Search Results (185)

This study aimed to evaluate cytokine profiling in a periapical lesion to provide a rationale for future treatment strategies for periapical lesions. Thirteen samples of exudative fluid were collected from such a lesion directly through the root canal. Cytokine profiling was performed using the Bio-Plex system. CXCL10 (C-X-C motif chemokine ligand 10, IP10) was found to be elevated in apical exudates of patients exhibiting favorable healing. To evaluate the role of CXCL10 in cell migration, a Transwell assay was conducted using bone marrow-derived mononuclear cells (BMMCs). Different types of cytokines were detected from the samples of periapical lesion at the initial visit. However, cytokine production varied across patient samples. Release of interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and tumor necrosis factor (TNF)-α showed differential expression. Comparison of cytokine profiles indicated that cytokine production was variable before and after root canal treatment. In vitro, CXCL10 significantly improved BMMC migration in a dose-dependent manner, supporting clinical findings that elevated CXCL10 levels are associated with favorable healing in apical lesions. Although this study was limited by the small sample size and exploratory design, the cytokine profile of periapical lesions may be useful for assessing the condition of periapical lesions and modulating the immune response to bacterial infection. Full article

Periodontitis is a prevalent condition that leads to the destruction of periodontal tissue, making the regeneration of the periodontium a key focus in dental research. In this context, periodontal ligament cells (PDLCs) are particularly interesting due to their stem cell-like properties, including the ability to differentiate into various cell types and further contribute to tissue repair. This study aimed to isolate and characterize primary human PDLCs and examine the effects of both indirect and direct treatment with the blood concentrate platelet-rich fibrin (PRF), with particular focus on how PRF influences cell proliferation and differentiation. PDLCs were treated with PRF prepared using a low relative centrifugal force (600 rpm) either directly through a conditioned medium or indirectly using trans-well filter systems. The impact of PRF on PDLC proliferation and differentiation was assessed through viability assays, alkaline phosphatase assays, gene and protein expression analyses, and immunofluorescence. PDLCs exhibited cellular markers characteristic of stem cell-like cells. In addition, PRF treatment was found to suppress cell proliferation while concurrently promoting osteogenic differentiation and increase factors important for tissue regeneration. These effects were more pronounced when the cells were directly treated with PRF-conditioned medium compared to indirect treatment. Our findings support the hypothesis that PRF serves as a biologically active reservoir of growth factors that modulate PDLC behavior and might create a microenvironment favorable for periodontal repair. Full article

Bone remodeling relies on the coordinated activity of osteoblasts (OBs) and osteoclasts (OCs). Disruptions in OB-OC balance can lead to diseases such as periodontitis, a chronic microbial-induced inflammatory disease. To investigate how inflammation affects OB-OC interactions, we standardized an in vitro 2D indirect co-culture system using primary human OB and OC precursors from peripheral blood mononuclear cells in a transwell setup, which allows paracrine signaling and separate analysis of each cell type. When exposed to bacterial lipopolysaccharides (Aa LPS and E. coli LPS) and proinflammatory cytokines (IL-6 and TNF-α), we observed that inflammatory stimuli significantly increased OC differentiation, particularly TNF-α, while E. coli LPS specifically suppressed OB activity as observed by the expression of key markers and cellular staining. These results demonstrate that microbial and host-derived inflammatory factors can differentially modulate bone cell behavior. This approach offers a physiologically relevant and ethically advantageous alternative to animal models to screen dual-targeted bone therapies to restore perturbed metabolism. Full article

