Search Results (83)

Porcine circovirus type 2 (PCV2) is the primary causative agent of a spectrum of porcine circovirus-associated diseases (PCVDs) and remains a major threat to the global swine industry. In this study, ten monoclonal antibodies (mAbs) targeting the Cap protein of PCV2 were generated and characterized. One mAb, designated 4C4, which exhibited high reactivity, strong neutralizing activity, and superior blocking efficacy, was selected for horseradish peroxidase (HRP) labeling. After optimizing the reaction parameters, a blocking ELISA was developed for the detection of the anti-PCV2 antibody. Using receiver operating characteristic (ROC) curve analysis, a cutoff value of 40% was established to distinguish positive from negative serum samples. The sensitivity and specificity of this blocking ELISA method were 98.66% and 100%, respectively. No cross-reactivity was observed with serum antibodies against classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus (PRRSV), or pseudorabies virus (PRV). Intra-assay and inter-assay repeatability tests yielded coefficients of variation (CVs) all below 10%, confirming the assay's excellent reproducibility. Simultaneous testing of 312 clinical porcine serum samples using the developed bELISA and a commercial indirect ELISA kit revealed an overall coincidence rate of 99.04%. In addition, the percentage inhibition (PI) in the bELISA was strongly correlated with serum anti-PCV2 neutralizing antibody titers. In conclusion, the blocking ELISA developed herein demonstrates high sensitivity, strong specificity, and good reproducibility, serving as a potentially effective tool for the detection of the anti-PCV2 antibody and epidemiological investigation. Full article

Cervical cancer is a common gynecological malignancy, with a 5-year survival rate of only 17% for recurrent or metastatic cases. Increased extracellular matrix stiffness, a key change in the tumor mechanical microenvironment, promotes tumor metastasis via mechanotransduction. Piezo1, a mechanosensitive cation channel, senses matrix stiffness and converts mechanical signals into intracellular chemical signals. Neutrophil extracellular traps (NETs) are overformed in tumors, but the mechanism by which matrix stiffness regulates NETs in cervical cancer remains unclear. We detected matrix stiffness and related protein expression in cervical cancer tissues using atomic force microscopy and histochemical staining. Polyacrylamide gel models were used to culture HeLa/SiHa cells, with transcriptome sequencing and ELISA to analyze IL-8 expression. NETs were induced from human peripheral blood neutrophils, and their effect on lymphatic endothelial cells was evaluated. A TC-1 mouse model was used to verify in vivo effects, and Western blot/ELISA explored the Piezo1/NF-κB pathway. Higher Young’s modulus, increased α-SMA/Collagen I expression and collagen content in metastatic cervical cancer tissues. High matrix stiffness activated Piezo1/NF-κB, upregulated IL-8, induced NETs, and enhanced lymphatic endothelial cell tube formation/migration. BAPN reduced tumor stiffness, inhibited metastasis, and decreased NETs in mice. Knocking down Piezo1 blocked NF-κB activation and IL-8 upregulation. High matrix stiffness activates Piezo1/NF-κB to promote IL-8 secretion and NETs formation, enhancing lymphangiogenesis and cervical cancer metastasis, providing a new target for advanced cervical cancer treatment. Full article

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is an acute, febrile, and highly contagious disease that has led to significant economic losses in the global swine industry. Although the attenuated lapinized CSF vaccine (C-strain) has effectively controlled CSF outbreaks in China since the 1950s, it remains challenging to serologically differentiate infected from vaccinated animals (DIVA). Currently, the application of E2 subunit vaccines allows for DIVA by detecting antibodies against the E^rns protein. Therefore, this study aimed to develop a blocking ELISA for CSFV E^rns antibody detection using porcine monoclonal antibodies (mAbs) derived from single B cell technology. Peripheral blood mononuclear cells (PBMCs) were isolated from immunized pigs, and single CD21⁺IgM⁻E^rns-His tag⁺ B cells were sorted via flow cytometry. Using one-step PCR, full-length genes of porcine IgG heavy and light chains were amplified separately, yielding 11 porcine mAbs against the CSFV E^rns protein. Among these, three mAbs (E0S3, E0S5, and E0S10) exhibited broad reactivity, while two (E0S1, E0S4) showed no cross-reaction with bovine viral diarrhea virus (BVDV). Using mAb E0S4 as the blocking antibody, a blocking ELISA was established and optimized. The assay demonstrated a detection limit of 1:128, no cross-reactivity with other swine viruses or BVDV, and intra- and inter-assay coefficients of variation below 10%. ROC curve analysis determined an optimal cut-off value of 48.4%, with high sensitivity and specificity. In conclusion, the developed blocking ELISA provides a reliable tool for high-throughput serological surveillance, facilitating the DIVA strategy and contributing to CSF eradication programs. Full article

