Search Results (2,270)

Background: Claudin-18.2 (CLDN18.2) has emerged as a promising therapeutic target for gastric cancer due to its frequent and specific expression in malignant lesions. Dendritic cell (DC)-based vaccines represent a potent strategy for inducing antitumor immunity; however, their efficacy against solid tumors, such as gastric cancer, remains challenging. Methods: We developed a lentiviral vector encoding human CLDN18.2 (Lv-CLDN18.2) to generate antigen-loaded DC vaccines. In vitro, human monocyte-derived DCs were transduced and co-cultured with autologous T cells to induce cytotoxic T lymphocytes (CTLs). CTL function was assessed by flow cytometry, cytokine ELISA, and cytotoxicity assays against CLDN18.2-positive gastric cancer cells. In vivo, the therapeutic efficacy of the DC vaccine was evaluated in a syngeneic mouse model subcutaneously inoculated with MFC-CLDN18.2 cells. Results: We successfully produced high-titer Lv-CLDN18.2 and established stable CLDN18.2-positive gastric cancer cell lines. Lv-CLDN18.2-transduced DCs exhibited a mature phenotype with upregulated co-stimulatory (CD80/CD86) and antigen-presenting molecules (HLA-ABC/DR). These DCs potently stimulated CTLs, leading to a significantly higher proportion of activated CD8⁺CD25⁺ T cells, enhanced secretion of IFN-γ and TNF-α, and potent, specific lysis of CLDN18.2-positive target cells in vitro. In mouse models, vaccination with Lv-CLDN18.2-DCs significantly suppressed tumor growth, which was associated with robust CD8⁺ T cell infiltration, reduced tumor cell proliferation (Ki-67), and decreased CLDN18.2-positive tumor cells in vivo. Conclusions: Our study demonstrates that a CLDN18.2-targeting DC vaccine can effectively induce potent antigen-specific CTL responses and elicit significant antitumor immunity in a preclinical model. These findings provide a strong rationale for the clinical development of CLDN18.2-directed DC-based immunotherapy for gastric cancer. Full article

Cancer is one of the leading causes of death worldwide, and the majority of cancer-related deaths are caused by cancer that has spread to other organs. Precision cancer medicine (PCM) holds potential to improve outcomes and relies on molecularly matched therapies based on cancer cell specific molecular alterations. The tumor immune microenvironment plays an important role beyond response to therapy; however, this is generally not considered in current PCM platforms. We established patient-matched organoids and immune cell cultures for drug testing in mono- and co-culture treatment setups using three distinct treatment strategies (pretreatment, co-culture treatment, and T-cell bispecific antibody testing). Response to treatment and impact of immune cells were evaluated by tumor cell viability assays and flow cytometry analysis. Phenotypic analysis showed high heterogeneity of tumor-infiltrating lymphocytes (TILs) across the patients and low immune cell portions of organoids, emphasizing the need for a patient-matched co-culture PCM approach. Our in-depth study of three patients revealed an effect of the patients’ immune cells on drug response and T-cell bispecific antibody treatment in vitro. Here, we illustrate a state-of-the-art co-culture PCM pipeline for patient-matched organoids and immune cells replicating patient response to treatment at the time of biopsy. Full article

The intestine–liver–muscle axis plays an essential role in drug and nutrient absorption, metabolism, and energy balance. Yet in vitro models capable of recapitulating this inter-organ communication remain limited. Here, we present a pump-free, 3D-printed multi-organ-on-a-chip device that enables dynamic co-culture of Caco-2 intestinal epithelial cells, HepG2 hepatocytes, and primary human skeletal myoblasts (HSkMs) under gravity-driven oscillatory flow. The device consists of five interconnected chambers designed to accommodate Transwell cell culture inserts for intestine and muscle compartments and hydrogel-embedded hepatocyte spheroids in the central hepatic compartment. The device was fabricated by low-cost fused deposition modeling (FDM) using acrylonitrile butadiene styrene (ABS) polymers. Under dynamic rocking, oscillatory perfusion promoted inter-organ communication without the need for external pumps or complex tubing. Biological assessments revealed that dynamic co-culture significantly enhanced the characteristics of skeletal muscle, as indicated by increased myosin heavy chain expression and elevated lactate production, while HepG2 spheroids exhibited improved hepatic function with higher albumin expression compared with monoculture. Additionally, Caco-2 cells maintained stable tight junctions and transepithelial electrical resistance, demonstrating preserved intestinal barrier integrity under dynamic flow. These results establish the device as a versatile, accessible 3D-printed platform for modeling the intestine–liver–muscle axis and investigating metabolic cross-talk in drug discovery and disease modeling. Full article

