Search Results (729)

Background/Objectives: Three-dimensional (3D) in vitro cell culture models have recently stimulated great interest since they may have more pre-clinical value than conventional in vitro 2D models. In fact, 3D culture models may mimic the in vivo biophysical 3D structure of tumors and cell-to-cell interaction, thereby representing a more useful approach to testing drug responses. In this study we have developed a 3D culture model of an EBV+/CD30+cell line, D430B, previously characterized as an Anaplastic Large Cell Lymphoma of B phenotype (B-ALCL), to determine the cytotoxic activity of the antibody–drug conjugate Brentuximab Vedotin. Methods: By using of ultra-low attachment plates, we developed D430B spheroids that appeared particularly homogenous in terms of growth and size. Results: Brentuximab Vedotin treatment (1 to 20 μg/mL) turned out to be significantly cytotoxic to these cells, while the addition of the anti-CD20 chimeric antibody Rituximab (10 μg/mL) appeared almost ineffective, even though these cells express CD20. Moreover, when we co-cultured D430B cells with stromal cells (HS5), to re-create a microenvironment representative of neoplastic cell/mesenchymal cell interactions within the lymph node, we observed a significant, although faint, protective effect. Conclusions: This simple and reproducible method of generating D430B-ALCL spheroids to evaluate their response to Brentuximab Vedotin treatment, as here described, may provide a valuable preliminary tool for the future pre-clinical screening of patients’ primary lymphoma cells or the development of novel therapies for this type of pathology and related diseases. Full article

Three-dimensional (3D) bioprinting is increasingly explored as a strategy for myocardial repair and regenerative medicine. Conventional 3D casting often yields heterogeneous cellularization, slow electromechanical maturation, and inadequate vascularization; by contrast, bioprinting places cells and biomaterials in predefined architectures to program alignment, stiffness, vascular pathways, and electrical coupling that better recapitulate native myocardium. This review focuses on cardiac-specific advances in 3D bioprinting. We compare major platforms (jetting, light-based, extrusion, and volumetric) and their trade-offs for cardiac applications; distill bioink design principles trending toward natural–synthetic hybrids, including conductive and shape-morphing components; and outline practical characterization readouts spanning rheology, print fidelity, swelling/degradation, and cardiac function. We also summarize cell sources and co-culture strategies. Applications surveyed include cardiac patches, engineered tissues, chambered constructs, and organoids. Finally, we discuss current limitations and potential future directions for 3D bioprinting cardiac tissues. Collectively, recent advances position 3D bioprinting to accelerate the realization of in vivo-like engineered heart tissues. Full article

Background/Objectives: Juzentaihoto (JTT), a traditional Kampo formula composed of ten medicinal herbs, is widely prescribed in Japan for immune enhancement and general health maintenance. This exploratory, open-label pilot study aimed to evaluate the feasibility and immunomodulatory effects of JTT in cancer patients and to explore its potential mechanisms of action. Methods: Ten cancer patients received oral JTT (7.5 g/day) for 14 days, while healthy volunteers served as a reference group. Peripheral natural killer (NK) cell phenotypes and CD95 expression were analyzed by flow cytometry, and serum Fas ligand (FasL) concentrations were measured by ELISA. Complementary in vitro assays using PBS-extracted, autoclaved JTT were conducted to assess Fas/FasL-mediated apoptosis in Jurkat and primary T cells by flow cytometry and Western blotting for cleaved caspase-8 and -3. Additional experiments with staurosporine (intrinsic apoptosis) and TRAIL in OSC-19 carcinoma cells were performed to determine pathway specificity. Results: In patients, most NK-cell markers showed no statistically significant within-subject changes, although a trend-level increase in NKp46 and a significant reduction in NK-cell CD95 expression (paired p = 0.014) were observed. Between-group differences primarily reflected baseline disparities between cancer patients and healthy controls. In vitro, JTT (50–100 µg/mL) partially attenuated FasL-induced apoptosis and reduced cleaved caspase-3 without affecting cleaved caspase-8, suggesting selective downstream modulation of the extrinsic pathway. Conclusions: Within the limitations of a small, non-randomized cohort without placebo, these findings are hypothesis-generating and indicate that JTT selectively modulates Fas-mediated lymphocyte apoptosis without promoting tumor immune evasion. Further randomized trials and mechanistic studies incorporating co-culture or 3D tumor–immune models are warranted to confirm these observations and identify active constituents. Full article

