinal fluid (CSF) production. CP also produces numerous neurotrophic factors (NTF) which circulate to different brain regions. Regulation of NTFs in the CP during natural aging is largely unknown. Here, we investigated the age and gender-specific transcription of NTFs along with the changes in the tight junctional proteins (TJPs) and the water channel protein Aquaporin (AQP1).MethodsMale and female mice were used for our study. Age-related transcriptional changes were analyzed using quantitative PCR at three different time points: mature adult, middle-aged, and aged. Transcriptional changes during aging were further confirmed with digital droplet PCR. Additionally, we used immunohistochemical analysis (IHC) for the evaluation of in vivo protein expression. We further investigated the cellular phenotype of these NTFS, TJP, and water channel proteins in the mouse CP by co-labeling them with the classical vascular marker, Isolectin B4, and epithelial cell marker, Plectin.ResultsAging significantly altered NTF gene expression in the CP. Brain-derived neurotrophic factor (BDNF), Midkine (MDK), VGF, Insulin-like growth factor (IGF1), IGF2, Klotho (KL), Erythropoietin (EPO), and its receptor (EPOR) were reduced in the aged CP of males and females. Vascular endothelial growth factor (VEGF) transcription was gender-specific; in males, gene expression was unchanged in the aged CP, while females showed an age-dependent reduction. Age-dependent changes in VEGF localization were evident, from vasculature to epithelial cells. IGF2 and klotho localized in the basolateral membrane of the CP and showed an age-dependent reduction in epithelial cells. Water channel protein AQP1 localized in the tip of epithelial cells and showed an age-related reduction in mRNA and protein levels. TJP’s JAM, CLAUDIN1, CLAUDIN2 and CLAUDIN5 were reduced in aged mice.ConclusionsOur study highlights transcriptional level changes in the CP during aging. The age-related transcriptional changes exhibit similarities as well as gene-specific differences in the CP of males and females. Altered transcription of the water channel protein AQP1 and TJPs could be involved in reduced CSF production during aging. Importantly, reduction in the neurotrophic factors and longevity factor Klotho can play a role in regulating brain aging.Graphical

The Protective Potential of Butyrate against Colon Cancer Cell Migration and Invasion Is Critically Dependent on Cell Type.

Short-chain fatty acids such as butyrate are produced through the fermentation of dietary fiber by colonic bacteria. Preclinical studies indicate an anticancer potential of butyrate, but clinical evidence shows greater variability. The study hypothesizes the effectiveness of butyrate on reducing colon cancer cell migration and invasion may vary due to the cell-type. The study determines the efficacy of butyrate (0-4mM) to inhibit cancer cell migration, invasion, and related signaling proteins in three distinct human colorectal cancer (CRC) cell lines: HCT116, HT-29, and Caco-2.Butyrate exhibits a dose-dependent inhibitory effect on cancer cell migration and invasion. This inhibitory potential on oncogenic focal adhesion kinase (FAK) and sarcoma (Src) proteins is greater in HCT116 cells (1.1 and 0.8-fold) and HT-29 cells (0.9 and 0.4-fold) compared to Caco-2 cells, respectively. Conversely, E-cadherin protein, a classical epithelial cell marker and potential tumor suppressor, is 2.3-fold greater in HCT116 cells than in HT-29 cells and Caco-2 cells. Moreover, survival analysis from a public cancer database demonstrates that CRC patients with high E-cadherin expression have a 13% greater 5-year survival rate than those with low expression. Collectively, butyrate's anti-cancer efficacy on CRC cells varies depending on cell-type and is linked to the FAK/Src/E-cadherin pathway.

SSPH I, A Novel Anti-cancer Saponin, Inhibits EMT and Invasion and Migration of NSCLC by Suppressing MAPK/ERK1/2 and PI3K/AKT/ mTOR Signaling Pathways.