The blood–brain barrier (BBB) is a structure that regulates the exchange of substances between the peripheral circulation and the central nervous system (CNS), thereby protecting this environment. An increase in BBB permeability may lead to the influx of inflammatory cells, resulting in neuroinflammation and neurodegeneration. The integrity of the BBB is maintained due to the specific properties of brain endothelial cells. Considering the importance of brain endothelial cells in the BBB during inflammatory processes, these cells may be a target for anti-inflammatory agents. Polyphenols are substances exhibiting the ability to decrease inflammation; therefore, in our research, we aimed to examine their effectiveness in a brain endothelial cell culture stimulated with the pro-inflammatory cytokine TNF-α. The tested polyphenols were myricetin, chrysin, resveratrol, and curcumin. ELISA tests revealed that myricetin and chrysin decreased the concentrations of the pro-inflammatory cytokines IL-1ß, IL-6, and IL-8 secreted by brain endothelial cells. The results of flow cytometry indicate that chrysin and resveratrol are the most potent in downregulating the expression of VCAM-1 on the surface of brain endothelial cells. The obtained results confirm the anti-inflammatory potential of polyphenols in brain endothelial cells. The selected polyphenols also contribute to increasing brain endothelial cell viability and act as antioxidants. Full article

Dilated cardiomyopathy (DCM) is a major contributor to heart failure and cardiac transplantation. This study investigated the metabolic potential of human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and subsequently cardiac sphere-derived cells (SDCs) obtained from healthy (non-dilated) and pathological (dilated) myocardial tissues. hmMSCs were isolated using the explant outgrowth method and expanded in monolayer culture. Small round cells loosely attached on hmMSCs were harvested and cultivated as cardiac spheroids for 1–3 days, subsequently obtaining SDCs. The cell morphology, proliferation rate, mitochondrial activity, and intracellular calcium levels were analyzed using flow cytometry, Seahorse metabolic assays, and spectrophotometry, while expression of cell progenitor and cardiac commitment genes were analyzed by quantitative PCR. Both healthy and pathological SDCs demonstrated significantly enhanced mitochondrial function—reflected by increased maximal respiration, ATP production, and coupling efficiency—along with reduced steady-state intracellular calcium levels compared with hmMSCs. SDCs from both healthy and dilated myocardium showed marked upregulation of several cardiac progenitor and lineage-commitment genes relative to hmMSCs. SDCs derived from both healthy and dilated myocardiums possess a more favorable metabolic, progenitor and cardiac commitment profile than conventional hmMSCs. hmMSCs and SDCs from dilated myocardium retain residual metabolic potential, which may be further enhanced under 3D culture conditions. Full article

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, a burden further exacerbated by HIV co-infection and the increasing prevalence of drug-resistant strains. Although a wide range of laboratory diagnostic methods are currently available, their applicability, implementation, and clinical impact vary substantially across healthcare settings with different levels of complexity and resources. This review provides a comprehensive overview of the main laboratory diagnostic methods for active and latent TB, emphasizing their clinical applicability, implementation challenges, and role within integrated diagnostic strategies. Conventional approaches, such as smear microscopy and culture, are discussed alongside modern diagnostic technologies, including automated nucleic acid amplification tests (NAATs), loop-mediated isothermal amplification (LAMP), line probe assays (LPAs), next-generation sequencing (NGS), and lateral flow assays, highlighting their strengths and limitations in distinct epidemiological and operational contexts. Unlike existing WHO guidelines and prior reviews that predominantly focus on test performance and recommendation status, this review adopts an implementation-oriented perspective, critically examining diagnostic methods in light of real-world constraints, regional disparities, and evidence gaps. Particular attention is given to limitations related to laboratory infrastructure, biosafety, workforce capacity, and sustainability, as well as to under-addressed areas such as latent TB, metagenomic approaches, and the investigation of co-pathogens. By integrating WHO guidance with contextual and operational considerations, this review aims to support rational test selection and the development of flexible, integrated diagnostic workflows tailored to local health system capacity, patient populations, and clinical scenarios, thereby strengthening the effectiveness and equity of TB diagnostic strategies. Full article

