Search Results (202)

Background/Objectives: Seasonal waves of respiratory viruses—including SARS-CoV-2, influenza A virus (IAV), and respiratory syncytial virus (RSV)—continue to pose a global health burden and highlight the need for antiviral agents that are effective, safe, broadly active, affordable, and widely accessible. Current interventions are limited by the need for their early administration, the risk of resistance, their costs, and the restricted availability in large parts of the world. For certain natural products, such as European black elderberry (Sambucus nigra L.) fruit extract (ElderCraft^®; EC) and the seaweed-derived sulfated polymer iota-carrageenan (IC), antiviral activities against respiratory viruses, particularly IAV and SARS-CoV-2, have previously been shown. Here, we assessed the antiviral activity of IC and an anthocyanin-standardized EC extract against SARS-CoV-2, IAV, and RSV, either as monotherapy or in multiple-dose combinations. Methods: MDCKII cells were infected with IAV_PR8, human Calu-3 lung epithelial cells with the SARS-CoV-2 Omicron variant, and HEp-2 cells with RSV (A2 strain). Inhibitors were administered either by pre-incubation of cell-free virions prior to infection or, in separate time-of-addition experiments, during or post-infection. Viral replication was quantified by qRT-PCR or intracellular immunostaining. Cytotoxicity was evaluated using a neutral red uptake assay. Results: Most intriguingly, both EC and IC are able to neutralize virions derived from SARS-CoV-2, IAV, or RSV extracellularly in a dose-dependent manner. Notably, EC and IC alone exhibited strong anti-RSV activity, which was not reported previously. Most importantly, combined treatment with IC and EC caused a pronounced synergistic antiviral effect against the tested viruses, as confirmed by the Bliss independence model, without any detectable impact on cell viability. Finally, solutions prepared from matrix-standardized mono- or combi-lozenges, containing IC and/or EC in high or low doses, reproduced the antiviral and synergistic combination effects observed with the pure compounds. Conclusions: In summary, these findings support further development of EC and IC as a topically accessible, virion-neutralizing combination (e.g., lozenges) to provide additional protection against major respiratory viruses and potentially strengthen pandemic preparedness. Full article

Airway remodeling is a process that occurs in chronic obstructive diseases, such as asthma and COPD. It is associated with adverse changes in the structure and function of the airways. An increasing amount of literature points to the potential protective effects of vitamin D and caffeine against inflammation and fibrosis. The aim of the study is to evaluate the effect of calcitriol and caffeine on the expression of genes and proteins associated with airway remodeling. The Calu-3 cell line was treated with TGF-β, calcitriol, and caffeine in different combinations. Subsequently, the expression of VDR, CDH1, VIM, MMP-2, and MMP-9 were examined at the mRNA and protein levels using real-time PCR and Western blot, respectively. One-way analysis of variance was used to determine differences in several groups. Both calcitriol and caffeine were associated with a decrease in the expression of MMP-2 and VIM in TGF-β-treated cells (p = 0.01 and p = 0.006, respectively). Both compounds also reduced the expression of MMP-9 in comparison to TGF-β alone (p = 0.03), though the changes in MMP-9 protein levels did not reach statistical significance. Calcitriol was associated with a decrease in CDH1 expression at both levels in comparison to TGF-β (p < 0.0001 and p = 0.02, respectively). A potential synergistic effect was demonstrated for CDH1 at the mRNA level and for the vitamin D receptor at the protein level. Both vitamin D and caffeine may influence the pathways involved in airway remodeling. Preliminary in vitro findings suggest a potential role of these substances for future therapeutic strategies targeting obstructive diseases; however, the observations require confirmation in further in vivo studies. Full article

Klebsiella pneumoniae (K. pneumoniae, KP) is a significant opportunistic pathogen responsible for both nosocomial and community-acquired infections. Bacterial adhesion is the critical initial step for host colonization and the establishment of disease. In this study, we utilized a mariner transposon mutagenesis system to construct a mutant library from the clinical KP strain KP20, identifying a mutant with significantly impaired epithelial cell adhesion due to an insertion in the uspF gene. Genetic knockout experiments confirmed that uspF deletion markedly reduced the adhesion to human airway epithelial cells (Calu-3) and downregulated the transcription of type III pili-encoding genes (mrkABDF). Furthermore, uspF deficiency compromised antioxidant stress and serum resistance and increased susceptibility to dendritic cell and macrophage phagocytosis. In vivo challenge experiments further demonstrated that uspF deletion significantly attenuated K. pneumoniae virulence in mice. These findings provide important insights into the molecular pathogenesis of K. pneumoniae and identify UspF as a potential target for therapeutic intervention. Full article

