The Bacillus Calmette-Guérin (BCG) vaccine, a live-attenuated derivative of Mycobacterium bovis, has long been central to global tuberculosis prevention. Although it protects well against severe childhood TB, its efficacy against adult pulmonary TB is variable. At the same time, epidemiological and clinical observations suggest that BCG may reduce all-cause mortality and protect against infections beyond TB. Randomised trials have reported lower neonatal all-cause mortality and fewer sepsis-related deaths, supporting the idea of broader immunological benefits. These heterologous effects are proposed to be derived from trained immunity, a form of functional reprogramming of innate immune cells driven by epigenetic and metabolic changes. In some settings, BCG may also induce trained tolerance, leading to a more suppressive immune state, based mainly on animal and in vitro evidence. Clinically, intravesical BCG is an established local immunotherapy for non-muscle-invasive bladder cancer, with current evidence and emerging data suggesting that its effects may extend beyond the bladder through systemic immune training. However, repurposing BCG for other cancers, non-oncological autoimmune diseases, and respiratory tract infections remains established in experimental animal models but is represented with mixed efficacy accompanied by inconclusiveness in human trials and mostly in preclinical or early-phase evidence. Major barriers to translation include strain variability, lack of standardised dosing, uncertain durability, and unresolved long-term safety concerns. Future progress will depend on engineered BCG derivatives, improved delivery systems, rational combination therapies, and well-designed controlled clinical trials.Methodology: Literature was identified through searches of PubMed, Google Scholar, and the Cochrane Library, from database inception to 2026, with a primary focus on studies published between 2011 and 2026. The Bacillus Calmette-Guérin (BCG) vaccine was developed over a century ago, in 1921, to combat tuberculosis. Beyond its primary role, researchers are uncovering its capacity for “trained immunity” a form of innate immune memory. This process involves the functional reprogramming of the body’s natural defenses, which may allow them to respond to a diverse spectrum of pathogens and diseases.Clinical observations have established that BCG administration may reduce neonatal mortality from non-specific infections, such as sepsis, by enhancing the underlying immune reprogramming and granulopoiesis. This broad-spectrum immunomodulation remains investigational and preclinical for oncological applications with mixed and negative results. While BCG is already a foundational immunotherapy for non-muscle-invasive bladder cancer, emerging evidence suggests it possesses epigenetic reprogramming potential that may work to some extent in fighting respiratory tract infections and to some extent slow down progression of autoimmune conditions, including type 1 diabetes and multiple sclerosis, with mixed/biased outcomes.However, the transition from successful preclinical models to widespread clinical use requires addressing several complex variables. These include the nuances of strain variability, standardized dosing protocols, and the long-term systemic safety of these therapies. The future of BCG-based vaccines lies in the development of engineered derivatives and novel delivery mechanisms designed to optimize this “trained” response.For patients and advocates, this research represents a noble pursuit: repurposing the BCG vaccine by overcoming translational challenges and by bridging a century of vaccine history with modern epigenetic science, we can enter a new era of multi-targeted disease prevention and treatment.Methodology: Literature searches were conducted in PubMed, Google Scholar, and the Cochrane Library, with additional screening of relevant reviews and reference lists from independent journals and reputable scientific websites, from database inception to 2026, to identify English-language articles, with a primary focus on recent developments from 2011 to 2026. while integrating a few from foundational records of earlier years. Search terms included combinations of “Bacillus Calmette-Guérin,” “BCG,” “trained immunity,” “innate immune memory,” “heterologous effects,” “epigenetic reprogramming,” “metabolic reprogramming,” “histone modification,” “lactylation,” “tolerance,” and disease-specific terms such as “tuberculosis,” “bladder cancer,” “autoimmunity,” and “viral infection,” chosen to capture the major biological mechanisms, clinical applications, and conflicting or context-dependent outcomes of BCG-induced immune modulation.