Cystic fibrosis (CF) is caused by loss-of-function variants in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride and bicarbonate channel and affects multiple organs, with pancreatic involvement showing very high penetrance. In pancreatic ducts, CFTR drives secretion of alkaline, bicarbonate-rich fluid that maintains intraductal patency, neutralises gastric acid and permits safe delivery of digestive enzymes. Selective impairment of CFTR-dependent bicarbonate transport, even in the presence of residual chloride conductance, is strongly associated with exocrine pancreatic insufficiency, recurrent pancreatitis and cystic-fibrosis-related diabetes. These clinical manifestations are captured by pharmacodynamic anchors such as faecal elastase-1, steatorrhoea, pancreatitis burden and glycaemic control, providing clinically meaningful benchmarks for CFTR-targeted therapies. In this review, we summarise the principal mechanisms underlying pancreatic pathophysiology and the current approaches to clinical management. We then examine in vitro pancreatic duct models that are used to evaluate small molecules and emerging therapeutics targeting CFTR. These experimental systems include native tissue, primary cultures, organoids, co-cultures and microfluidic devices, each of which has its own advantages and limitations. Intact micro-perfused ducts provide the physiological benchmark for studying luminal pH control and bicarbonate (HCO₃⁻) secretion. Primary pancreatic duct epithelial cells (PDECs) and pancreatic ductal organoids (PDO) preserve ductal identity, patient-specific genotype and key regulatory networks. Immortalised ductal cell lines grown on permeable supports enable scalable screening and structure activity analyses. Co-culture models and organ-on-chip devices incorporate inflammatory, stromal and endocrine components together with flow and shear and provide system-level readouts, including duct-islet communication. Across this complementary toolkit, we prioritise bicarbonate-relevant endpoints, including luminal and intracellular pH and direct measures of HCO₃⁻ flux, to improve alignment between in vitro pharmacology and clinical pancreatic outcomes. The systematic use of complementary models should facilitate the discovery of next-generation CFTR modulators and adjunctive strategies with the greatest potential to protect both exocrine and endocrine pancreatic function in people with CF. Full article

Recurrent pregnancy loss (RPL) is defined as the loss of two or more pregnancies before the 22nd gestational week and affects 10–15% of clinical pregnancies. Despite extensive diagnostics, over 50% of RPL cases remain unexplained, suggesting an important role for immunological mechanisms. Sex hormones (SH) are key regulators of immune responses during pregnancy; however, their influence on immune checkpoint proteins (ICPs) is poorly understood. This study evaluated the effects of progesterone, β-estradiol, and dihydrotestosterone (DHT) on ICP expression on immune cells, including Treg, NK, NKT, TC, Th, and T cells, collected from pregnant women and patients with unexplained RPL (uRPL). Peripheral blood mononuclear cells from 20 pregnant women and 20 uRPL patients were cultured for 48 h with SH. The expression of the first generation of ICPs—PD-1 and TIM-3—and the second—LAG-3, TIGIT, and VISTA—on T, NK, and NKT cells was analyzed by the flow cytometry method. In pregnant women, SH exerted modest effects, with DHT increasing VISTA and LAG-3 expression, while progesterone and estradiol mainly upregulated LAG-3 and TIM-3 on cytotoxic cells. In contrast, uRPL immune cells showed pronounced SH sensitivity, characterized by increased TIM-3 and VISTA expression and reduced TIGIT expression, particularly after DHT stimulation. In conclusion, SH modulates ICP expression in a cell-specific manner, with stronger effects observed in uRPL patients’ lymphocytes. These findings highlight a potential role for hormonal and ICP-targeted strategies in RPL management. Full article

The first step to selecting interesting lactic acid bacteria for commercial use is testing their resistance to different physicochemical stresses. In this study, we evaluated the viability of Enterococcus faecium and Enterococcus durans, obtained from two traditional fermented cheeses, subjected to several stresses (thermal, osmotic, acidic, alkaline, oxidative, detergent, and alcoholic). The assessment of cell viability was conducted via flow cytometry (FCM) combined with nucleic-acid double staining (NADS) and was compared to the conventional plate count method (CFU). The findings from the two approaches indicated that Enterococcus faecium and Enterococcus durans demonstrated a substantial proportion of viable cells following exposure to osmotic, thermal, and acidic stress. The alkaline stress treatment does not diminish the proportion of viable cells. Both strains exhibited extensive sensitivity to SDS, oxidative stress, and experienced total cell death under alcoholic stress. We observed a satisfactory correlation between cell viability as measured by FCM and CFU under all stress conditions. These data demonstrate the existence of indigenous strains of Enterococcus spp. that exhibit notable stress resistance. FCM for viability enumeration is better than the conventional plate counting method due to its rapid results and precision, which offer an effective evaluation of live, dead, and permeabilised cells. This technique holds promise for physiological state research in dairy applications to evaluate the quality of fermented products and the viable cell count for probiotic manufacturing. Full article