Saponin of Schizocapsa plantaginea Hance I (SSPH I).a bioactive saponin found in Schizocapsa plantaginea, exhibits significant anti-proliferation and antimetastasis in lung cancer. To explore the anti-metastatic effects of SSPH I on non-small cell lung cancer (NSCLC) with emphasis on epithelial-mesenchymal transition (EMT) both in vitro and in vivo. The effects of SSPH I at the concentrations of 0, 0.875,1.75, and 3.5 μM on A549 and PC9 lung cancer cells were evaluated using colony formation assay, CCK-8 assay, transwell assay and wound-healing assay. The actin cytoskeleton reorganization of PC9 and A549 cells was detected using the FITC-phalloidin fluorescence staining assay. The proteins related to EMT (N-cadherin, E-cadherin and vimentin), p- PI3K, p- AKT, p- mTOR and p- ERK1/2 were detected by Western blotting. A mouse model of lung cancer metastasis was established by utilizing 95-D cells, and the mice were treated with SSPH I by gavage. The results suggested that SSPH I significantly inhibited the migration and invasion of NSCLC cells under a non-cytotoxic concentration. Furthermore, SSPH I at a non-toxic concentration of 0.875 μM inhibited F-actin cytoskeleton organization. Importantly, attenuation of EMT was observed in A549 cells with upregulation in the expression of epithelial cell marker E-cadherin and downregulation of the mesenchymal cell markers vimentin as well as Ncadherin. Mechanistic studies revealed that SSPH I inhibited MAPK/ERK1/2 and PI3K/AKT/mTOR signaling pathways. SSPH I inhibited EMT, migration, and invasion of NSCLC cells by suppressing MAPK/ERK1/2 and PI3K/AKT/mTOR signaling pathways, suggesting that the natural compound SSPH I could be used for inhibiting metastasis of NSCLC.

Calprotectin is regulated by IL-17A and induces steroid hyporesponsiveness in asthma.

Calprotectin, a calcium-binding protein, plays a crucial role in inflammation and has been associated with various inflammatory diseases, including asthma. However, its regulation and impact on steroid hyporesponsiveness, especially in severe asthma, remain poorly understood. This study investigated the regulation of calprotectin proteins (S100A8 and S100A9) by IL-17 and its role in steroid hyporesponsiveness using in vitro and in vivo models. Calprotectin expression was assessed in primary bronchial fibroblasts from healthy controls and severe asthmatic patients, as well as in mouse models of steroid hyporesponsive lung inflammation induced by house dust mite (HDM) allergen and cyclic-di-GMP (cdiGMP) adjuvant. The effects of IL-17A stimulation on calprotectin expression and steroid response markers in bronchial epithelial and fibroblast cells were examined. Additionally, the therapeutic potential of paquinimod, a calprotectin inhibitor, in mitigating airway inflammation and restoring steroid response signatures in the mouse model was evaluated. The results demonstrated upregulation of calprotectin expression in asthmatic bronchial fibroblasts compared to healthy controls, as well as in refractory asthma samples compared to non-refractory asthma. IL-17 stimulation induced calprotectin expression and dysregulated glucocorticoid response signatures in lung epithelial and fibroblast cells. Treatment with paquinimod reversed IL-17-induced dysregulation of steroid signatures, indicating the involvement of calprotectin in this process. In the HDM/cdiGMP mouse model, paquinimod significantly attenuated airway inflammation and hyperresponsiveness, and restored steroid response signatures, whereas dexamethasone showed limited efficacy. Mechanistically, paquinimod inhibited MAPK/ERK and NF-κB pathways downstream of calprotectin, leading to reduced lung inflammation. These findings highlight calprotectin as a potential therapeutic target regulated by IL-17 in steroid hyporesponsive asthma. Targeting calprotectin may offer a promising approach to alleviate airway inflammation and restore steroid responsiveness in severe asthma. Further investigations are warranted to explore its therapeutic potential in clinical settings and elucidate its broader implications in steroid mechanisms of action.