Background: Predicting oral drug absorption in humans is critical during early drug development. Current in vitro systems to predict absorption (e.g., PAMPA and MDCK cells) are lacking for certain classes of drugs. Intestinal organoids are emerging as a promising alternative that offers several potential advantages. In this study, we utilized human intestinal organoid-derived monolayers to predict oral absorption of lenalidomide. Methods: Human jejunal organoids (RepliGut^®) were cultured as monolayers on transwell plates and differentiated into intestinal epithelial cells. Lenalidomide permeability in the organoid system was compared with the permeability in the conventional Madin-Darby Canine Kidney cell (MDCK) monolayer system, as well as P-gp knockout, human P-gp overexpressing, and human BCRP overexpressing MDCK cells across a concentration range of 1 to 500 µM. Male Sprague Dawley rats were administered lenalidomide orally/intravenously, and concentrations in the serum, urine, and feces were measured and modeled in Phoenix WinNonlin. Results: Orally administered lenalidomide was well absorbed by rats at all doses (bioavailability = 68–120%). In the human jejunal organoid model, lenalidomide apparent permeability (Papp) was approximately 0.6 × 10⁻⁶ cm/s independent of the concentration used (1–500 µM). In contrast, lenalidomide Papp was significantly lower in gMDCK cell monolayers, approximately 0.2 × 10⁻⁶ cm/s. Additionally, lenalidomide was identified as a P-gp/BCRP substrate in intestinal organoids and gMDCK P-gp and BCRP overexpressing cells. Conclusions: Lenalidomide Papp was significantly lower in gMDCK monolayers than expected based on its high bioavailability. Our results suggest that organoid systems can better capture transporter and paracellularly mediated effects on drug permeability, which may allow for more accurate predictions of in vivo absorption. Full article

The major event that leads to death from breast cancer (BrCa) is the emergence of micrometastases into lethal growing metastases. While it is still uncertain what regulates the cell fate decision between remaining in dormancy and aggressive proliferative progression, accumulating evidence demonstrates a major role for the metastatic microenvironment. One area of interest is that of tissue and circulating mesenchymal stem cells (MSCs), which have been shown to alter the proliferative and metastatic potential of BrCa. Herein, we investigate how these cells impact the phenotype of metastatic BrCa. As the disseminated BrCa cells initially adopt an epithelial phenotype in ectopic organs, one that is dormant in having limited proliferation and being immune-silent, interactions that revert the disseminated metastatic BrCa to aggressive mesenchymal phenotypes, would be a driver of metastatic progression. BrCa cells exhibited phenotypic changes including increased E-cadherin expression, altered proliferation, and differential sensitivity to TRAIL-induced apoptosis when directly co-cultured with immortalized human MSCs, compared to the BrCa cells not co-cultured. These regulatory effects were dependent upon the BrCa cell’s epithelial–mesenchymal status and involved distinct juxtacrine and paracrine signaling mechanisms, as evidenced by differing responses in direct co-culture, conditioned medium, and Transwell systems. Our findings highlight the complex and context-dependent roles of MSCs in BrCa progression, improving our understanding of tumor-stroma interactions and laying groundwork for future therapeutic exploration. Full article

Osteosarcopenia is a widespread geriatric condition resulting from the coexistence of osteoporosis and sarcopenia, where the connection between bone and muscle is, in part, driven by bone–muscle crosstalk. Given the close, reciprocal influence of muscle on nerve, and vice versa, it is not surprising that there are corresponding aging changes in the biochemistry and morphology of the neuromuscular junction (NMJ). Indeed, degeneration of motor neurons and progressive disruption of the neuromuscular connectivity were observed in old age. Extracellular vesicles (EVs) derived from human amniotic fluid stem cells (hAFSC), exhibiting antioxidant properties, which can also explain their anti-aging and cytoprotective effects, can be considered as potential treatment for age-related diseases. To study cell interactions under both healthy and pathological conditions occurring in musculo–skeletal apparatus, we developed a three-culture system exploiting the use of well-known transwell supports. This system allows both myotubes and neurons, eventually treated with EVs, and osteoblasts, induced to osteoporosis, to interact physically and biochemically. Collectively, this method allowed us to understand how the modifications induced in osteoblasts during bone disorders trigger a cascade of detrimental effects in the muscle and neuron parts. Moreover, we demonstrated the efficacy of hAFSC-EVs in preventing NMJ dysfunction, muscle atrophy, and osteoblast impairment. Full article