Bioreactors used for the maturation of cell-seeded tissue-engineered scaffolds should essentially mimic the dynamic in vivo environments experienced by the native tissues they intend to substitute. In addition to perfusion of growth medium to facilitate continuous mass transfer, application of appropriate mechanical stimulation is important to enhance cellular responses in scaffolds for tissues such as tendons, skin, and cardiac muscle that experience dynamic loading. This study focuses on the development of a multi-modal custom bioreactor capable of applying cyclic tensile stimulation and perfusion within physiologically relevant ranges while minimizing shear stress detrimental to cells seeded on scaffolds. To validate the bioreactor design and operation, we assessed the effects of tensile stimulation (0.1 Hz, 2000 cycles/day) and perfusion (media flow rate = 0.15 mL/min) over 21 days on the biofunctional performance of 3D-bioplotted polycaprolactone (PCL) auxetic scaffolds with a representative design (missing-rib pattern) characterized by negative Poisson’s ratio similar to the aforementioned soft tissues. The scaffold had a tensile yield strain of 9.14%, yield strength of 0.25 MPa, elastic modulus of 2.85 MPa, and ultimate tensile strength (UTS) of 1.32 MPa. The application of perfusion and tensile stimulation (0–5% cyclic strain) for 21 days did not adversely affect the yield strength and elastic modulus of the scaffold but affected its UTS (22.5% decrease) compared to the control cultured without perfusion or stimulation. Notably, it resulted in significantly improved fibroblast cellular responses (DNA = 29 µg/g sample and collagen = 371.78 µg/g sample) compared to the control (7.52 µg/g sample and 163.51 µg/g sample, respectively). These results validate the bioreactor system operation and the ability of multi-modal stimulation to control biofunctional responses of auxetic scaffolds, which will serve as the basis for future studies that will optimize auxetic scaffold design and dynamic culture parameters for NPR tissue-specific applications. Full article

The widespread use of antibiotics has led to the persistence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in drinking water systems, posing potential public health risks at the point of use. In this study, a residential secondary water supply system (SWSS) in eastern China was investigated over one year to characterize microbial communities, ARB and ARG occurrence, and their associations with water quality in bulk water and biofilms. Culture-based methods, flow cytometry, quantitative PCR, and high-throughput 16S rRNA and ITS sequencing were applied. Although conventional treatment removed 94.8% of total bacteria, significant microbial regrowth occurred during secondary distribution, with the highest heterotrophic plate counts observed in rooftop storage tanks (up to 4718 CFU/mL). ARG concentrations increased along the distribution line, and the class 1 integron intI1 was enriched in downstream locations, indicating enhanced horizontal gene transfer potential. Sulfonamide resistance genes dominated the resistome, accounting for more than 60% of total ARG abundance in water samples. Seasonally, ARG levels were higher in autumn and winter, coinciding with elevated disinfectant residuals and lower temperatures. Chlorine was negatively associated with total bacterial abundance, while positive correlations were observed with the relative abundance of several ARGs when normalized to bacterial biomass, suggesting selective pressure under oxidative stress. Turbidity and bacterial abundance were positively correlated with ARB, particularly sulfonamide-resistant bacteria. Biofilms exhibited more stable microbial communities and provided microhabitats that facilitated microbial persistence. Notably, fungal abundance showed strong positive correlations with multiple ARGs, implying that microbial interactions may indirectly contribute to ARG persistence in SWSSs. These findings highlight the role of secondary distribution conditions, disinfectant pressure, and microbial interactions in shaping resistance risks in residential water supply systems, and provide insights for improving microbial risk management at the point of consumption. Full article

Museums are key institutions for cultural communication and public education, and their operating concept is shifting from exhibit-centered to experience-centered. As expectations for exhibition experience rise, museum fatigue has become a major constraint on visitors. Existing studies rely on questionnaires and other subjective measures, which makes it difficult to locate fatigue in specific spaces. At the same time, body pose detection and fatigue recognition techniques remain hard to apply in museums because of complex spatial configurations and dense visitor flows. Effective methods for quantifying and mitigating museum fatigue are still lacking. This study proposes a contact-free sensing scheme based on computer vision and builds a coupled analytical framework with three stages: Human Pose Estimation (HPE) for visitor posture detection, fatigue assessment, and fatigue mitigation. A Fatigue Index (FI) quantifies bodily fatigue. Applying this index to the exhibition space in both the baseline and adjusted configurations guides the formulation of mitigation strategies and shows a consistent reduction in FI, which indicates that the adopted measures are effective. The proposed approach establishes a complete frame from fatigue quantification to fatigue mitigation, supports evaluation of exhibition space design, and provides theoretical and methodological support for future improvements to museum experience. Full article