Central lipid metabolism disorders are crucial for the development of Alzheimer’s disease (AD). Phlorizin (PHZ) improved lipid metabolism abnormalities in AD nematodes, but its mechanism of action in improving AD-related symptoms and whether it can alleviate AD cognitive impairment remain unclear. To elucidate the effects and mechanisms of PHZ on lipid metabolism disorders in an AD model, gavage administration of PHZ for 8 weeks improved cognitive dysfunction and lipid disorders in APPswe/PSEN1dE9 (APP/PS1) mice. Concurrently, in astrocytes induced by palmitic acid (PA)- mediated lipid metabolic disorder, PHZ treatment improved astrocytic lipid accumulation by upregulating the target peroxisome proliferator-activated receptor α (PPARα) and its downstream pathways, thereby promoting astrocytic fatty acid oxidation. We validated PHZ’s strong in vitro binding affinity with PPARα. Co-culture systems of lipid-metabolically disordered astrocytes and neurons further demonstrated that PHZ significantly improved neuronal cell viability and reduced intracellular lipid accumulation, thereby decreasing the expression of enzymes associated with β-amyloid protein (Aβ) production. This study demonstrates that gavage administration of PHZ for 2 months improves cognitive deficits and pathological markers in AD mice. Furthermore, at the cellular level, PHZ may exert its effects by enhancing astrocytic lipid metabolism, thereby preventing neuronal lipotoxicity and mitigating AD progression. Full article

Background: Pancreatic ductal adenocarcinoma (PDAC) exhibits marked resistance to immunotherapy. Beyond its characteristically low tumor mutational burden, post-translational modifications (PTMs) remodel the immunopeptidome and promote immune escape through reversible, enzyme-driven programs. Subject Matter: We synthesize evidence that aberrant glycosylation, O-GlcNAcylation, phosphorylation, and citrullination constitute core determinants of antigen visibility operating within spatially discrete tumor niches and a desmoplastic stroma. In hypoxic regions, HIF-linked hexosamine metabolism and OGT activity stabilize immune checkpoints and attenuate antigen processing; at tumor margins, sialylated mucins engage inhibitory Siglec receptors on innate and adaptive lymphocytes; within the stroma, PAD4-dependent NET formation enforces T cell exclusion. We also delineate technical barriers to discovering PTM antigens labile chemistry, low stoichiometry, and method-embedded biases and outline practical solutions: ETD/EThcD/AI-ETD fragmentation, PTM-aware database searching and machine-learning models, and autologous validation in patient-derived organoid–T cell co-cultures. Finally, we highlight therapeutic strategies that either immunize against PTM neoepitopes or inhibit PTM machinery (e.g., PAD4, OGT, ST6GAL1), with stromal remodeling as an enabling adjunct. Conclusions: PTM biology, spatial omics, and patient sample models can uncover targetable niches and speed up PDAC vaccination, TCR, and enzyme-directed treatment development. Full article

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that contaminate agricultural commodities, posing risks to food safety, animal productivity, and human health. The gastrointestinal tract is the first and most critical site of exposure, where the intestinal epithelium functions as both a physical and immunological barrier against luminal toxins and pathogens. While extensive research has demonstrated that mycotoxins disrupt epithelial integrity through tight junction impairment, oxidative stress, apoptosis, and inflammation, their effects on the intestinal stem cell (ISC) compartment and epithelial regeneration remain insufficiently understood. This review integrates recent findings from in vivo, cell culture, and advanced 3D intestinal organoid and gut-on-chip models to elucidate how mycotoxins such as deoxynivalenol and zearalenone impair ISC proliferation, alter Wnt/Notch signaling, and compromise mucosal repair. We also discuss dose relevance, species differences, and the modulatory roles of the microbiome and short-chain fatty acids, as well as emerging evidence of additive or synergistic toxicity under co-exposure conditions. By bridging well-established mechanisms of barrier disruption with the emerging concept of ISC-driven regenerative failure, this review identifies a critical knowledge gap in mycotoxin toxicology and highlights the need for integrative models that link epithelial damage to impaired regeneration. Collectively, these insights advance understanding of mycotoxin-induced intestinal dysfunction and provide a foundation for developing nutritional, microbial, and pharmacological strategies to preserve gut integrity and repair. Full article