Conbercept reverses TGF-β2-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway

To investigate the mechanism by which conbercept reverses transforming growth factor-β2 (TGF-β2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). Cultured HLEC SRA01/04 cells were treated with TGF-β2, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway. Conbercept significantly reduced TGF-β2-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-β2+conbercept group than in TGF-β2 group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β2-induced EMT (P <0.01). Conbercept inhibits TGF-β2 induced EMT by downregulating the expression of pSmad2/3 in TGF-β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling.

Ischemia/reperfusion (I/R) is inevitable during kidney transplantation and causes acute kidney injury (AKI), which affects immediate outcome and leads to chronic changes such as fibrotic remodeling of the graft. We investigated pro-fibrotic signaling after I/R, focusing on the complement component and receptor C5a/C5aR1 and macrophage/tubule crosstalk. Male Dark Agouti rats were subjected to I/R and their kidneys were harvested 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after reperfusion. The development of renal fibrosis was assessed by the detection of Vimentin (VIM), α-smooth muscle actin (α-SMA) and collagen by immunohistochemistry and Sirius Red staining, respectively. The characterization of C5a/C5aR1 activity and C5aR1+ cells was performed by multiplex mRNA analysis, ELISA, immunofluorescence flow cytometry and in situ hybridization in animal models and cell culture analyses. In the cell culture experiments, we focused on macrophage/tubule cell crosstalk in co-culture experiments and mimicked in vivo conditions by hypoxia/reoxygenation and supplementation with C5a. Already 6-24 h after the induction of I/R in the rat model, C5a concentration in the plasma was significantly increased compared to the control. The matrix components VIM and α-SMA peaked on day 5 and declined after 8 weeks, when an increase in collagen was detected using Sirius Red. In contrast to early I/R-induced C5a activation, renal C5ar1 expression was maximal at day 5 and C5 expression increased until week 8, indicating that the renal upregulation of expression is not required for early complement activation. C5aR1 mRNA was detected in neutrophils and macrophages, but not in proximal tubular cells in the injured kidneys. The macrophage/tubular cell co-culture experiments showed that macrophages were mainly responsible for the increased expression of fibrosis-associated genes in tubule cells (ACTA2, VIM, SNAI1, TGFB1 and FGF-2), and hypoxia/reoxygenation had a partially enhancing effect. A direct pro-fibrotic effect of C5a was not observed. Increased TGF-ß levels were dependent on the differentiation of macrophages to the M2 subtype. In conclusion, the early activation of mesenchymal markers in tubular epithelial cells leads to long-term fibrotic remodeling characterized by VIM expression and driven by TGF-ß-dependent macrophage/tubular crosstalk. The chemoattractive properties of complement C5a may contribute to the recruitment of pro-fibrotic macrophages.

Pasteurized form of a potential probiotic lactobacillus brevis IBRC-M10790 exerts anti-inflammatory effects on inflammatory bowel disease in vitro

BackgroundInflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder of the gastrointestinal system. So far, no treatment has been identified that can completely cure IBD. Lactobacillus brevis is hypothesized to be beneficial in preventing inflammation. This study aimed to evaluate the potential probiotic effects of live and pasteurized L. brevis IBRC-M10790 on the in vitro cell co-culture model of IBD.MethodsAn in vitro intestinal model was established using a transwell co-culture system of Caco-2 intestinal epithelial cells and RAW264.7 macrophages. Inflammatory conditions were induced in RAW264.7 cells using lipopolysaccharide. The effects of live and pasteurized L. brevis IBRC-M10790 on inflammatory mediators and epithelial barrier markers were investigated.ResultsL. brevis IBRC-M10790 was able to significantly decrease the proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and increase the anti-inflammatory cytokine (IL-10) in the in vitro co-culture system. In addition, L. brevis increased adherens and tight junction (TJ) markers (ZO-1, E-cadherin, and Occludin) in Caco-2 intestinal epithelial cells. Based on the results, pasteurized L. brevis showed a higher protective effect than live L. brevis.ConclusionsOur findings suggest that live and pasteurized forms of L. brevis possess probiotic properties and can mitigate inflammatory conditions in IBD.

Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper)and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology.

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4°C) but replicated at high temperatures (30°C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

Urine-derived renal epithelial cells isolated after kidney transplant are sensitive to neutrophil gelatinase-associated lipocalin exposure during in vitro culture

Urine-derived renal epithelial cells (URECs) are highly voided after kidney transplant and express typical kidney markers, including markers of kidney epithelial progenitor cells. Recently URECs have shown promising immunomodulatory properties when cultured with Peripheral Blood Mononuclear Cells (PBMCs), promoting an increase in the T regulatory cells. In vivo, kidney cells are highly exposed to damage associated molecules during both acute and chronic kidney injury. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most -known early marker of acute and chronic kidney damage. However, its role on the evolution of renal damage has not yet been fully described, nor has its impact on the characteristics of renal-derived cells during in vitro culture. The aim of this study is to investigate the effect of NGAL on the characteristics of URECs isolated after kidney transplant, by exposing these cells to the treatment with NGAL during in vitro culture and evaluating its effect on UREC viability, proliferation, and immunomodulatory potential. The exposure of URECs to NGAL reduced their viability and proliferative capacity, promoting the onset of apoptosis. The immunomodulatory properties of URECs were partially inhibited by NGAL, without affecting the increase of Treg cells observed during UREC-PBMCs coculture. These results suggest that the exposure to NGAL may compromise some features of kidney stem and specialized cell types, reducing their viability, increasing apoptosis, and partially altering their immunomodulatory properties. Thus, NGAL could represent a target for approaches acting on its inhibition or reduction to improve functional recovery.

An easy-to-perform protocol for culturing primary murine lung tumor cells as organoids

Cancer research involves significant animal consumption and suffering. Tumor cells can be differentiated in vitro into three-dimensional organoids that resemble the primary tumor. In basic cancer research, however, tumor organoids are usually only used alongside animal experiments. We have established an easy-to-perform protocol that allows to culture KRAS-driven lung tumor cells as organoids for extended periods of time. Like the corresponding tumors in mice, the organoids produce surfactant protein C but no markers of airway epithelial cells (e.g. SCGB1A1, KRT5). The organoids can be passaged as single cell suspensions. Our organoid model contributes to replace animal experiments with cell culture systems and can be used for drug testing or functional studies in cancer research.

Establishment and characterization of a novel multidrug-resistant pancreatic ductal adenocarcinoma cell line, PDAC-X1

Drug resistance remains a significant challenge in the treatment of pancreatic cancer. The development of drug-resistant cell lines is crucial to understanding the underlying mechanisms of resistance and developing novel drugs to improve clinical outcomes. Here, a novel pancreatic cancer cell line, PDAC-X1, derived from Chinese patients has been established. PDAC-X1 was characterized by the immune phenotype, biology, genetics, molecular characteristics, and tumorigenicity. In vitro analysis revealed that PDAC-X1 cells exhibited epithelial morphology and cell markers (CK7 and CK19), expressed cancer-associated markers (E-cadherin, Vimentin, Ki-67, CEA, CA19-9), and produced pancreatic cancer-like organs in suspension culture. In vivo analysis showed that PDAC-X1 cells maintained tumorigenicity with a 100% tumor formation rate. This cell line exhibited a complex karyotype, dominated by subtriploid karyotypes. In addition, PDAC-X1 cells exhibited intrinsic multidrug resistance to multiple drugs, including gemcitabine, paclitaxel, 5-fluorouracil, and oxaliplatin. In conclusion, the PDAC-X1 cell line has been established and characterized, representing a useful and valuable preclinical model to study the underlying mechanisms of drug resistance and develop novel drug therapeutics to improve patient outcomes.

Drug-Resistance Biomarkers in Patient-Derived Colorectal Cancer Organoid and Fibroblast Co-Culture System.