CD26 (dipeptidyl peptidase-4) is a marker of colorectal cancer stem cells with high metastatic potential and resistance to therapy. Although CD26 expression is known to be associated with tumor progression, its functional involvement in epithelial-mesenchymal transition (EMT) and metastasis remains to be fully elucidated. In this study, we aimed to investigate the effects of a monoclonal anti-CD26 antibody on EMT-related phenotypes and metastatic behavior in colorectal cancer cells. We evaluated changes in EMT markers by quantitative PCR and Western blotting, assessed cell motility and invasion using scratch wound-healing and Transwell assays, and examined metastatic potential in vivo using a splenic injection mouse model. Treatment with the anti-CD26 antibody significantly increased the expression of the epithelial marker E-cadherin and reduced levels of EMT-inducing transcription factors, including ZEB1, Twist1, and Snail1, at the mRNA and protein levels. Functional assays revealed that the antibody markedly inhibited cell migration and invasion in vitro without exerting cytotoxic effects. Furthermore, systemic administration of the anti-CD26 antibody significantly suppressed the formation of liver metastases in vivo. These findings suggest that CD26 may contribute to the regulation of EMT and metastatic behavior in colorectal cancer. Our data highlight the potential therapeutic utility of CD26-targeted antibody therapy for suppressing EMT-associated phenotypes and metastatic progression. Full article

Disruption of the intestinal epithelial barrier is a key driver of gut-derived inflammation in various disorders, yet strategies to preserve or restore barrier integrity remain limited. To address this, we evaluated a four-strain Bifidobacterium mixture—selected for complementary anti-inflammatory potency and industrial scalability—in lipopolysaccharide (LPS)-challenged RAW 264.7 macrophages and a Caco-2/THP-1 transwell co-culture model. Pretreatment with the probiotic blend reduced nitric oxide (NO) release in a dose-dependent manner by 25.9–48.3% and significantly down-regulated the pro-inflammatory markers in macrophages. In the co-culture system, the formulation decreased these markers, increased transepithelial electrical resistance (TEER) by up to 31% at 10⁵ colony-forming unit (CFU)/mL after 48 h, and preserved the membrane localization of tight junction (TJ) proteins. Adhesion to Caco-2 cells (≈ 6%) matched that of the benchmark probiotic Lacticaseibacillus rhamnosus GG, suggesting direct epithelial engagement. These in vitro findings demonstrate that this probiotic mixture can attenuate LPS-driven inflammation and reinforce epithelial architecture, providing a mechanistic basis for its further evaluation in animal models and clinical studies of intestinal inflammatory disorders. Full article

Murine microglia exhibit rapid self-renewal upon removal from the postnatal brain. However, the signaling pathways that regulate microglial repopulation remain largely unclear. To address this knowledge gap, we depleted microglia from mixed glial cultures using anti-CD11b magnetic particles and cultured them for 4 weeks to monitor their repopulation ability in vitro. Flow cytometry and immunocytochemistry revealed that anti-CD11b bead treatment effectively eliminated >95% of microglia in mixed glial cultures. Following removal, the number of CX3CR1-positive microglia gradually increased; when a specific threshold was reached, repopulation ceased without any discernable rise in cell death. Cell cycle and 5-ethynyl-2′-deoxyuridine incorporation assays suggested the active proliferation of repopulating microglia at d7. Time-lapse imaging demonstrated post-removal division of microglia. Colony-stimulating factor 1 receptor-phosphoinositide 3-kinase-protein kinase B signaling was identified as crucial for microglial repopulation, as pharmacological inhibition or neutralization of the pathway significantly abrogated repopulation. Transwell cocultures revealed that resident microglia competitively inhibited microglial proliferation probably through contact inhibition. This in vitro microglial removal system provides valuable insights into the mechanisms underlying microglial proliferation. Full article