Endometriosis is a chronic inflammatory disorder affecting approximately 10% of reproductive-age women, yet non-hormonal therapeutic options remain limited. This study investigates the role of the TLR4/NF-κB/NLRP3 inflammasome axis in endometriosis pathogenesis and evaluates the therapeutic potential of pharmacologic TLR4 inhibition. Ectopic endometriotic tissues, eutopic endometrium, and peritoneal fluid were collected from 15 patients with ovarian endometriosis and 15 control subjects. The endometriotic epithelial cell line 11Z was stimulated with LPS and ATP with or without the TLR4 inhibitor TAK-242. A murine endometriosis model was established in wild-type C57BL/6 and TLR4⁻/⁻ mice treated with TAK-242. Expression of TLR4, p-p65, NLRP3, caspase-1, cleaved caspase-1 (p20), GSDMD-N, IL-1β, PCNA, and CD31 was assessed by qPCR, Western blot, IHC, and ELISA. Ectopic lesions showed significantly elevated TLR4/NF-κB/NLRP3/IL-1β signaling compared with eutopic and control endometrium (all p < 0.05). Peritoneal fluid IL-1β was increased in patients, indicating a localized pelvic inflammatory response. In vitro, TAK-242 suppressed LPS/ATP-induced NF-κB/NLRP3 activation, pyroptosis, and IL-1β secretion (p < 0.05). Furthermore, the NLRP3-specific inhibitor MCC950 confirmed the essential role of NLRP3 inflammasome activation in IL-1β maturation. In vivo, TLR4 deletion or TAK-242 treatment reduced lesion weight, PCNA proliferation, and CD31 microvessel density (all p < 0.05). TLR4 inhibition blocks NF-κB nuclear translocation and subsequent inflammasome activation, suggesting a potential role in attenuating inflammation and angiogenesis. The TLR4/NF-κB/NLRP3 axis may drive endometriosis progression by linking innate immunity, inflammasome activation, pyroptosis, with possible involvement in angiogenesis warranting further investigation. Pharmacological inhibition of TLR4 attenuates lesion growth, supporting TLR4 as a promising non-hormonal therapeutic target for endometriosis. Full article

Inflammation is a key feature in breast cancer progression, with neutrophil extracellular traps (NETs) playing an important role. NETs are DNA-based structures released by neutrophils that can promote tumor adhesion, invasion, and immune evasion. Another crucial mechanism is the inflammasome, a multiprotein complex that drives inflammation through cytokine release. Both mechanisms are present in tumors and may act synergistically. In this study, we evaluated how isolated NETs modulate the NLRP3 inflammasome in a human breast cancer model. Exposure of MDA-MB-231 cells to NETs increased the expression of NLRP3, CASP1, and IL1B. Blocking IL-1R with Anakinra reduced IL1B expression, while inhibition of the P2X7 receptor with A740003 decreased NLRP3 and IL1B. ELISA confirmed that NETs stimulate IL-1β release, which was reduced by MCC950, Anakinra, and A740003. Functionally, NETs accelerated tumor cell migration, and this effect was inhibited by MCC950 and Anakinra. Bioinformatics analysis of TCGA breast cancer samples showed differential inflammasome gene expression among subtypes and a positive correlation between inflammasome components and NET-related genes. These findings highlight the interplay between inflammatory and immune mechanisms in breast cancer progression and may support the development of new therapeutic strategies. Full article