The development of effective inhalable drugs remains a key challenge in the treatment of pulmonary diseases, due to the physiological barriers of the respiratory tract and the lack of predictive models that accurately reproduce the human lung environment. In this context, liposomes (LP) have emerged as promising nanocarriers for pulmonary drug delivery due to their high biocompatibility, surfactant-like composition, capacity to encapsulate both hydrophilic and lipophilic drugs, and potential to provide sustained drug release while reducing systemic toxicity. This study evaluates the influence of size and PEGylation on their physicochemical properties, cytotoxicity, interaction with the pulmonary mucus, and cellular internalisation. LP of 100 nm (LP 100), 200 nm (LP 200), and 600 nm (LP 600) were characterised physiochemically and evaluated in pulmonary cell lines (A549 and Calu-3) exposed in liquid–liquid interface (LLI) and air–liquid interface (ALI) by nebulisation. In addition, artificial pulmonary mucus (APM) was employed to analyse LP penetration through the pulmonary mucus barrier. Results indicate that LP 100 exhibits greater colloidal stability, lower cytotoxicity, and sustained migration through the APM over time with respect to larger particles. PEGylation of LP 100 (LP-PEG) further increases their stability and ability to penetrate the APM, although cellular internalisation is reduced due to the steric effect of the PEG coating. These findings highlight the importance of adjusting the size and surface modifications of LPs according to the therapeutic target of the drug, optimising their persistence on the epithelial surface or their cellular uptake. Full article

Background: Pancreatic adenocarcinoma (PAAD), often referred to as the “king of cancers,” remains poorly understood in terms of the regulatory mechanisms involving brown adipocytes (BAs). Methods: Bioinformatics approaches were employed to explore the role of BAs in PAAD progression, utilizing transcriptomic data from public databases. Prognostic genes were identified through differential expression analysis, univariate Cox regression, and machine learning. A risk model categorizing patients into high- and low-risk groups was developed, accompanied by a nomogram. Functional analysis, immune microenvironment profiling, somatic mutation analysis, and drug sensitivity testing were performed, with further validation via gene localization, immunohistochemistry, and clinical sample analysis. Results: Six prognostic genes (SERPINB5, CALU, TFRC, LY6D, SFRP1, and GBP2) were identified, with the model and nomogram exhibiting robust predictive performance. Notable differences between the high- and low-risk groups were found in immune pathways, cell infiltration, tumor mutational burden, and drug sensitivity (e.g., axitinib). Conclusions: SERPINB5, SFRP1, and TFRC were highly expressed in PAAD samples, providing new insights into potential therapeutic strategies in PAAD treatment. Full article

The presence of micro- and nano-plastics in the atmosphere has become evident, necessitating risk assessments for humans. Although submerged culture systems are often used to evaluate the safety of fine particles, some plastics float in culture media owing to their low density. Therefore, developing an air–liquid interface (ALI) system capable of assessing plastic exposure is essential. In this study, we developed a chamber for exposing nanoplastic aerosols to ALI cultures and evaluated their toxicological effects. A glass exposure chamber integrated with a donut-shaped culture plate was constructed. The aerosols were introduced through four upper inlets and discharged through five lower outlets. The culture temperature was controlled by circulating water through the inside space of the plate. A nano-polystyrene (PS) suspension was nebulized and introduced into the chamber. Exposure of co-culture of Calu-3 and U937 cells to nano-PS aerosols resulted in a spatial mass concentration-dependent increase in hydrogen peroxide concentration in the culture medium, elevated expression of inflammatory cytokines and chemokines (including IL-6 and IL-8) in Calu-3 cells and decreased trans-epithelial electrical resistance. These findings indicate that nano-PS aerosol exposure induces oxidative stress and inflammatory responses, leading to alveolar barrier dysfunction. Overall, the developed ALI exposure system provides a useful in vitro culture system for evaluating the safety of nanomaterials, including nanoplastics, and highlights the importance of aerosol-based approaches in assessing the toxicity of respirable particles. Full article