Colorectal cancer, the third most commonly occurring tumor worldwide, poses challenges owing to its high mortality rate and persistent drug resistance in metastatic cases. We investigated the tumor microenvironment, emphasizing the role of cancer-associated fibroblasts in the progression and chemoresistance of colorectal cancer. We used an indirect co-culture system comprising colorectal cancer organoids and cancer-associated fibroblasts to simulate the tumor microenvironment. Immunofluorescence staining validated the characteristics of both organoids and fibroblasts, showing high expression of epithelial cell markers (EPCAM), colon cancer markers (CK20), proliferation markers (KI67), and fibroblast markers (VIM, SMA). Transcriptome profiling was conducted after treatment with anticancer drugs, such as 5-fluorouracil and oxaliplatin, to identify chemoresistance-related genes. Changes in gene expression in the co-cultured colorectal cancer organoids following anticancer drug treatment, compared to monocultured organoids, particularly in pathways related to interferon-alpha/beta signaling and major histocompatibility complex class II protein complex assembly, were identified. These two gene groups potentially mediate drug resistance associated with JAK/STAT signaling. The interaction between colorectal cancer organoids and fibroblasts crucially modulates the expression of genes related to drug resistance. These findings suggest that the interaction between colorectal cancer organoids and fibroblasts significantly influences gene expression related to drug resistance, highlighting potential biomarkers and therapeutic targets for overcoming chemoresistance. Enhanced understanding of the interactions between cancer cells and their microenvironment can lead to advancements in personalized medical research..

GDF6 in gastric cancer upregulated by helicobacter pylori induces epithelial-mesenchymal translation via the TGF-β/SMAD3 signaling pathway

ObjectiveTo investigate the association between Helicobacter pylori infection and GDF6 expression in gastric cancer patients, and to determine its influence on prognosis and resistance to capecitabine. MethodsTumor and adjacent non-tumor tissues were collected from 148 gastric cancer patients who underwent surgery in our department from October 2019 to June 2022. Of these patients, 78 tested positive for Helicobacter pylori and 70 tested negative. Hematoxylin-eosin (HE) and immunofluorescence staining were utilized to quantify GDF6 expression in cancerous and adjacent tissues. Patient prognosis was monitored via follow-up. Western blotting analyzed GDF6 expression in common gastric cancer cell lines. HGC27 cells exhibiting high GDF6 expression and BGC823 cells with low expression were used to create GDF6-silenced and overexpressed cell lines. The impact of GDF6 on the proliferation, migration, invasion, and cloning abilities of gastric cancer cells was evaluated using the CCK-8 assay, scratch test, Transwell assay, and plate colony formation assay. Fluorescent quantitative PCR and Western blotting assessed the effects of GDF6 levels on epithelial-mesenchymal transition (EMT) and tumor cell stemness. ResultsGDF6 expression in gastric cancer tissues was significantly correlated with cancer grading and staging (P<0.05). Helicobacter pylori-positive tissues exhibited significantly higher GDF6 expression levels than negative samples (P<0.05). Kaplan-Meier survival analysis indicated that high GDF6 expression was associated with poor survival prognosis. Overexpressed GDF6 enhanced the proliferation, migration, and invasion abilities of gastric cancer cells, while silencing GDF6 yielded opposite results. Increased GDF6 expression upregulated TGF-β expression and the phosphorylation levels of SMAD3, leading to an elevation in mesenchymal cell markers N-cadherin, vimentin, and a reduction in epithelial cell markers cytokeratins, E-cadherin. Moreover, high GDF6 levels contributed to increased resistance to capecitabine and enhanced the expression of tumor stem cell markers Nanog, Sox-2, Oct-4, CD44, amplifying tumor cell stemness. ConclusionHelicobacter pylori infection is associated with increased GDF6 expression in gastric cancer tissue, correlating with poor survival prognosis. Elevated GDF6 expression promotes the proliferation, migration, and invasion abilities of gastric cancer cells, facilitates EMT via the TGF-β/SMAD3 pathway, and intensifies cell stemness and capecitabine resistance. Consequently, GDF6 presents itself as a potential new target for gastric cancer treatment. Data availability statementThe data that support the findings of this study are available from the corresponding author upon reasonable request.