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

Accurate in vitro models of intestinal permeability are essential for predicting oral drug absorption. Standard models like Caco-2 cells have well-known limitations, including lack of segment-specific physiology, but are widely used. Emerging models such as organoid-derived monolayers and microphysiological systems (MPS) offer enhanced physiological relevance but require comparative validation. We performed a head-to-head evaluation of Caco-2 cells, human jejunal (J2) and duodenal (D109) enteroid-derived cells, and EpiIntestinal^TM tissues cultured on either static Transwell and flow-based MPS platforms. We assessed tissue morphology, barrier function (TEER, dextran leakage), and permeability of three model small molecules (caffeine, propranolol, and indomethacin), integrating the data into a physiologically based gut absorption model (PECAT) to predict human oral bioavailability. J2 and D109 cells demonstrated more physiologically relevant morphology and higher TEER than Caco-2 cells, while the EpiIntestinal^TM model exhibited thicker and more uneven tissue structures with lower TEER and higher passive permeability. MPS cultures offered modest improvements in epithelial architecture but introduced greater variability, especially with enteroid-derived cells. Predictions of human fraction absorbed (F_abs) were most accurate when using static Caco-2 data with segment-specific corrections based on enteroid-derived values, highlighting the utility of combining traditional and advanced in vitro gut models to optimize predictive performance for F_abs. While MPS and enteroid-based systems provide physiological advantages, standard static models remain robust and predictive when used with in silico modeling. Our findings support the need for further refinement of enteroid-MPS integration and advocate for standardized benchmarking across gut model systems to improve translational relevance in drug development and regulatory reviews. Full article

Cell migration assays provide valuable insights into pathological conditions, such as tumor metastasis and immune cell infiltration, and the regenerative capacity of tissues. In vitro tools commonly used for cell migration studies exploit commercial transwell systems, whose functionalities can be improved through engineering of the pore pattern. In this context, we propose the fabrication of a transwell-like device pursued by combining the proton beam writing (PBW) technique with wet etching onto thin layers of polydimethylsiloxane (PDMS). The resulting transwell-like device incorporates a PDMS membrane with finely controllable pore patterning that was used to study the arrangement and migration behavior of HCMEC/D3 cells, a well-established human brain microvascular endothelial cell model widely used to study vascular maturation in the brain. A comparison between commercial polycarbonate membranes and the PBW-holed membranes highlights the impact of the ordering of the pattern and porosity on cellular growth, self-organization, and transmigration by combining fluorescent microscopy and advanced digital processing. Endothelial cells were found to exhibit distinctive clustering, alignment, and migratory behavior close to the pores of the designed PBW-holed membrane. This is indicative of activation patterns associated with cytoskeletal remodeling, a critical element in the angiogenic process. This study stands up as a novel approach toward the development of more biomimetic barrier models (such as organ-on-chips). Full article

Background/Objectives: Non-melanoma skin cancer (NMSC) is among the most common cancers in the United States, with solar ultraviolet (UV) radiation being a primary etiological factor. T-LAK cell-originated protein kinase (TOPK), a serine/threonine kinase activated by solar UV, has been implicated in skin carcinogenesis. This study aimed to investigate the mechanistic role of TOPK in solar UV-induced skin damage and tumor development. Methods: RNA sequencing (RNA-seq) was performed on skin tissues from wild-type (WT) and TOPK knockout (KO) mice, with or without solar UV exposure, to identify TOPK-regulated genes and pathways. Follow-up experiments using Western blotting, immunofluorescence, and luciferase assays were conducted in vitro and in vivo. Functional assays included 3D spheroid and Transwell co-culture systems involving cutaneous squamous cell carcinoma (cSCC) and fibroblast cells. Results: TOPK deletion altered gene expression profiles and inhibited solar UV-induced activation of multiple signaling pathways, including cytokine–cytokine receptor interaction, PI3K/AKT, MAPKs, PKG, cAMP, and calcium signaling. RNA-seq and protein analyses identified interleukin-19 (IL19) as a key downstream effector suppressed by TOPK deletion. In cSCC and fibroblast cells, TOPK knockdown reduced IL19 expression and secretion. IL19 promoted cSCC growth and activated PI3K/AKT, ERK, and TOPK pathways. Additionally, chronic TGFβ exposure increased IL19 expression and activated fibroblasts, as indicated by elevated αSMA and FAPα levels. Conclusions: These findings establish TOPK as a central regulator of solar UV-induced skin carcinogenesis, partially via modulation of IL19 signaling and fibroblast activation. Targeting TOPK may offer a novel strategy for the prevention and treatment of NMSC. Full article