To establish a rapid, sensitive, and reproducible method for evaluating the immunogenic performance of Tembusu virus (TMUV) vaccines, we developed and optimized a blocking enzyme-linked immunosorbent assay (bELISA) using the TMUV envelope (E) protein as the coating antigen. By systematically screening the coating antigen concentration, mAb dilution, serum dilution, and chromogenic reaction time, we determined the optimal reaction conditions for this assay. The results showed that bELISA exhibited high specificity, yielding positive reactions only with TMUV-positive sera and no cross-reactivity with sera against other common duck viruses; the cutoff value for positivity was 48.89%, and the lowest detectable serum dilution was 1:10. Neutralization assays confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In a comparative investigation, this assay was used to assess five TMUV vaccines, including both inactivated and attenuated variants, in Cherry Valley ducks. The DF2 inactivated vaccine was found to elicit the highest antibody levels and blocking rates. This was followed by the WF100 attenuated vaccine, which also demonstrated a strong immune response. The TC2B inactivated vaccine, although effective, showed a comparatively lower response, whereas the FX2010-180P strain and mosquito cell-derived WF100 attenuated vaccine showed weaker immunogenicity. Neutralization assays further confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In summary, the bELISA described here demonstrates high specificity, sensitivity, and reproducibility and is suitable for evaluating the immune efficacy of different TMUV vaccines, providing a reliable technical platform for vaccine immunology studies and optimization of immunization strategies. Full article

Acinetobacter baumannii represents a critical-priority organism due to its multidrug resistance. The emergence of carbapenem-resistant strains poses a major clinical challenge, underscoring the urgent need for novel antibacterial agents with alternative mechanisms. As peptide nucleic acids (PNAs) have recently gained attention as antisense therapeutics, we aimed to validate their potential as novel antimicrobial strategies against multidrug-resistant A. baumannii. We synthesized a cell-penetrating peptide (CPP)–PNA conjugate targeting pdxA, an essential gene involved in vitamin B6 biosynthesis. Among several candidate genes tested, the pdxA-targeting PNA exhibited the strongest inhibitory activity, achieving complete growth suppression of A. baumannii at 1.56 μM. Although quantitative real-time polymerase chain reaction did not reveal significant reductions in pdxA transcript levels, ELISA quantification revealed an approximately 80% reduction in intracellular vitamin B6, indicating translational inhibition rather than mRNA degradation. The pdxA-targeting CPP–PNA showed negligible activity against other Gram-negative or Gram-positive species, indicating high target specificity; no detectable cytotoxicity in human cells was observed even at relatively high concentrations. CPP–PNA conjugates targeting pdxA interfere with vitamin B6 biosynthesis, leading to growth inhibition of A. baumannii. These findings support PNA as a promising antisense antimicrobial platform that inhibits multidrug-resistant A. baumannii by blocking vitamin B6 biosynthesis. Full article

MICB-targeting CAR-NK (chimeric antigen receptor-modified natural killer cells) therapy may serve as off-the-shelf immunotherapy. We designed soluble Anti-MICB-scFv blocks tumor immune evasion targeting the MICB antigen, thereby enhancing CAR-NK cytotoxicity while reactivating endogenous immune attacks against malignancies. The Anti-MICB-CAR includes two Anti-MICB-scFv connected by an F2A linker, the CD8 hinge and transmembrane domain, the 4-1BB co-stimulatory domain, the CD3ζ activation domain, and IL-15. The expression efficiency of Anti-MICB-CAR in NK cells was investigated by flow cytometry; ELISA demonstrated that Anti-MICB-CAR-NK secreted free Anti-MICB-scFv and detected IL-15 secretion. Flow cytometry and CCK8 were utilized to study Anti-MICB-CAR-NK on tumor cell viability. The PANC-1 xenograft model was established in order to elucidate the anti-tumor effects of Anti-MICB-CAR-NK in vivo. In vitro investigations have demonstrated that the treatment of tumor cells with Anti-MICB-CAR-NK supernatant + NK cells or Anti-MICB-CAR-NK cells not only significantly increased the cytotoxic activity of tumor cells, but also secreted and produced higher levels of IL-15, IFN-γ, TNF-α, perforin, and granzyme B compared with NK cells. Anti-MICB-CAR-NK cells exhibit strong cytotoxic activity against tumor cells with high MICB expression. In vivo, Anti-MICB-CAR-NK cells exhibited a substantial inhibitory effect on tumor growth. The IHC results reveal that Anti-MICB-CAR-NK cells show a more pronounced ability to infiltrate the tumor. We demonstrated the successful expression of Anti-MICB-CAR in NK cells, which enhances the anti-tumor activity of NK cells both in vitro and in vivo. This stress ligand-targeting approach provides a promising strategy for solid tumors. Full article