Lung cancer is one of the malignant tumors with the highest mortality in the world. Quercetin (Qu) is a flavonoid compound isolated from Tetrastigma hemsleyanum Diels & Gilg (SYQ), which has multi-target anti-tumor activity. However, there are problems such as low extraction purity, poor water solubility, and difficulty in targeted delivery. Its anti-lung cancer mechanism remains to be elucidated. To investigate the enrichment of Qu by molecularly imprinted polymers (MIPs) and to construct targeted micelles to improve its solubility, tumor targeting, and bioavailability and to explore its anti-lung cancer mechanism by in vitro cell experiments and in vivo animal models. The static adsorption performance of Qu in SYQ extract was evaluated by MIPs. Key targets and pathways were predicted by network pharmacology and molecular docking. The targeted micelles were constructed, and their particle size, morphology, drug loading, and release behavior were characterized. The cytotoxicity and migration inhibition of free Qu and drug-loaded micelles on H1299 and A549 cells were evaluated by MTT assay, crystal violet staining, live/dead cell staining, and scratch healing assay. The uptake efficiency of Qu and the change of intracellular reactive oxygen species level were observed by confocal microscopy. The expression of the PI3K/AKT pathway protein was detected by Western blot, and the H1299 tumor-bearing mouse model was established to evaluate the in vivo distribution and anti-tumor effect. Qu was isolated and identified from SYQ extracts using ¹HNMR, MALDI-TOF, Q-TOF LC/MS, and FTIR. When the ratio of Qu to acrylamide was 1:7, the equilibrium adsorption capacity for Qu reached 393.26 μg/g. In network pharmacology, Qu and non-small cell lung cancer (NSCLC) shared 111 common target proteins, with a binding energy of -8.71 kcal/mol between Qu and the BCL-2. The HPP@Qu micelles exhibited a particle size of 107.75 nm, a drug loading capacity of 6.57%, and a cumulative release rate of 55.15% at 72 h. HPP@Qu significantly inhibited the viability and migration of H1299 cells, increased intracellular reactive oxygen species (ROS) levels, downregulated p-PI3K, p-AKT, and BCL-2 proteins, and upregulated cleaved caspase-3. Furthermore, HPP@Qu effectively accumulated in tumor tissues, suppressed tumor growth in tumor-bearing mice, and showed no obvious toxicity to major organs. MIPs selectively enrich high-purity Qu from SYQ. Targeted micelles enhance Qu's solubility and tumor targeting, effectively inhibiting tumor growth. Qu induces H1299 cell apoptosis by elevating ROS levels, suppressing the PI3K/AKT pathway, downregulating BCL-2, and activating Caspase-3. These findings reflect the research value of Qu as a potential natural anticancer candidate drug.
Brucellosis remains one of the most significant zoonotic diseases worldwide, and the absence of licensed human vaccines highlights the need for novel vaccine platforms and reproducible laboratory-scale manufacturing strategies. In the present study, the replication efficiency of a recombinant capripoxvirus expressing a Brucella antigen was evaluated in Vero cells cultured under serum-free conditions using two 3D carrier-based cultivation systems. Vero cells were cultivated in serum-free medium either on Cytodex 1 microcarriers in a 500-mL reusable spinner flask operated in repeated-batch mode or on BioNOC II® macrocarriers in single-use 500-mL BelloCell™ 500AP vessels integrated into the BelloStage™-3000 system and operated according to the Tide Motion principle with medium recirculation. Cells were infected with recombinant SPPV(TKΔ)-OMP16 at a multiplicity of infection of 0.1, and total cell yield, metabolic parameters, and virus production were subsequently monitored throughout cultivation. The microcarrier and macrocarrier systems achieved maximum total cell yields of 7.0 × 10⁸ and 3.1 × 10⁹ cells, respectively. Peak virus titers reached 5.75 log₁₀ TCID₅₀/mL at 120 h post-infection in the microcarrier system and 7.25 log₁₀ TCID₅₀/mL at 168 h post-infection in the macrocarrier system, corresponding to a 1.5 log₁₀ increase in virus titer (p < 0.01). After normalization to the working culture volume, the macrocarrier system exhibited 4.4-fold higher volumetric cell productivity. In addition, virus productivity normalized to the total number of cells present at the time of infection was approximately sevenfold higher than that observed in the microcarrier system. These findings demonstrate that the macrocarrier-based dynamic cultivation system enables superior total cell yield and enhanced recombinant virus production under serum-free laboratory-scale conditions. KEY POINTS: • BioNOC II® macrocarriers increased volumetric cell productivity by 4.4-fold compared with Cytodex 1. • BioNOC II® macrocarriers increased peak virus titers by 1.5 log₁₀ TCID₅₀/mL. • Cell-specific viral productivity was approximately sevenfold higher in the macrocarrier-based bioreactor system.
Vaccination is widely regarded as the most effective strategy for controlling infectious diseases that compromise the sustainability and productivity of global aquaculture. Although inactivated and subunit vaccines are commonly applied, their protective efficacy often depends on potent adjuvants to induce robust and durable immune responses. In aquatic settings, environmental exposure and physiological barriers in fish-including enzymatic degradation, pH fluctuations, osmotic stress, and mucosal and integumentary defenses-pose substantial challenges to antigen stability and delivery efficiency. To address these constraints, advanced delivery systems have been developed to enhance antigen protection, facilitate transport to lymphoid tissues, and improve antigen presentation to immune cells. Among these, biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA)-based microparticles (MPs) and nanoparticles (NPs) have emerged as versatile and promising platforms. PLGA has been extensively utilized in human pharmaceutical applications and is increasingly being investigated for veterinary purposes, including fish vaccines. This review summarizes the physicochemical properties of PLGA, elucidates its controlled degradation behavior and antigen release kinetics, and examines PLGA particle-mediated antigen uptake, processing, and presentation mechanisms that contribute to immune activation in teleost fish. Recent advances in PLGA-based vaccines targeting major aquatic pathogens are also discussed. By enhancing antigen stability, prolonging immune stimulation, and enabling non-invasive administration routes such as oral and immersion vaccination, PLGA-based delivery systems represent a promising strategy for aquatic immunoprophylaxis. Their broader implementation may further reduce antibiotic reliance in aquaculture, thereby mitigating antimicrobial resistance and environmental impacts while supporting sustainable industry development.