Head and neck cancer (HNC) patients frequently experience alterations in the oral environment following radiotherapy, including xerostomia and impaired mucosal integrity, which may favour fungal overgrowth. This study aimed to characterise oral Candida colonisation in radiotherapy-treated HNC patients and compare it with that of healthy individuals. Unstimulated saliva samples from 61 HNC patients and 100 controls were cultured on chromogenic agar, and isolates were identified using API 20C AUX or MALDI-TOF. Salivary flow was measured to quantify xerostomia. A representative subset of isolates (10 per group) underwent antifungal susceptibility testing by disk diffusion according to CLSI/EUCAST criteria. Candida colonisation was significantly higher in HNC patients than in controls (64.6% vs. 20%, p < 0.001), with greater species diversity and increased detection of non-albicans yeasts, including C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei. All HNC patients exhibited reduced salivary flow. Azole resistance was more frequent among HNC isolates (26%) than among controls (10%), whereas all isolates remained susceptible to amphotericin B and nystatin. These findings indicate that radiotherapy-associated xerostomia substantially alters the oral mycobiota and underscore the importance of routine species-level identification and antifungal susceptibility testing in HNC patients to guide clinical decision-making. Full article

Atopic allergy is rising globally and placing a significant strain on healthcare systems, yet the understanding of the underpinning mechanisms of allergic sensitization remains incomplete. Extracellular vesicles (EVs) have recently emerged as important mediators of immune modulation, due to their diverse cargo, and therefore may play a mechanistic role in allergic sensitization development. Thus, this study investigated whether EVs released by activated dendritic cells (DCs) contribute to allergic sensitization of the common egg allergen, ovalbumin (OVA). DCs were generated from human monocytes cultured with GM-CSF and IL-4, then stimulated with LPS and/or OVA. EVs were subsequently isolated using size-exclusion chromatography and added to freshly isolated naive T cells at defined time points. T cell responses were then analyzed using spectral flow cytometry. The results highlight that EVs derived from LPS or LPS + OVA-stimulated DCs enhanced IL-4 production and reduced IFN-γ production in naive T cells from egg-allergic donors, indicating a shift toward a Th2 profile. In healthy donors, LPS-induced DC EVs also suppressed IFN-γ expression. Notably, EVs alone were insufficient to activate T cells without CD3/CD28 co-stimulation, suggesting that EVs may function as a “third signal” shaping T cell polarization. These findings highlight a potential role for DC-derived EVs in initiating allergic sensitization. Full article

Probiotics are live microorganisms that provide health benefits when administered in adequate amounts. Due to the increasing popularity of probiotic supplements, concerns have arisen regarding their quality, microbial composition, and safety. This study aimed to evaluate the quantitative and qualitative characteristics of the selected probiotics available on the Polish market, including both dietary supplements and foods for special medical purposes, and to compare the obtained results with the information provided on the product labels. Fifteen commercial probiotic products were analysed. Viable microorganism counts were determined using the traditional culture-based plate count method and by flow cytometry for selected products. Species identification was performed using MALDI-TOF MS and qPCR, whereas microbiological purity testing was conducted to confirm the absence of pathogenic bacteria. Significant differences were observed between the declared and experimentally determined numbers of viable microorganisms. Only a few products maintained bacterial counts consistent with label claims, while most contained considerably low viable cells. Flow cytometry revealed higher viable cell counts than plate counting, indicating the presence of viable but non-culturable bacteria. The declared species composition of the strains was mostly confirmed, although in several cases, undeclared probiotic microorganisms were identified. All tested products were free from pathogens. The study indicates significant discrepancies in the quality of probiotic supplements available on the Polish market. From a consumer perspective, these findings highlight the importance of verifying probiotic quality and suggest that not all commercial products may guarantee the full range of claimed health benefits. The implementation of standardised analytical procedures and enhanced quality control measures is therefore essential to ensure the product safety, strain authenticity, and reliability of health-related claims. Full article