Background/Objectives: Hepatocellular carcinoma (HCC) therapies are limited by poor response, rapid resistance, and recurrence of aggressive disease. Sorafenib, a multi-tyrosine kinase inhibitor, can trigger β-catenin stabilization and activation, contributing to resistance. Overexpression of the chemokine receptor CXCR6 and its ligand CXCL16 and hyperactivation are implicated in HCC progression and β-catenin stabilization. We hypothesized that SBI-457, a small-molecule CXCR6 antagonist we developed, could disrupt CXCR6/β-catenin crosstalk and enhance sorafenib sensitivity. Methods: We tested SBI-457 alone and in combination with sorafenib in SK-Hep-1 xenograft models and a panel of human HCC cell lines. Tumor burden, β-catenin activation, and CXCR6 expression were assessed by tumor volume measurements, immunohistochemistry, Western blotting, and immunofluorescence. Soluble CXCL16 levels were quantified by ELISA, and cell death responses were evaluated using MTT assays. Results: In vivo, SBI-457 combined with sorafenib reduced normalized tumor volume by 55% compared to vehicle controls, modestly exceeding monotherapy effects, and attenuated sorafenib-induced β-catenin upregulation. In vitro, SBI-457 blocked nuclear accumulation of β-catenin and reversed sorafenib-induced increases in β-catenin levels. Enhanced cell death was observed in specific “responder” HCC cell lines (Hep-3B, SNU-398, JHH-5), which correlated with high intracellular β-catenin, secretion of soluble CXCL16, and expression of a high molecular weight form of CXCR6. In contrast, “non-responder” cell lines with conventional CXCR6 expression and low CXCL16 secretion showed no enhanced cell death response. Conclusions: CXCR6 antagonism with SBI-457 can modulate β-catenin activation and may help overcome sorafenib resistance in selected HCC models. These findings support further development of CXCR6 antagonists as single agents or combination therapies to improve treatment outcomes in HCC. Full article

Background/Objectives: Pinostrobin is a natural flavonoid compound, a specific type of monohydroxyflavanone, found in various plants like fingerroot and honey. It displays various biological activities such as antioxidant, antimicrobial, anti-adaptogenic and anti-inflammatory. However, the anti-inflammatory effect on human macrophages has not yet been investigated. Methods: In this study, we reported the effect of pinostrobin on inhibiting production of pro-inflammatory cytokines and chemokines in human THP-1 macrophages exposed to lipopolysaccharide. In addition, the responsible mechanisms of pinostrobin at the molecular level were identified. Results: Results from ELISA demonstrated that pinostrobin significantly inhibited production of pro-inflammatory cytokines and chemokines, including IL-6, TNF-α, IL-8, MCP-1/CCL2, and CXCL10/IP10. In addition, data from Western blot analysis revealed that pinostrobin suppressed LPS-induced NF-κB activation, in part through blocking IκB-α phosphorylation and degradation as well as NF-κB phosphorylation and nuclear translocation. Results from immunofluorescence study verified that pinostrobin effectively inhibited IκB-α degradation and NF-κB nuclear translocation. Additionally, pre-treatment of pinostrobin alone retained the existence of IκB-α in the cytoplasm of human macrophages, and this may contribute to the enhanced inhibitory activity of pinostrobin in suppressing NF-κB activation. Conclusions: This study provided accumulated evidence that pinostrobin exhibits anti-inflammatory activities, and these properties of pinostrobin in human macrophages stem from the inhibition of the NF-κB signal transduction pathway. Our findings suggest that pinostrobin may be useful for the development of therapeutic treatment of inflammation-related diseases or conditions in which NF-κB is overactive. Full article