Emerging and re-emerging infectious diseases (EIDs) represent an escalating threat to global public health, as exemplified by outbreaks of COVID-19, Ebola, Zika, and other zoonotic viruses. Traditional pathogen-specific vaccines, although effective for individual diseases, face significant limitations in addressing EIDs due to long development timelines, resource intensity, and restricted adaptability to novel pathogens or variants. Pan-pathogen vaccines-designed to provide broad-spectrum immunity across multiple related or unrelated pathogens-offer a transformative approach to pandemic preparedness. This review presents a comprehensive overview of pan-pathogen vaccinology within the One Health framework, emphasizing the integration of human, animal, and environmental health for proactive disease prevention. We provide explicit definitions for "universal", "broad-spectrum", and "pan-pathogen" vaccines and embed the One Health principle into the full vaccine development lifecycle through real-world case studies, including Nipah virus and rVSV-Ebola. The review highlights strategies for epitope discovery using comparative genomics, evolutionary biology, and immunoinformatics to identify conserved antigenic regions, and details mechanisms of cross-protective immunity mediated by T cells, broadly neutralizing antibodies, mucosal responses, and trained innate immunity with emphasis on their interplay. Advanced vaccine platforms-mRNA, viral vectors, protein subunits, and nanoparticle-based systems-are evaluated for their capacity to deliver multivalent, chimeric, and mosaic antigens. Preclinical and clinical advances against influenza, coronaviruses, flaviviruses, filoviruses, and critically, bacterial, fungal, parasitic, and DNA virus targets are summarized. Ethical, regulatory, and global health considerations for equitable vaccine distribution are discussed, with expanded safety analysis addressing pan-pathogen-specific hazards such as antigenic competition, autoimmunity, and regulatory adaptation. Persistent gaps, including antigenic variability, immune imprinting, safety concerns, and challenges in clinical validation, are identified, alongside controversies surrounding the balance between broad coverage and potential immune escape. Integrating genomic surveillance, predictive modeling, and emerging technologies such as artificial intelligence, systems biology, and synthetic vaccinology is essential to optimize vaccine design and accelerate translational implementation. Collectively, pan-pathogen vaccines represent a proactive, adaptive, and globally coordinated strategy to mitigate future pandemics and strengthen long-term health security.
Ovarian cancer (OC) is a globally prevalent malignancy associated with a high mortality rate and marked biological heterogeneity, with most cases arising from epithelial cells and presenting as serous, endometrioid, or clear cell subtypes. Standard management is largely stage dependent and primarily involves cytoreductive surgery followed by platinum- and taxane-based chemotherapy, with modifications based on disease extent and patient factors. In recent years, the therapeutic landscape of OC has evolved with the introduction of maintenance strategies and targeted therapies, particularly driven by advances in molecular profiling and the identification of biomarkers such as BRCA mutations and homologous recombination deficiency (HRD). These developments have led to the clinical integration of PARP inhibitors and anti-angiogenic agents such as bevacizumab, which have improved disease control and survival outcomes as part of standard treatment strategies in OC, whereas immunotherapeutic approaches remain largely investigational and are currently limited to clinical settings. Metronomic chemotherapy (MCT), characterized by the continuous administration of low-dose chemotherapeutic agents, has emerged as a promising alternative to conventional maximum tolerated dose regimens. MCT offers reduced systemic toxicity while exerting sustained antitumor effects through modulation of the tumor microenvironment, inhibition of angiogenesis, and enhancement of antitumor immune responses, thereby addressing key limitations of standard chemotherapy, including resistance and cumulative adverse effects. Furthermore, the integration of artificial intelligence (AI) into metronomic treatment strategies holds significant potential for optimizing drug selection, dosing schedules, and patient stratification. AI-driven tools can facilitate predictive modeling, high-throughput data analysis, and personalized treatment planning, ultimately enhancing therapeutic efficacy while minimizing toxicity. This review summarizes current management strategies in OC with particular emphasis on maintenance therapies, targeted approaches, and emerging evidence supporting MCT and AI-enabled approaches as potential future directions in OC therapy.