Background: Multidrug-resistant (MDR) pulmonary infections represent a critical global health challenge, necessitating innovative therapeutic approaches. Green synthesis methodologies offer sustainable alternatives for nanoparticle fabrication while addressing antimicrobial resistance. Methods: Stimuli-responsive chitosan–silver hybrid nanoparticles (CS–Ag HNPs) were biosynthesized using Pseudomonas fluorescens bacterial extracts and loaded with ciprofloxacin for targeted pulmonary delivery. Comprehensive characterization included dynamic light scattering, transmission electron microscopy, UV–visible spectroscopy, and aerodynamic assessment via next-generation impactor. Antimicrobial efficacy was evaluated against MDR Pseudomonas aeruginosa and Klebsiella pneumoniae, including biofilm disruption studies, and biocompatibility was assessed. Molecular docking analysis elucidated binding mechanisms. Cytotoxicity and epithelial barrier integrity were evaluated using Calu-3 cell models. Results: The biosynthesized NPs exhibited optimal physicochemical properties (180 ± 20 nm, PDI 0.21 ± 0.04, ζ-potential + 32.4 ± 3.1 mV) with high encapsulation efficiency (68.2 ± 4.0%). Aerodynamic analysis revealed excellent inhalation characteristics (MMAD 2.6 μm, FPF 65 ± 5%). The hybrid system demonstrated 4-fold enhanced antimicrobial activity against MDR pathogens and significant biofilm disruption (70% for P. aeruginosa, 65% for K. pneumoniae) compared to free ciprofloxacin. Cell viability remained ≥85% at therapeutic concentrations. Molecular docking revealed enhanced drug-target binding affinity (−11.2 vs. −9.3 kcal/mol) and multi-residue interactions. Conclusions: Green-synthesized CS–Ag HNPs represent a promising sustainable platform for combating pulmonary MDR infections through enhanced antimicrobial efficacy and optimal aerodynamic properties. Full article

Lung cancer remains the most prevalent and deadliest malignancy worldwide. According to the European Society for Medical Oncology guidelines for non-oncogene-addicted metastatic non-small-cell lung cancer (NSCLC), patients with metastatic squamous-cell carcinoma (LUSC) or metastatic non-squamous NSCLC with performance status 2 and PD-L1 < 50% may receive single-agent chemotherapy with gemcitabine (GEM), docetaxel (DOC), or vinorelbine. Herein, we investigated the cellular effects of GEM/DOC as monotherapies in NSCLC cell lines—lung adenocarcinoma, A549 and CALU6; LUSC, H520 and H1703. Treatment with GEM/DOC may induce apoptotic cell death in all NSCLC cell lines at 48 h. GEM/DOC can affect cancer cell migration assessed by scratch assay. Both GEM/DOC may produce distinct effects on cell cycle arrest, consistent with their particular pharmacodynamic effects. Furthermore, GEM/DOC induced signals consistent with autophagic activity in LUSC cell lines, but only GEM triggered signals consistent with autophagic activity in the CALU6 cell line. Analysis of three key long non-coding RNAs (lncRNAs)—MALAT1, NEAT1, and HOTAIR—showed variable expression in the studied cell lines as a potential response to DOC and GEM treatment. Our findings indicate different cellular effects of GEM and DOC in NSCLC cell lines and provide an overview of how currently used chemotherapeutics may influence the expression of lncRNAs. Full article

Influenza virus is one of the most frequent causes of respiratory infection in humans. Recent studies suggest that extracellular vesicles (EVs)—small particles released by cells during influenza virus infection—can influence the immune response and viral pathogenesis. However, during viral replication, infected cells can also release EVs, which may include different subtypes. This study aimed to purify and characterize viral preparations and EVs using sequential ultracentrifugation methods. Influenza A/H1N1, A/H3N2, and B virus strains were produced in human Calu-3 and A549 cell lines. Viral supernatants then underwent a series of differential ultracentrifugation steps at 3000× g, 17,000× g, and 100,000× g. Dynamic light scattering analysis (DLS) validated size heterogeneity for all three types of EVs. Measurement of infectious virus particles for all three pellets showed virus enrichment at 17,000× g and 100,000× g. Dot blot analysis confirmed the enrichment of virus particles in these fractions and the presence of EV protein. This study demonstrates the presence of EVs in virus preparations and highlights the need for improved separation methods to characterize them better and explore their role in viral infection pathogenesis. Full article

Background/Objectives: Local delivery of gene therapy products through the airways shows great promise for the treatment of a number of serious lung diseases, but its effectiveness is hampered by the mucus layer protecting the lung epithelium in the trachea and bronchi. Methods: To overcome this barrier, we engineered carbon dots (CDs) with mucus penetrating properties. Results: The CDs were synthesized by solvothermal treatment of citric acid and branched polyethyleneimine, and functionalized with maleamic acid groups to create cationic mucoinert nanoparticles with tunable charge. We characterized their interactions with a mucus model through turbidity and transport measurements, and assessed their impact on the viscoelastic properties of the biopolymer. We then demonstrated that the carriers are effective at delivering pDNA to a variety of cell models in vitro. In particular, mucus-producing Calu-3 cells cultured at the air–liquid interface (ALI) were used as a discriminating model to evaluate intracellular delivery of the genetic cargo through a thick layer of mucus at the cell surface. Conclusions: The functionalization of CDs with maleamic acid groups resulted in a 1000- to 10,000-fold increase in transfection efficiency in the mucus-producing model, offering new opportunities for lung gene therapy. Full article