In 2014, Duck Adenovirus type 3 (DAdV-3) emerged in Muscovy ducks and has since spread rapidly across China, causing significant economic losses to the duck industry. Given this situation, the development of reliable diagnostic tools is crucial for effective disease control. In this study, a neutralizing monoclonal antibody (mAb) 2F12 specific to DAdV-3 was generated, which showed a blocking rate of over 70% and a neutralization titer of up to 1:794. A blocking enzyme-linked immunosorbent assay (b-ELISA) was further developed based on mAb 2F12 to efficiently detect neutralizing antibodies against DAdV-3. The cut-off values of percent inhibition (PI) were set based on testing 84 negative duck serum samples, with a value below 16.79% (mean ($\overline{X}$) + 2 standard deviations (SD)) for negative sera and over 21.62% ($\overline{X}$ + 3SD) for positive sera. The b-ELISA exhibited a high specificity, reacting exclusively with DAdV-3 positive serum and showing no cross-reactivity with other representative positive sera tested. Additionally, the b-ELISA showed significantly higher sensitivity than the serum neutralization test (SNT), detecting antibodies 16-fold greater than the endpoint dilution of the SNT. The established b-ELISA, validated with 90 field serum samples from six duck farms, was well-suited for clinical detection of DAdV-3 antibodies and for monitoring post-vaccination antibody levels, representing a significant advancement in DAdV-3 detection and prevention. Full article

Polyporusterone B, a triterpene carboxylic acid isolated from Polyporus umbellatus Fries, exhibits anti-cancer and anti-hemolytic activities; however, its anti-inflammatory properties and underlying mechanisms remain unelucidated. We studied the anti-inflammatory effects of Polyporusterone B using lipopolysaccharide (LPS)-stimulated Raw264.7 murine macrophages (in vitro) and LPS-induced endotoxin shock in C57BL/6 mice (in vivo). Results showed that Polyporusterone B (1, 5, and 10 μM) had no cytotoxicity toward Raw264.7 cells, but significantly inhibited LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines (tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6)) in a concentration- and time-dependent manner, as demonstrated by Griess assay, qPCR, and ELISA. Western blot analysis revealed that Polyporusterone B suppressed LPS-induced phosphorylation of mitogen-activated protein kinases (ERK, P38, and NK) and reduced phosphorylation-mediated degradation of inhibitor of κBα (IκBα). Immunofluorescence and immunohistochemical staining further confirmed that Polyporusterone B blocked nuclear translocation of nuclear factor kappa-B (NF-κB)/Rel A in both Raw264.7 cells and mouse tissues. In the in vivo model, Polyporusterone B pretreatment significantly mitigated LPS-induced multi-organ pathological damage (e.g., lung edema, hepatic inflammation, renal hemorrhage) and downregulated tissue levels of TNF-α, IL-1β, and IL-6. These findings suggest that Polyporusterone B exerts anti-inflammatory effects by inhibiting the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, suggesting its potential as a therapeutic candidate for inflammatory diseases. Full article

Chronic inflammation driven by Cyclooxygenase-2 (COX-2) overexpression plays a key role in lung cancer (LC) progression, making it a critical therapeutic target. This study explores thymoquinone (TQ), a potent bioactive phytochemical derived from Nigella sativa, known for its anti-inflammatory and anti-cancer effects, focusing on its ability to suppress lipopolysaccharide (LPS)-induced COX-2 expression via microRNA hsa-miR-199a-3p modulation in LC cells. Using A549 and SHP-77 LC cells, we tested the effect of TQ under LPS stimulation and miRNA inhibition. Advanced techniques like TaqMan qPCR, luciferase reporter gene constructs, and anti-miRNA transfection confirmed that miR-199a-3p directly silences COX-2. Western blot and ELISA assays revealed that TQ dramatically reduces COX-2 protein and PGE2 levels by boosting miRNA-199a-3p. Importantly, TQ also blocked MAPK (p38, JNK, ERK) and NF-κB activation, even when miR-199a-3p was suppressed, proving its multi-targeted action beyond miRNA regulation. These findings reveal a novel anti-inflammatory mechanism, where TQ curbs COX-2-driven inflammation by enhancing miR-199a-3p, simultaneously shutting down pro-cancer MAPK/NF-κB signaling pathways. Given the strong link between chronic inflammation and LC aggressiveness, this study positions TQ as a promising therapeutic candidate, especially for inflammation-mediated lung cancer progression. Its dual ability to modulate miRNA and key signaling cascades makes it a compelling option for future LC treatment strategies. Full article