Background: The blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) but also limits drug delivery. Insufficient knowledge of how the CNS promotes the onset and maintenance of peripheral neuropathic pain limits therapeutic methods for the treatment of persistent neuropathic pain. Thus, this study aimed to evaluate the ability of a novel combination of Palmitoylethanolamide (PEA) and Equisetum arvense L. (Equisetum A.L.) to cross the BBB and modulate nociceptive pathways. Methods: Using a humanised in vitro BBB tri-culture model, the permeability, cytotoxicity, and integrity of the barrier were assessed after exposure to two different PEA forms, PEA ultramicronized (PEA-um) and PEA80mesh, Equisetum A.L., and a combination of the last two samples. The samples exhibited no cytotoxicity, maintained tight junction integrity, and efficiently crossed the blood–brain barrier (BBB), with the combination displaying the highest permeability. The eluate from the BBB model was then used to stimulate the co-culture of CCF-STTG1 astrocytes and SH-SY5Y neurons pre-treated with H₂O₂ 200 µM. Results: Treatment with the combination significantly increased cell viability (1.8-fold, p < 0.05), reduced oxidative stress (2.5-fold, p < 0.05), and decreased pro-inflammatory cytokines (TNFα, IL-1β) compared to single agents. Mechanistic analysis revealed modulation of key targets involved in pain pathways, including decreased FAAH and NAAA activity, increased levels of endocannabinoids (AEA and 2-AG), upregulation of CB2 receptor expression, enhanced PPARα activity, and reduced phosphorylation of PKA and TRPV1. Conclusions: These findings suggest that the combination of PEA and Equisetum A.L. effectively crosses the BBB and exerts combined anti-inflammatory and analgesic effects at the CNS level, suggesting a possible role in modulating neuroinflammatory and nociception responses. Full article

Esophageal squamous cell carcinoma (ESCC), one of the most frequent malignant tumors of the digestive system, is marked by a poor prognosis and high mortality rate. There is a critical need for effective therapeutic strategies with minimal side effects. Isoquercitrin (IQ) is a natural compound with potent antioxidant properties in cancer and cardiovascular diseases. However, its specific effects and mechanisms in ESCC remain largely unexplored. This study aims to investigate the effects of IQ in ESCC cells and elucidate the mechanisms underlying its therapeutic effects. Specifically, its impact on cell proliferation, colony formation, migration, and invasion was assessed using cell viability assay, morphology, transwell, and colony formation assays. The effects on apoptosis were evaluated by flow cytometry, while immunofluorescence (IF) staining and Western blotting were performed to confirm the underlying mechanisms. The in vivo anti-cancer effects of IQ were then evaluated using a xenograft tumor model. Our results demonstrate that IQ inhibits ESCC cell growth and colony formation while promoting its apoptosis by enhancing caspase activation and downregulating Bcl-2 expression. Furthermore, IQ suppresses cell migration by modulating the epithelial–mesenchymal transition-related proteins. Additionally, IQ induces excessive autophagy by promoting reactive oxygen species accumulation and inhibiting the AKT/mTOR signaling pathway. Importantly, IQ effectively reduces tumor growth in vivo, highlighting its potential as a therapeutic agent for ESCC. Full article