Despite numerous research efforts and several effective vaccines and therapies developed against coronavirus disease 2019 (COVID-19), drug repurposing remains an attractive alternative approach for treatment of SARS-CoV-2 variants and other viral infections that may emerge in the future. Cellular polyamines support viral propagation and tumor growth. Here we tested the antiviral activity of two polyamine metabolism-targeting drugs, an irreversible inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), and a non-steroidal anti-inflammatory drug (NSAID), Sulindac, which have been previously evaluated for colon cancer chemoprevention. The drugs were tested as single agents and in combination in the human Calu-3 lung adenocarcinoma and Caco-2 colon adenocarcinoma cell lines and the K18-hACE2 transgenic mouse model of severe COVID-19. In the infected human cell lines, the DFMO/Sulindac combination significantly suppressed SARS-CoV-2 N1 Nucleocapsid mRNA by interacting synergistically when cells were pretreated with drugs and additively when treatment was applied to the infected cells. The Sulindac alone and DFMO/Sulindac combination treatments also suppressed the expression of the viral Spike protein and the host angiotensin-converting enzyme 2 (ACE2). In K18-hACE2 mice, the antiviral activity of DFMO and Sulindac as single agents and in combination was tested as prophylaxis (drug supplementation started 7 days before infection) or as treatment (drug supplementation started 24 h post-infection) at the doses equivalent to patient chemoprevention trials (835 ppm DFMO and 167 ppm Sulindac). The drugs’ antiviral activity in vivo was evaluated by measuring the clinical (survival rates and clinical scores), viral (viral load and virus infectivity), and biochemical (plasma polyamine, Sulindac, and Sulindac metabolite levels) endpoints. Prophylaxis with DFMO and Sulindac as single agents significantly increased survival rates in the young male mice (p = 0.01 and p = 0.027, respectively), and the combination was effective in the aged male mice (p = 0.042). Young female mice benefited the most from the prophylaxis with Sulindac alone (p = 0.001) and the DFMO/Sulindac combination (p = 0.018), while aged female mice did not benefit significantly from any intervention. Treatment of SARS-CoV-2-infected animals with DFMO or/and Sulindac did not significantly improve their survival rates. Overall, our studies demonstrated that DFMO and Sulindac administration as the prophylaxis regimen provided strong protection against the lethal outcome of SARS-CoV-2 infection and that male mice benefited more from the polyamine-targeted antiviral treatment than female mice. Our findings underscore the importance of evaluation of the antiviral activity of the drugs in the context of sex and age. Full article

COVID-19 has triggered the rapid adoption of human organoid-based tissue culture models to overcome the limitations of the commonly used Vero cell line that did not fully recapitulate SARS-CoV-2 infection of human tissues. As the primary site of SARS-CoV-2 infection, the human nasal epithelium (HNE) cultivated in vitro and differentiated at air–liquid interface (ALI) is an ideal model to study infection processes and for testing anti-viral antibodies and drugs. However, the need for primary basal cells to establish the ALI-HNE limits the scalability of this model system. To try and bypass this bottleneck, we devised an ALI-differentiated form of the human adenocarcinoma cell line Calu-3, reported to model most aspects of authentic SARS-CoV-2 infection, including viral entry. The ALI-Calu-3 were tested for infection by a panel of SARS-CoV-2 variants, including ancestral (VIC01) and early pandemic lineages (VIC2089, Beta, Delta), and Omicron subvariants (BA2.75, BA4, BA5, XBB1.5). All tested lineages infected the ALI-HNE. In stark contrast, infection of the ALI-Calu-3 by Omicron subvariants BA4 and XBB1.5 was reduced. These data support the use of ALI-Calu-3 as a complementary, intermediary model for most but not all SARS-CoV-2 lineages, and places the ALI-HNE as the benchmark culture model for SARS-CoV-2 infection. Full article