Sanitization is a critical foundation for the safe treatment of animal manure. High-temperature anaerobic digestion (AD) is often recommended for pathogen inactivation. However, heat stress may induce microorganisms into a viable-but-non-culturable (VBNC) state rather than truly inactivating them. This study therefore investigated the fate of Escherichia coli, Salmonella, and Enterococcus in pig manure during biothermal hydrolysis-enhanced AD (55 °C followed by 37 °C) and compared it with single-phase mesophilic AD (37 °C). In the two-phase process, culturable E. coli and Salmonella were reduced to below the detection limit (< 1 CFU/g fresh matter) at 55 °C, while Enterococcus achieved a > 5 log10 reduction. Conversely, high levels of culturable E. coli and Enterococcus persisted in the single-phase mesophilic AD. However, propidium monoazide-quantitative PCR revealed substantial VBNC counts (5.6 log10 - 7.8 log10 cells/g fresh matter) in both systems' effluents. Imaging flow cytometry, excitation-emission matrix fluorescence spectroscopy, and ATP measurements further confirmed that these VBNC cells retained membrane integrity and energy metabolism. VBNC cells also showed reduced cell size and amino acid/protein-related signals, indicating a low-metabolic but persistent state. During 129-day storage at ambient temperatures rising from 24 to 30 °C, culturable Salmonella increased to 2 log10-4 log10 CFU/g fresh matter in both digestates while culturable E. coli increased only in the single-phase digestate, likely due to proliferation of residual culturable cells or resuscitation of VBNC cells. These findings reveal that conventional culture-based methods underestimate the actual pathogen load. Optimizing the AD process is therefore necessary to truly eliminate VBNC pathogens for safe land application.
While the natural flavonoid Kaempferol (Kae) possesses promising anti-inflammatory and immunomodulatory properties for treating rheumatoid arthritis (RA), it is challenged by poor aqueous solubility and low bioavailability, which impede its targeting of key effector cells like macrophages and limit its clinical utility. To overcome Kae's pharmaceutical limitations in RA therapy, We developed mannose-modified PLGA nanoparticles (Kae-NPs) for macrophage-targeted delivery and investigated their therapeutic efficacy and underlying mechanisms. Integrated bioinformatic analyses of GEO datasets identified macrophage-related pathways as central to rheumatoid arthritis (RA) pathogenesis. Kae-NPs were synthesized and evaluated for their morphology, size distribution, surface charge, and drug-release profile. Cellular uptake and macrophage polarization were assessed in LPS-treated RAW264.7 cells by flow cytometry, ELISA, and immunofluorescent staining. In a collagen-induced arthritis (CIA) rat model, therapeutic efficacy and mechanisms were evaluated through small-animal imaging, micro-CT, histopathology, and serum immune profiling. Kae-NPs showed uniform size (∼136 nm) and sustained release. They were efficiently internalized by macrophages and promoted M1-to-M2 polarization in vitro. In CIA rats, Kae-NPs accumulated in inflamed joints, reduced swelling, cartilage damage, and bone erosion. Mechanistically, Kae-NPs scavenged ROS, modulated cytokine production by suppressing IL-6, IL-1β and TNF-α while elevating TGF-β and IL-10, restored Treg/Th17 balance, and inhibited fibroblast-like synoviocyte (FLS) proliferation, with no systemic toxicity observed. Kae-NPs enable targeted Kae delivery to joint macrophages, ameliorating RA through ROS clearance, macrophage reprogramming, and immune homeostasis restoration, offering a promising nanotherapeutic strategy.
Antiviral immunity profoundly impacts host metabolism, which can, in turn, modulate immune responses and influence disease pathology. The liver orchestrates systemic bile acid (BA) metabolism, a pathway disrupted in chronic liver diseases such as viral hepatitis. BAs are increasingly recognized for their immunomodulatory properties. Thus, improved understanding of the interplay between immunity and BA metabolism may reveal novel therapeutic avenues. Using lymphocytic choriomeningitis virus (LCMV) as a model, we investigated the interplay between chronic virus infection, BA metabolism, and immunity. Chronic LCMV infection increased BA levels and shifted circulating and liver BA composition toward host-derived, conjugated BAs. Hepatic BA transport and synthesis genes were broadly downregulated, in part depending on CD8+ T cells. Pharmacological inhibition of the main hepatic transporter of conjugated BAs, NTCP (Slc10a1), increased hepatic damage, while combined genetic disruption of the BA transporters Slco1a1, Slco1a4, and Slco1b2, responsible for the hepatic reuptake of unconjugated BA, reduced liver pathology and impaired antiviral CD8+ T cell responses. These findings reveal a reciprocal interplay between BA metabolism and CD8+ T cells, expanding our understanding of adaptive immunity in viral hepatitis. They also highlight how immunometabolic changes in liver disease may affect adaptive immune responses against infections.