Sjögren’s disease (SjD) targets the salivary and lacrimal glands and is characterized by autoantibody production and glandular lymphocytic infiltrate with ectopic germinal centers (EGCs). The chemokine CCL25 recruits CCR9⁺ CD4⁺ T cells to the salivary glands to promote B cell activation. However, the therapeutic potential of targeting the CCL25–CCR9 axis to limit glandular inflammation and lymphoid neogenesis remains largely unexplored. Evaluate whether blocking the CCL25–CCR9⁺ T cell axis with a monoclonal antibody could reduce immune infiltration, ectopic germinal center (EGC) formation, and local autoantibody production in the NOD.H2(h4) mouse model of SjD. Female NOD.H2(h4) mice were administered anti-CCL25 antibody, isotype control, or PBS intraperitoneally for 12 weeks. Sera and saliva were collected to evaluate anti-Ro52 antibodies via ELISA across treatment groups. Salivary glands were harvested and processed for H&E staining to assess lymphocytic infiltration and focus scores. Treatment with α-CCL25 was well tolerated, with no significant differences in body weight or stimulated salivary flow between treatment groups. Histopathological evaluation revealed no reduction in lymphocytic infiltration, focus scores, or percentage of inflamed tissue in α-CCL25-treated mice compared to controls. Anti-Ro52 antibodies were undetectable in plasma or saliva across all groups and timepoints. Systemic CCL25 blockade did not significantly alter salivary gland inflammation, function, or autoantibody production in NOD.H2(h4) mice. These findings suggest that monotherapy targeting the CCL25–CCR9 axis may be insufficient to resolve glandular autoimmunity in this model and that additional or combinatorial strategies may be necessary for effective intervention. Full article

Background: Cucurbitacin B (CuB) is a relatively unique and valuable component in plants of the Cucurbitaceae family due to its diverse and remarkable physiological activities, but its specific mechanisms in regulating tumor metabolism and immune response remain unclear. The hypoxic tumor microenvironment (TME) of pancreatic cancer induces metabolic reprogramming in cancer cells, causing them to rely on glycolysis for energy. LDHA, a key enzyme in glycolysis, can suppress glycolysis and tumor growth when inhibited. Objective: The objective of this study was to investigate the mechanism of CuB against pancreatic cancer and its effect on the immune system. Methods: In this study, cell migration/invasion assays, immunofluorescence, ELISA, Western blot, CETSA, flow cytometry, mouse models, and metabolomic and transcriptomic analyses were utilized to systematically elucidate the mechanism by which CuB inhibits pancreatic cancer and activates the immune system. Results: This study confirms that CuB inhibits pancreatic cancer by suppressing the PI3K/Akt/mTOR pathway and activating PINK1/Parkin to induce mitophagy, thereby inhibiting cell migration, invasion, and proliferation. It downregulates the expression of LDHA to block glycolysis, reduce lactate production and efflux, and improve the acidic TME. CuB also induces ICD to activate dendritic cells, promote CD8+ T-cell and M1 macrophage infiltration, and reduce the levels of regulatory T cells. Metabolomic and transcriptomic analyses validate CuB’s dual effects on metabolic reprogramming and immune activation. Conclusions: This study, for the first time, reveals that CuB induces mitophagy via the PI3K/Akt/mTOR and PINK1/Parkin pathways to selectively eliminate damaged mitochondria and suppress tumor energy metabolism. CuB inhibits pancreatic cancer through a triple mechanism—inducing mitophagy, inhibiting glycolysis, and activating immunity—which provides innovative insights for pancreatic cancer therapy. Full article