Non-small cell lung cancer (NSCLC) is a predominant form of lung cancer that is often diagnosed at an advanced metastatic stage. The processes of cancer cell migration and invasion involve epithelial-to-mesenchymal transition (EMT), which is crucial for metastasis. Targeting cancer aggressiveness with effective plant compounds has gained attention as a potential adjuvant therapy. Eurycomanone (ECN), a bioactive quassinoid found in the root of Eurycoma longifolia Jack, has demonstrated anti-cancer activity against various carcinoma cell lines, including human NSCLC cells. This study aimed to investigate the in vitro effects of ECN on the migration and invasion of human NSCLC cells and to elucidate the mechanisms by which ECN modulates the EMT in these cells. Non-toxic doses (≤IC20) of ECN were determined using the MTT assay on two human NSCLC cell lines: A549 and Calu-1. The results from wound healing and transwell migration assays indicated that ECN significantly suppressed the migration of both TGF-β1-induced A549 and Calu-1 cells. ECN exhibited a strong anti-invasive effect, as its non-toxic doses significantly suppressed the TGF-β1-induced invasion of NSCLC cells through Matrigel and decreased the secretion of MMP-2 from these cancer cells. Furthermore, ECN could affect the TGF-β1-induced EMT process in various ways in NSCLC cells. In TGF-β1-induced A549 cells, ECN significantly restored the expression of E-cadherin by inhibiting the Akt signaling pathway. Conversely, in Calu-1, ECN reduced the aggressive phenotype by decreasing the expression of the mesenchymal protein N-cadherin and inhibiting the TGF-β1/Smad pathway. In conclusion, this study demonstrated the anti-invasive activity of eurycomanone from E. longifolia Jack in human NSCLC cells and provided insights into its mechanism of action by suppressing the effects of TGF-β1 signaling on the EMT program. These findings offer scientific evidence to support the potential of ECN as an alternative therapy for metastatic NSCLC. Full article

Angiotensin-converting enzyme 2 (ACE2) is a cell-surface receptor that helps the body regulate blood pressure and endocrine secretions. Transmembrane serine protease 2 (TMPRSS2) is a cell surface protein expressed mainly by endothelial cells of the respiratory and digestive tract, which participates in the cleavage of protein peptide bonds with serine as the active site. These two proteins have been studied to be highly associated with infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Soybean trypsin inhibitor (SBTI) has special bioactivities such as anticarcinogenic and anti-inflammatory functions, which can be widely used in functional foods or drugs. Our study involved in vitro and in vivo experiments to elucidate the effect of SBTI on SARS-CoV-2 host invasion. First, it was confirmed that being under 250 μg/mL of SBTI was not toxic to HepG2, HEK293T, and Calu-3 cells. The animal study administered SBTI to mice once daily for 14 days. In the lungs, liver, and kidneys, the histopathologic findings of the SBTI group were not different from those of the control group, but the expression of ACE2, TMPRSS2, and CD147 was reduced. Thus, our findings suggest that the inhibition of ACE2, TMPRSS,2 and CD147 proteins by SBTI shows promise in potentially inhibiting SARS-CoV-2 infection. Full article

Klebsiella pneumoniae, a zoonotic pathogen of global concern, poses significant threats to both veterinary and public health. Here, a comparative study characterized 14 clinical isolates (7 avian-derived, 7 human-derived) from Jiangsu, China, through integrated genomic and phenotypic analyses. Firstly, multilocus sequence typing (MLST) revealed distinct epidemiological patterns: the same ST type in avian isolates was circulating between different species and different regions, whereas it was not found in human isolates. In addition, hypervirulent Klebsiella pneumoniae (hvKP) phenotypes confirmed by string test were exclusive to two human isolates (KP15, KP20). Secondly, biofilm detection demonstrated 78.6% (11/14) of isolates possessed biofilm-forming capacity, with cellulose but not curli as the predominant matrix component. Human-derived KP15 and KP20 had the strongest biofilm formation ability in all isolates. Antimicrobial susceptibility profiling identified serious multidrug resistance in both avian and human isolates. Virulence gene analysis revealed striking disparities, with human isolates harboring 10–20 virulence factors (median 15) versus 6–7 (median 6.5) in avian counterparts. Finally, functional pathogenesis assessments demonstrated human-derived strains exhibited stronger epithelial cell adhesion (2-fold higher) and invasion (1.97-fold higher) in Calu-3 cell models and paradoxically showed reduced macrophage phagocytosis (2.85-fold lower at 2 h) for immune escape. In vivo models confirmed dose-dependent mortality, with human isolates demonstrating higher lethality in both Galleria mellonella and mice. Virulence gene burden positively correlated with mortality outcomes. These findings delineate critical host adaptation differences in Klebsiella pneumoniae populations and provide empirical evidence for pathogen transmission dynamics at the human-animal interface. Full article