Fatty liver is highly prevalent in dairy cows and poses a significant challenge to the dairy industry by reducing both milk yield and quality, thereby inflicting substantial economic losses. Although dietary betaine supplementation has been demonstrated to enhance milk protein content, the underlying molecular mechanisms remain to be fully elucidated. This study aimed to elucidate the role of betaine in milk protein synthesis and the underlying mechanisms. Blood, liver and mammary gland samples were obtained from healthy and fatty liver-affected bovines. The serum NEFA levels, oxidation and antioxidant enzyme systems and milk protein synthesis-related proteins expression were determined. In vitro, the bovine mammary epithelial cells (BMECs) were pretreated with 25 mM betaine/10 mM N-acetyl-L-cysteine (NAC) and then stimulated with NEFA. The results indicated an elevation in serum NEFA levels accompanied by enhanced oxidative stress (OS) and reduced protein levels related to milk protein synthesis (phosphorylated (p)-mechanistic target of rapamycin (mTOR)/mTOR, p-ribosomal protein S6 kinase 1 (S6K1)/S6K1, p-Janus kinase 2 (JAK2)/JAK2, p-signal transducer and activator of transcription 5 (STAT5)/STAT5 and β-casein) in dairy cows with fatty liver. In vitro, the results revealed betaine decreased the hydrogen peroxide (H2O2), malondialdehyde (MDA), and oxygen free radicals (OFR) contents, and lactate dehydrogenase (LDH) activity in the supernatant, but increased the glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) activities; the reduced glutathione-to-oxidised glutathione (GSH/GSSG) ratio; and the total antioxidant capacity (T-AOC); and elevated protein levels related to milk protein synthesis in NEFA-treated BMECs. These findings indicate that betaine reduces NEFA-induced OS and enhances milk protein synthesis, suggesting its potential as a nutritional intervention for enhancing milk protein content in dairy cows with metabolic disorders.
Epigenetic mechanisms profoundly regulate gene expression, developmental trajectories, and phenotypic variation, extending biological influence beyond DNA sequence alone. A growing body of evidence suggests that environmental exposures, including pollutants, drugs, stress, and diet, can induce germline and early embryonic epimutations that alter developmental programs with lasting consequences for neurodevelopmental and cognitive outcomes. However, the fields most relevant to these processes have largely developed independently. These include germline epigenetics, early embryonic patterning, neurodevelopment and cognitive regulation, and intergenerational or transgenerational inheritance. Each field has its own conceptual frameworks and mechanistic models. This fragmentation obscures the biological reality that these systems are tightly interconnected: environmentally induced epigenetic perturbations in gametes can reshape the epigenetic landscape of the early embryo, influence lineage allocation during gastrulation, and ultimately modify the molecular architecture of the developing central nervous system. A systems-biology perspective capable of linking germline epimutations and early embryonic epigenetic instability to later neurodevelopmental and cognitive phenotypes and their potential inheritance is therefore required. This review synthesizes current evidence across these traditionally isolated domains and proposes a coherent mechanistic framework linking germ cell epimutations and early embryonic epigenetic instability to the emergence of neurodevelopmental and cognitive phenotypes. By bridging these conceptual gaps, we aim to establish a cohesive foundation for understanding how early epigenetic disruptions generate long-lasting and in some cases heritable effects on brain development and cognitive function.
Listeria monocytogenes (LM) is found in various environmental sources, including animal intestinal tracts, soil, and sewage. LM can contaminate various food products, including meat, seafood, cheese, fruits, and vegetables. Individuals with underlying medical conditions, the elderly, pregnant women, and infants, may develop meningitis and sepsis upon consuming LM-contaminated food. A rapid and standardized quantification method for evaluating initial contamination levels of LM in food remains to be established. This study aimed to develop a rapid quantification method to monitor LM contamination in food handling facilities. A regression equation was developed to determine the concentrations of LM cells before and after enrichment. Regression equations developed to estimate LM cell concentrations in food samples (ground poultry and green salad) were evaluated. The concentrations of LM cells in ground poultry and green salad before enrichment were 2.44 and 2.92 log10 CFU/g, respectively, and the concentrations obtained using a rapid qPCR-based detection and quantification method were 2.05 and 3.27 log10 CFU/g, respectively. The proposed method can semi-quantitatively estimate LM contamination at approximately 102 CFU/g of food, which is the maximum allowable level of LM contamination in foods. This study may serve as a basis for establishing and improving hygiene management systems and preventing listeriosis.