The role of the tumor microenvironment in papillary thyroid microcarcinoma nodal metastasis.

The genetic alterations currently identified in papillary thyroid microcarcinomas (PTMCs) are insufficient for distinguishing tumors with aggressive features. We aimed to identify candidate markers associated with lateral lymph node metastasis (LLNM, N1sb disease) in patients with PTMC using transcriptomic analysis. RNA sequencing was performed on 26 matched tumor and normal thyroid tissue samples (N0, n = 14; N1b, n = 12), followed by functional enrichment analyses of differentially expressed genes (DEGs). EcoTyper was used to explore the distinct tumor microenvironment (TME). We identified 631 DEGs (213 upregulated and 418 downregulated) between N1b and N0 PTMCs. The most significantly upregulated genes in N1b were associated with tumorigenesis, adhesion, migration, and invasion. DEGs were mainly enriched in the pathways of idiopathic pulmonary fibrosis, TME, wound healing, and inhibition of matrix metalloproteases. We predicted the activation of these pathways in N1b PTMCs. N1b PTMCs had a unique TME with abundant fibroblasts and epithelial cells, associated with an increased risk of disease progression. Fibroblast marker genes, including POSTN, MMP11, TNFAIP6,and FN1, and epithelial cell marker genes, including NOX4, MFAP2, TGFVBI,and TNC, were selected. POSTN and FN1, fibroblast cell-specific genes, and NOX4 and TNC, epithelial cell-specific genes, were promising biomarkers for predicting LLNM development and recurrence in patients with PTMC. We delineated the cellular ecotypes within the TME of patients with N1b PTMC and revealed potential markers for predicting LLNM and the prognosis of PTMC. These findings provide valuable insights into the contributions of cancer-associated fibroblasts and epithelial cells to PTMC progression and metastasis.

Testing of Anti-EMT, Anti-Inflammatory and Antibacterial Activities of 2',4'-Dimethoxychalcone.

Chalcone (1,3-diaryl-2-propen-1-one) is an α, β-unsaturated ketone that serves as an active constituent or precursor of numerous natural substances, exhibiting a broad spectrum of pharmacological effects. In this study, the classical Claisen-Schmidt condensation method was used to synthesize the chalcone derivative 2',4'-dimethoxychalcone (DTC) and evaluate its pharmacological activity. By upregulating the expression of the epithelial cell marker E-cadherin and downregulating the expression of the mesenchymal cell marker vimentin, DTC was found to inhibit transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) process in A549 cells, maintaining the cells' epithelial-like morphology and reducing the ability of the cells to migrate. Additionally, DTC demonstrated the ability to decrease the expression levels of nitric oxide (NO), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in RAW264.7 cells, suggesting a possible anti-inflammatory effect. Furthermore, DTC was found to exhibit bacteriostatic activity against Staphylococcus aureus (S. aureus), Proteus vulgaris (P. vulgaris), methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans (C. albicans), indicating that this chemical may possess broad-spectrum antibacterial activity.

A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery.

The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.

Lung fibroblast-derived extracellular vesicles and soluble factors alleviate elastase-induced lung injury