Epithelial tissues form protective barriers throughout the body, covering external surfaces and lining internal cavities. Nanofibrous scaffolds have emerged as leading platforms in tissue engineering because of their ability to mimic the nanoscale fibrillar architecture of the native extracellular matrix. Thus, they support the optimal microstructure and cellular functions that facilitate the generation of epithelial tissues. This review focuses on the pivotal role of nanofibrous scaffolds in the development of physiologically relevant three-dimensional (3D) culture systems for various types of epithelial cells. Nanofiber proper ties, including diameter, alignment, and surface chemistry, can be tailored to modulate epithelial cell attachment and growth on scaffolds. Fabrication techniques and optimized scaffold properties for culturing epithelial cells from various epithelial tissues on nanofibrous scaffolds have been examined. The key 3D culture methodologies and coculture systems that incorporate fibroblasts, endothelial cells, and immune cells, which are essential for achieving functional differentiation into an epithelium, are elucidated. Finally, the current challenges in this field and potential future directions, including the integration of scaffolds into organ-on-a-chip systems, development of “smart” bioactive materials, and pursuit of personalized medicine through patient-derived cells, are discussed. Full article

Cells of Serratia liquefaciens were grown on trypticase soy agar (TSA) for 28 d under Martian conditions of 7 mbar, 0 °C, and CO₂-enriched anoxic atmospheres (called Mars low-PTA conditions). Earth controls were maintained for 24 h at 1013 mbar, 30 °C, and a standard pN₂/pO₂ gas composition. Cells were harvested at either 24 h or 28 d from TSA surfaces and processed for SEM and TEM imaging. Cells of S. liquefaciens grown under Earth conditions were uniform in shape and size, averaging approximately 1.25 µm in length and 0.5 µm in width. Fimbriae were observed on 10–20% of cells grown under Earth conditions. Key features of low-PTA grown cultures were (1) cells exhibited swollen blunt ends at sites of cell division tapering to unusually constricted points on the distal ends of progeny cells, (2) cell division appeared disrupted with division planes occurring at odd angles often forming right-angle oriented daughter cells, (3) some cells failed to form divisional planes resulting in long spiral and oddly shaped cells measuring up to 6–8 µm in length, and (4) fimbriae were lacking. Cell walls were found to be approx. 17% thinner when cells were grown in low-PTA environments compared to lab-standard conditions. Full article

Over the past decade, the interest in using 3D cell culture models for studying the mammary gland in biomedical and veterinary fields has increased, but a fully functional in vitro model for domestic species is still lacking. Multiple cellular components, including epithelial cells, vascular endothelial cells, and stromal/stem cells, sustain the secretory mammary gland tissue in a well-organized 3D architecture. Considering the Göttingen Minipigs widely used for translational lactation studies, this work aimed to establish a 3D culture protocol to generate mammary heterogeneous multicellular spheroids composed of three different Göttingen Minipigs primary cells: mammary epithelial cells (mpMECs), aortic endothelial cells (mpAECs), and vascular-wall mesenchymal stem cells (mpVW-MSCs). Cells were cultured with hanging-drop (HD) and ultra-low-adherence plate (ULA) methods, evaluating aggregate formation in both monocultures and co/triple co-cultures. Brightfield area, eccentricity, viability, and cell distribution were analyzed. Results showed mpMECs formed irregular aggregates in both HD and ULA, while more compact and viable spheroids were formed when co-cultured with mpVW-MSCs and mpAECs by ULA. A well-organized cellular distribution was demonstrated by cytokeratin-18, vimentin, and e-NOS immunofluorescence analysis. In conclusion, this study established a stable 3D mammary multicellular spheroid model, representing a promising tool for future studies on hormonal modulation and mammary gland physiology. Full article