Feeding various probiotic lactic acid bacteria, including Enterococcus faecium, can alleviate intestinal inflammation and improve gut health in animals. Recently, postbiotics-non-living preparations derived from microbial cells or their metabolites-have gained attention. However, studies on the effects of these postbiotics on immune markers and changes in the gut microbiota of chickens are limited. In this study, we evaluated the effects of the probiotic strain E. faecium JB00008 on the chicken intestinal tract and characterized immune markers and gut microbiota following viral vaccination. Chicks were divided into three groups (Control, DH5α, and JB00008) and administered the respective supernatants in drinking water from days 1-12 at a 3:7 ratio. Samples were collected on days 13 and 28 for microbiota and gene expression analyses. To immunize against infectious bursal disease (IBD), the chicks received an oral vaccine on day 13. Growth, immune, and gut parameters were measured. Body weights did not differ among groups (p = 0.380). Several intestinal immune markers-mucin 2 (MUC2, p = 0.001), occludin (OCLN, p < 0.001), and interleukin-10 (IL-10, p < 0.001)-were significantly higher in the JB00008 group. Annexin A5 (ANXA5, p = 0.005) and interleukin-6 (IL-6, p < 0.001) also differed among groups. After IBD vaccination, IBD-specific immunoglobulin A (IgA, p = 0.200) and IgG (p = 0.065) responses were comparable; however, the alpha (p < 0.001) and beta diversities (p = 0.001) were significantly different among the groups. The JB00008 group showed higher Enterococcus and Bifidobacterium, with enrichment of pathways associated with iron complex transport systems (p < 0.050). These findings suggest that JB00008 postbiotics may enhance intestinal barrier function and microbiota health without affecting growth, thereby supporting gut stability after vaccination. Furthermore, these results highlight the potential use of E. faecium JB00008 as a feed additive and vaccine adjuvant.
Neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) exhibit pro-survival and homeostatic properties, but their clinical translation is limited by protein instability and rapid clearance. We evaluated a PEGylated human serum albumin (HSA) nanoparticle system for BDNF/NT3 co-delivery, focusing on physicochemical stability, ocular biodistribution in the rabbit eye, intracellular protein delivery, and protection against oxidative stress-associated cellular damage in human cells. PEGylated HSA-BDNF-NT3 nanoparticles with nominal neurotrophin concentrations of 5 µg/mL (NeO5) or 10 µg/mL (NeO10) were generated by spontaneous self-assembly and characterized using multiangle dynamic light scattering, electrophoretic light scattering, and atomic force microscopy. In vivo performance was assessed after intravitreal injection in rabbits by enzyme-linked immunosorbent assay (ELISA)-based protein quantification and exploratory reverse transcription quantitative polymerase chain reaction (RT-qPCR) profiling of survival-, proliferation-, and apoptosis-related genes. Functional delivery was examined in sodium iodate-stressed ARPE-19 and 6-hydroxydopamine-stressed retinoic acid-differentiated SH-SY5Y cells using ELISA assays, JC-1 analysis, Annexin V/ propidium iodide flow cytometry, high-performance liquid chromatography for malondialdehyde quantification, and RT-qPCR. Both formulations formed stable, spherical nanoparticles (5.9-54.2 nm) with low polydispersity index (≈ 0.18) and preserved colloidal integrity over 28 days. In vivo, BDNF was detectable in ocular tissues up to 72 h and RT qPCR did not reveal a coordinated pro-apoptotic response under the tested conditions. In vitro, nanoparticle treatment significantly increased intracellular BDNF and NT3 levels, improved viability, reduced apoptotic cell fractions, and markedly decreased lipid peroxidation, particularly for NeO10. Increased tropomyosin receptor kinase B TRKB and cAMP response element-binding protein CREB expression provided supportive molecular evidence consistent with neurotrophin-related cellular responses. PEGylated HSA nanoparticles enable stable neurotrophin loading, efficient intracellular delivery, and attenuation of oxidative stress-induced cytotoxicity. These findings support further development of albumin-based nanocarriers for translational nanomedicine applications.
Avian influenza virus (AIV) is a rapidly evolving zoonotic pathogen that causes significant economic loss in the poultry industry and threatens public health. In the development of a broadly protective influenza vaccine, the extracellular domain of matrix protein 2 (M2e) is a promising antigen candidate as it is highly conserved across most influenza A subtypes. M2e-induced immune protection is primarily mediated through antibody-dependent cellular cytotoxicity (ADCC), which enables the clearance of infected cells before viral release. However, the immunogenicity of M2e is limited due to its small size and low abundance on the viral surface, necessitating effective antigen delivery systems to enhance its presentation. We developed a PLGA-based nanoshell vaccine co-encapsulating M2e peptide and a STING agonist and evaluated its immunogenicity and protective efficacy in specific-pathogen-free (SPF) chickens. Immunization of SPF chickens with the M2e nanoshell vaccine significantly enhanced immune-related gene expression and B-cell expansion, leading to a robust and durable M2e-specific antibody response. In addition, the induced antibodies demonstrated cross-reactivity by effectively recognizing the M2e protein expressed on MDCK cells infected with seven different strains of influenza viruses. Following H6N1 challenge, vaccinated chickens showed attenuated lesions in the kidneys and decreased viral load and viral antigen in the respiratory and intestinal lymphoid tissues, suggesting reduced viral replication. Furthermore, to explore potential Fc-mediated effector functions, an in vitro killing assay demonstrated that chicken splenic natural killer cells were capable of lysing influenza-infected target cells in the presence of serum from M2e nanoshell-immunized chickens, providing initial functional evidence of ADCC activity in the avian system. Collectively, these results demonstrate that the M2e nanoshell vaccine elicits durable and cross-reactive immune responses and reduces tissue damage, potentially involving ADCC-mediated effector mechanisms. The M2e nanoshell vaccine shows potential as a broadly protective avian influenza vaccine and holds value for controlling AIV outbreaks in poultry.