One of the main pathological features of chronic obstructive pulmonary disease (COPD) is the loss of functional alveolar tissue as a consequence of impaired regenerative capacities (emphysema). Recent research suggests that the secretome from mesenchymal cells, particularly extracellular vesicles (EVs), may possess regenerative properties beneficial for lung repair. However, the regenerative potential of the soluble factors (SFs) within the secretome remains largely unexplored in COPD. To this extent, we purified EVs and SFs secreted by lung fibroblasts to generate EV-enriched and SF-enriched fractions, and evaluated their effects on elastase-induced lung injury in both precision-cut lung slices (PCLS) and a mouse model. EV- and SF-enriched fractions were concentrated and purified from the conditioned medium of cultured MRC-5 lung fibroblasts using a combination of ultrafiltration and size exclusion chromatography, and were subsequently characterized according to the MISEV guidelines. Treatment with EV- or SF-enriched concentrates prevented and improved elastase-induced emphysema in PCLS, leading to reduced lung injury and upregulated markers of alveolar epithelial cells (aquaporin 5 and surfactant protein C), indicating potential parenchymal regeneration. Accordingly, prophylactic intratracheal treatment with lung fibroblast-derived EV- and SF-enriched concentrates in vivo attenuated elastase-induced lung tissue destruction, improved lung function, and enhanced gene expression of alveolar epithelial cell markers. Here, alveolar repair not only serves the purpose of facilitating gas exchange, but also by reinstating the essential parenchymal tethering required for optimal airway mechanics. In conclusion, this study highlights the therapeutic potential of both lung fibroblast-derived EV- and SF-enriched concentrates for the treatment of lung injury and emphysema.

Comparative analysis of SEC61A1 mutant R236C in two patient-derived cellular platforms

SEC61A1 encodes a central protein of the mammalian translocon and dysfunction results in severe disease. Recently, mutation R236C was identified in patients having autosomal dominant polycystic liver disease (ADPLD). The molecular phenotype of R236C was assessed in two cellular platforms. Cells were immortalized by retroviral transduction of an oncogene (UCi) or reprogrammed to induced pluripotent stem cells (iPSC) that were differentiated to cholangiocyte progenitor-like cells (CPLC). UCi and CPLC were subjected to analyses of molecular pathways that were associated with development of disease. UCi displayed markers of epithelial cells, while CPLCs expressed typical markers of both cholangiocytes and hepatocytes. Cells encoding R236C showed a stable, continuous proliferation in both platforms, however growth rates were reduced as compared to wildtype control. Autophagy, cAMP synthesis, and secretion of important marker proteins were reduced in R236C-expressing cells. In addition, R236C induced increased calcium leakiness from the ER to the cytoplasm. Upon oxidative stress, R236C led to a high induction of apoptosis and necrosis. Although the grade of aberrant cellular functions differed between the two platforms, the molecular phenotype of R236C was shared suggesting that the mutation, regardless of the cell type, has a dominant impact on disease-associated pathways.

A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

The biological characteristics of chicken embryo mesenchymal stem cells isolated from chorioallantoic membrane.

Mesenchymal stem cells (MSCs) derived from fetal membranes (FMs) have the potential to exhibit immunosuppression, improve blood flow, and increase capillary density during transplantation. In the field of medicine, opening up new avenues for disease treatment. Chicken embryo chorioallantoic membrane (CAM), as an important component of avian species FM structure, has become a stable tissue engineering material in vivo angiogenesis, drug delivery, and toxicology studies. Although it has been confirmed that chorionic mesenchymal stem cells (Ch-MSCs) can be isolated from the outer chorionic layer of FM, little is known about the biological characteristics of MSCs derived from chorionic mesodermal matrix of chicken embryos. Therefore, we evaluated the characteristics of MSCs isolated from chorionic tissues of chicken embryos, including cell proliferation ability, stem cell surface antigen, genetic stability, and in vitro differentiation potential. Ch-MSCs exhibited a broad spindle shaped appearance and could stably maintain diploid karyotype proliferation to passage 15 in vitro. Spindle cells were positive for multifunctional markers of MSCs (CD29, CD44, CD73, CD90, CD105, CD166, OCT4, and NANOG), while hematopoietic cell surface marker CD34, panleukocyte marker CD45, and epithelial cell marker CK19 were negative. In addition, chicken Ch-MSC was induced to differentiate into four types of mesodermal cells in vitro, including osteoblasts, chondrocytes, adipocytes, and myoblasts. Therefore, the differentiation potential of chicken Ch-MSC in vitro may have great potential in tissue engineering. In conclusion, chicken Ch-MSCs may be an excellent model cell for stem cell regenerative medicine and chorionic tissue engineering.