While normal fibroblasts suppress tumor growth, during cancer initiation and progression, this capacity can be lost and even switched to tumor-promoting, for reasons that are not understood. In this study, we aimed to determine differences between patient-derived cancer-associated fibroblasts and fibroblasts from healthy breast tissue to identify if and how these changes stimulate Triple-negative breast cancer (TNBC). Two-dimensional and three-dimensional mono and co-cultures of TNBC cells with fibroblasts from healthy breast or TNBC were analyzed for cell contractility, migration, distribution, proliferation, and hyaluronan production by traction force microscopy, live cell imaging, flow cytometry, Western blot, and ELISA. In 3D spheroid co-culture, CAFs migrated into the tumor mass, mixing with tumor cells, whereas normal fibroblasts remained separate. In 2D, CAFs showed increased cell migration and contractile force, and, in both 2D and 3D co-culture, CAFs increased the proliferation of TNBC cells. CAFs showed increased production of hyaluronan, as compared to normal fibroblasts, and loss of hyaluronan synthase 2 reduced CAF-induced stimulation of TNBC proliferation. These findings suggest that increased production of hyaluronan by TNBC CAFs enhances their capacity to mix with and induce the proliferation of cancer cells, and that the production of hyaluronan by CAFs can be a future therapeutic target against TNBC. Full article

SARS-CoV-2 persists worldwide, driving the demand for effective antivirals that inhibit replication and limit the emergence of resistant variants. Lycorine, a non-nucleoside inhibitor of SARS-CoV-2 RNA-dependent RNA polymerase, exhibits antiviral activity without direct mutagenic effects. Here, we examine the occurrence of single-nucleotide variants (SNVs) and insertions/deletions (indels) in SARS-CoV-2 B.1.499 strain during serial passages in Vero cells, comparing lycorine-treated cultures (2.5 and 5 µg/mL) with untreated controls. Whole-genome sequencing was used to assess mutation patterns and frequencies. Lycorine-treated passages displayed greater variant diversity than controls, with fixed mutations mainly affecting non-structural proteins (Nsp3-F1375A, Nsp5-L50F, and Nsp14-G265D) and the envelope protein (E-S6L). A 15-nucleotide deletion in the spike gene (QTQTN motif) occurred in both groups but became fixed only in untreated passages, suggesting negative selection under lycorine pressure. Notably, the L50F mutation in Nsp5, previously linked to nirmatrelvir resistance, was found exclusively in lycorine-treated passages. Additionally, a 1-nucleotide deletion in the accessory gene ORF8, detected only under lycorine treatment, resulted in a frameshift mutation that added four amino acids, potentially altering the protein’s function. Overall, lycorine induces a distinct mutation profile, favoring replication-related variants while suppressing deleterious deletions. These findings suggest potential mechanisms of cross-resistance and highlight the importance of monitoring resistance during clinical use. Full article

Background: Chagas disease poses a significant public health challenge, particularly impacting socioeconomically vulnerable populations. Current treatment strategies still rely on two nitro heterocyclic compounds: benznidazole and nifurtimox. Both agents exhibit limited therapeutic efficacy during the chronic phase of the disease and are often linked to severe adverse effects that frequently lead to treatment discontinuation. This urgent need for safer, more effective oral treatments drives the development of novel chemotypes. Objective: In this study, we advanced the preclinical evaluation of 4-imidazoline-1H-pyrazole derivatives, which have been identified as promising candidates against Trypanosoma cruzi. Methods: The candidate compound identified from the reversibility assay underwent further evaluation for its efficacy using a three-dimensional (3D) culture model and a Transwell co-culture system, in addition to the in vivo assessment. Results: Our findings revealed that compound 3m (3-Cl, 4-CH₃) exhibited low cytotoxicity while substantially decreasing the parasite burden in 3Dcardiac spheroid models. The compound effectively permeated Caco-2 cell monolayers and demonstrated the ability to inhibit T. cruzi infection in Vero cell cultures within a co-culture system. Furthermore, the 3m derivative not only controlled parasite resurgence but also showed significant therapeutic benefits in a murine model of acute T. cruzi infection, resulting in marked reductions in parasitemia and tissue parasitism, associated with diminished inflammatory infiltrate and cardiac fibrosis. Treatment with 3m increased the survival rate of infected mice to 40%, comparable to the reference drug benznidazole in several key pathological endpoints. Conclusion: These findings highlight the potential of 4-imidazoline-1H-pyrazole derivatives, particularly compound 3m, in mitigating the pathological effects associated with T. cruzi infection. Full article

Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that influence cancer progression via extracellular matrix (ECM) remodeling and secretion of growth factors and cytokines. Endothelial-to-mesenchymal transition (EndMT) is emerging as an important axis among the heterogeneous origins of CAFs. This review introduces the diverse methods used to induce EndMT in cancer—mouse tumor models, conditioned-medium treatment, co-culture, targeted gene perturbation, ligand stimulation, exosome exposure, irradiation, viral infection, and three-dimensional (3D) culture systems—and summarizes EndMT cell-type evidence uncovered using transcriptomic and proteomic technologies. Hallmark EndMT features include spindle-like morphology, increased motility, impaired angiogenesis and barrier function, decreased endothelial markers (CD31, VE-cadherin), and increased mesenchymal markers (α-SMA, FN1). Reported mechanisms include signaling via TGF-β, cytoskeletal/mechanical stress, reactive oxygen species, osteopontin, PAI-1, IL-1β, GSK-3β, HSP90α, Tie1, TNF-α, HSBP1, and NOTCH. Cancer-induced EndMT affects tumors and surrounding TME—promoting tumor growth and metastasis, expanding cancer stem cell-like cells, driving macrophage differentiation, and redistributing pericytes—and is closely associated with poor survival and therapy resistance. Finally, we indicate each study’s stance: some frame cancer-induced EndMT as a source of CAFs, whereas others, from an endothelial perspective, emphasize barrier weakening and promotion of metastasis. Full article

Diabetic retinopathy (DR) is a serious complication of diabetes, leading to vision loss worldwide. The prevalence of DR has increased in recent decades. To understand the pathophysiology of DR, various experimental models have been developed and used. In this review article, we first outline what is currently known of the general pathology of DR, including the mechanisms involved in hyperglycemia, vascular dysfunction, retinal ischemia, retinal inflammation, and retinal degeneration. We next summarize various pathologies detected in experimental models in vivo, such as in chemically and genetically induced murine, rat, and monkey models, surgical methods in larger animals like cats, and a novel murine DR model using occlusion of the carotid artery under early diabetic conditions. A general overview of the in vitro models, including cell monocultures, co-cultures, and 3D models, is also provided. This current summary enables further research to obtain a more thorough understanding of DR pathogenesis and develop appropriate treatment measures. Full article

Respiratory viral infections are a major cause of morbidity and mortality worldwide. The COVID-19 pandemic has evidenced the need for broad-spectrum antivirals and improved preclinical models that more accurately recapitulate human respiratory disease. These new strategies should also involve the search for drug targets in the infected cell that hamper the development of resistance and of potential efficacy against diverse viruses. Since many viruses reprogram cellular metabolism to support viral replication, we performed a comparative analysis of inhibitors targeting the PI3K/AKT/mTOR pathway, central to virus-induced metabolic adaptations, using MRC5 lung fibroblasts and Huh7 hepatoma cells. HCoV-229E infection in MRC5 cells caused the expected shift in the energy metabolism but the inhibitors had markedly different effects on the metabolic profile and antiviral activity in these two cell lines. Dichloroacetate (DCA), a clinically approved inhibitor of aerobic glycolysis, showed antiviral activity against HCoV-229E in MRC5 cells, but not in Huh7 cells, underscoring that the screening model is more critical than previously assumed. We further tested DCA in polarized human small airway epithelial cells cultured in air–liquid interface, a 3D model that mimics the human respiratory tract. DCA reduced the viral progeny of HCoV-229E, SARS-CoV-2, and respiratory syncytial virus by 2–3 orders of magnitude, even when administered after infection was established. Our work reinforces the need for advanced human preclinical screening models to identify antivirals that target host metabolic pathways frequently hijacked by respiratory viruses, and establishes DCA as a proof-of-concept candidate. Full article