Efficient in vitro production of rabies virus is essential for vaccine development and quality control applications. High-density cultivation systems offer practical advantages for rabies virus production but also create culture conditions in which nutrient depletion, waste accumulation, and progressive deterioration of host-cell condition may limit infectious virus output. In this study, we evaluated the effects of sodium pyruvate supplementation on rabies virus CVS-11 production in Vero and BSR cells cultivated in a high-density macrocarrier-based tide-motion culture system under serum-containing and serum-free conditions, with complementary comparative observations in conventional monolayer cultures of BHK cells. Cultures were infected at a multiplicity of infection of 0.01, and infectious virus production was assessed over time, together with cell density, glucose consumption, and pH dynamics. Sodium pyruvate supplementation was associated with significantly higher infectious virus titers, delayed culture deterioration, prolonged maintenance of viable cell populations, and higher peak infectious titers in both Vero and BSR cultures. The highest infectious titers were observed under serum-free pyruvate-supplemented conditions, reaching 7.5 log10 FFU/mL in Vero cells and 7.2 log10 FFU/mL in BSR cells. Across the tested conditions, serum-free cultivation and pyruvate supplementation were both associated with significantly higher peak infectious titers. In contrast, exploratory correlation analysis based on condition-level summary values indicated an inverse association between minimum culture pH and peak infectious titer. Together, these findings show that sodium pyruvate supplementation can improve infectious rabies virus yield and prolong the productive phase in high-density macrocarrier-based cultures, supporting its use as a practical culture-modulation strategy for CVS-11 production in adherent cell systems.
Spermatogonial stem cells (SSCs) are responsible for lifelong spermatogenesis in adult males; however, their scarcity and inherent heterogeneity, coupled with the lack of robust SSC-specific surface markers, continue to impede isolation and characterization. In the present study, we found CD71, which corresponds to transferrin receptor (TfR1; encoded by Tfrc), a candidate marker capable of enriching for SSC populations from mouse testes. The immunohistochemistry detected co-localization of CD71 with the undifferentiated spermatogonia marker glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1) on the seminiferous basement membrane, with approximately 81% co-localization. Consistent with this finding, the expression of Tfrc was up-regulated in SSC-enriched germ cell populations relative to mouse germ cell lines (GC-1 and GC-2) and somatic cell lines from testis (TM3 and TM4). Fluorescence-activated cell sorting (FACS) analysis further showed that GFRα1+ cells exhibited approximately 1.4-fold higher Tfrc mRNA expression than GFRα1- cells. Similarly, CD71+ cells exhibited significantly higher expression of the undifferentiated spermatogonia markers Id4, Lhx1, Gfrα1, Zbtb16, and Etv5. Functional transplantation assays further demonstrate that CD71high cells give rise to approximately 5.5-fold more colonies than freshy isolated, FACS-unsorted donor cells. Moreover, peanut agglutinin (PNA) lectin staining confirmed the normal spermatogenic differentiation within colonies derived from CD71high donor cells. Our findings collectively indicate that CD71high cells represent an SSC-enriched population with enhanced spermatogenic regenerative capacity and support the use of CD71 as a complementary marker for SSC enrichment and fertility restoration.
Bluetongue (BT) virus (BTV) is an arthropod-borne pathogen that causes substantial economic losses in ruminants globally. Yunnan Province, located in China's tropical and subtropical border region, faces a high risk of BTV circulation. This study monitored BTV vectors, genetically characterized circulating BTV strains, and assessed seroprevalence to inform early warning efforts. In 2025, 46,000 Culicoides midges were collected from three counties, identified, and grouped into 460 pools. Additionally, 5934 cattle serum samples from 17 border counties were tested using C-ELISA. Midge pools were screened by RT-qPCR for BTV nucleic acids and then inoculated into C6/ 36 cells for virus isolation. Whole-genome sequencing and phylogenetic analysis of VP2 and VP5 genes were performed on the isolates. C. oxystoma and C. trithecoides were the dominant midge species at all sites. RT-qPCR identified 11 BTV-positive midge pools, primarily involving C. oxystoma, C. imicola, C. tainanus, and C. jacobsoni. Four BTV isolates were identified as serotypes 1, 4, 5, and 16. Phylogenetic analysis of VP2 and VP5 showed all four strains likely belong to the Eastern topotype and are closely related to historical BTVs from China, Japan, India, and Australia. The overall BTV seroprevalence in cattle was 41.37% (95% confidence interval [CI], 40.12%-42.62%), with significantly higher rates in autumn (58.07%, 95% CI, 56.29%-59.85%) than in spring (24.87%, 95% CI, 23.32%-26.42%). Several BTV serotypes have been detected and isolated from diverse Culicoides in the border regions of Yunnan. Additionally, BTV seroprevalence has been observed in local cattle populations, indicating that this area may represent a significant BTV endemic hotspot. These findings underscore the need for ongoing vector surveillance and early warning systems to prevent BT outbreaks in susceptible livestock.
Newcastle disease virus (NDV) has been the subject of extensive research as a potential oncolytic virus for various types of cancer. While clinical studies are ongoing, the manufacturing of high NDV doses on a large scale is challenging. It has previously been documented that Vero cells are capable of producing 2.4 × 108 TCID50/mL in suspension batch production. However, the requirement of higher input doses and the challenging nature of establishing high cell density processes with Vero cells are significant obstacles in the effective utilization of these therapies. In pursuit of enhanced process intensification and the generation of viral vectors at elevated cell densities, EB66 cells have been identified as a highly effective producer cell line. In this study, the characteristics of EB66 cells in regard to NDV production were examined, with particular reference to cell growth and cell-specific virus productivity in batch and semi-perfusion mode. Optimal infection conditions for producing NDV in batch and semi-perfusion modes were identified for cultivation parameters including temperature, protease concentration (TrypLE), and multiplicity of infection. The favorable production conditions were then transferred to different batch processes using a stirred tank bioreactor and an orbital shaken bioreactor. These processes yielded up to 4.2 × 108 TCID50/mL of the NDV LaSota strain with a cell-specific virus yield of 41 TCID50/cell. First semi-perfusion runs resulted in concentrations of 65 × 106 cells/mL and an infectious virus titer of 7.5 × 108 TCID50/mL. Finally, the potency of the produced viruses was evaluated, and a reduction in tumor size in mice after NDV injection was demonstrated. Overall, these results indicate that EB66 cells could be a viable host for producing oncolytic NDV.
Proteomic analysis revealed that Bacillus subtilis exhibits markedly different physiological adaptations under pellicle biofilm and swarming growth conditions, and that these lifestyles strongly influence the bacterial response to CeO₂ nanoparticles. In pellicle biofilms, proteins involved in respiration, amino acid acquisition, Mn/Fe uptake, and SUF-mediated iron‑sulfur cluster synthesis were upregulated, together with oxidative stress defense systems, indicating adaptation to microaerobic and heterogeneous biofilm conditions. In contrast, swarming cells displayed increased sporulation-associated processes and a stronger stringent response. Exposure to CeO₂ nanoparticles induced a pronounced response, particularly under swarming conditions, where central carbon metabolism enzymes were strongly repressed and stringent response pathways were reinforced. In biofilms, CeO₂ effects were more moderate, with limited metabolic perturbation and a slight stimulation of biofilm formation. The contrasting responses between lifestyles appear primarily linked to differences in metabolic state, oxidative stress physiology, and nanoparticle accessibility within the biofilm matrix rather than to direct nanoparticle toxicity alone. Overall, these findings demonstrate that nanoceria impacts B. subtilis physiology in a growth mode-dependent manner and highlight the importance of considering bacterial lifestyle when evaluating nanoparticle toxicity. SIGNIFICANCE: This research provides a biologically understanding of how Bacillus subtilis, an important bacterium for soil, plant, and animal health, adapts to environmental stress under more physiologically realistic growth conditions. Using shotgun proteomics, we demonstrated that pellicle biofilm and swarming lifestyles are associated with profoundly distinct physiological states, notably in metabolism, oxidative stress management, metal homeostasis, and developmental regulation. Extending this approach to exposure to cerium oxide nanoparticles (CeO₂ NPs), a widely distributed environmental nanomaterial, we showed that bacterial responses are strongly dependent on growth mode. Swarming cells exhibited pronounced metabolic repression and activation of stringent response pathways, whereas biofilms displayed more limited perturbations together with slight stimulation of biofilm formation. These contrasting responses appear to result primarily from lifestyle-dependent differences in metabolic activity, oxidative stress physiology, and nanoparticle accessibility within the biofilm matrix rather than from direct nanoparticle toxicity alone. Since regulatory toxicology frameworks such as REACH often overlook subtle physiological adaptations, this study highlights the importance of considering microbial lifestyle and physiological context when assessing the ecological risks of emerging chemicals and nanomaterials.