Search Results (4,436)

Coxsackieviruses B are single-stranded RNA viruses belonging to the Enterovirus genus and are associated with various clinical outcomes, ranging from acute infections to chronic diseases, such as type 1 diabetes (T1D). It was previously shown that inoculation of Swiss albino mice with CVB4 by the intraperitoneal route induced both anti-CVB4 neutralizing and enhancing activities of serum. This study aimed to investigate the humoral immune response of mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4, and the anti-CVB4 neutralizing activity of serum and saliva was assessed by a cell culture neutralization assay. Anti-enterovirus (EV) IgG and IgA antibodies were detected in serum and saliva, respectively, by ELISA. The serum-dependent enhancement of CVB4 infection in cultures of murine splenocytes was evaluated by detecting intracellular viral RNA using RT-qPCR. At day 45 post-inoculation, an anti-CVB4 neutralizing activity, the extent of which depends on the amount of inoculated infectious particles, was detected in the serum of mice exposed orally or intranasally. An increase in anti-CVB4 neutralizing activity was observed in the saliva of mice inoculated orally or intranasally during the follow-up. Oral or intranasal inoculation of CVB4 induced a systemic IgG and mucosal IgA response. In addition, serum from these mice harbored an anti-CVB4 enhancing activity in vitro. These data indicate that Swiss albino mice exposed to CVB4 via the mucosal route constitute a potentially useful model for testing strategies to promote the production of protective mucosal and systemic anti-CVB4 antibodies and for verifying whether or not enhanced antibodies are produced. Full article

Objectives: This study evaluated the analytical and clinical performance of a novel NS1 rapid diagnostic test in a dengue-endemic setting in Thailand. Methods: The K-Dengue NS1 Ag test (K.Bio Sciences, Pathumthani, Thailand) was developed. Analytical performance included determination of LOD, reproducibility, and evaluation against potentially cross-reactive pathogens and interfering substances. Unlike conventional assays employing 40 nm colloidal gold, this test incorporates 80 nm gold nanospheres to enhance detection sensitivity. The LOD was determined by serial dilution of recombinant NS1 proteins representing all four dengue virus serotypes. Clinical performance was assessed using 185 archived plasma samples collected between January 2024 and February 2025 from two tertiary care hospitals in Thailand, with a commercial NS1 ELISA serving as the reference standard. Results: The K-Dengue NS1 test demonstrated serotype-specific limits of detection (LODs) for recombinant NS1 antigen, 2.9 ng/mL (DENV-1), 0.5 ng/mL (DENV-2), 25.2 ng/mL 27 (DENV-3), and 4.5 ng/mL (DENV-4). Cross-reactivity testing revealed no false positives against closely related arboviruses or common co-infections, and no interference was observed from frequently encountered pathogens or biochemical substances. In clinical evaluation, the assay achieved a sensitivity of 98.08% (51/52), a specificity of 100% (133/133), and an overall accuracy of 99.37%. Importantly, sensitivity was significantly higher in primary infections (100.00%) than in secondary infections (93.3%, p = 0.288). Conclusions: In this clinically oriented evaluation, the K-Dengue NS1 rapid test showed high specificity and good sensitivity, particularly in primary dengue infections. While the assay may be useful as part of early diagnostic workflows in comparable healthcare settings, reduced sensitivity in secondary infections indicates that negative NS1 results should be interpreted with caution and, where appropriate, supplemented with additional diagnostic methods. Full article

Monkeypox virus (MPXV) is a zoonotic Orthopoxvirus responsible for Monkeypox (Mpox), historically associated with sporadic zoonotic transmission but increasingly characterised by sustained human-to-human spread. While vaccinia-based vaccination is known to confer cross-protection against MPXV, the durability of such immunity over a human lifetime remains incompletely characterised. Here, we assessed humoral and cellular immune responses to MPXV in octogenarians and nonagenarians vaccinated against smallpox during childhood. Twenty-three adults aged 79–94 years (median 83), who self-reported childhood vaccinia vaccination between 1925 and 1940, were recruited. MPXV-specific antibody responses were evaluated using ELISA, targeting homologous vaccinia and MPXV proteins, and live-virus neutralisation assays. Cellular immunity was assessed by IFN-γ ELISpot following stimulation with peptide pools derived from highly conserved vaccinia antigens. Responses were also obtained from younger, recently MVA–BN-vaccinated and unvaccinated control donors. All historically vaccinated participants exhibited MPXV-reactive IgG responses, with antibody binding and neutralisation levels comparable to recently vaccinated individuals. Functional neutralising activity against MPXV was detected in all donors, with ≥50% neutralisation observed in 78% of participants. Antibody concentrations correlated strongly with neutralisation capacity. T-cell responses were detectable in all historically vaccinated donors, most prominently against the major core protein A10L, although reduced magnitudes were observed in participants over 90 years of age. No MPXV-specific humoral or cellular responses were detected in unvaccinated controls. These findings demonstrate that childhood vaccinia vaccination induces durable humoral and cellular immunity against MPXV persisting for over seven decades. Historical smallpox vaccination status may therefore remain a relevant determinant of protection against Mpox. Full article

Background/Objectives: To investigate the antagonistic effect of probiotic Lactiplantibacillus plantarum GUANKE against respiratory syncytial virus (RSV) and its underlying molecular mechanisms. Methods: in vitro cell models (A549 and HEp2 cells) and an in vivo mouse model (BALB/c mice) were employed. RT-qPCR, TCID50 assay, immunofluorescence, ELISA, Western blot, and histopathological analysis were used to investigate the effects of GUANKE on RSV replication, inflammatory responses, and the type I interferon pathway. Results: Oral administration of GUANKE effectively cleared RSV and alleviated RSV-induced pulmonary inflammatory responses. GUANKE inhibited viral replication. The GUANKE intervention group exhibited significantly reduced pathological damage to lung tissue and decreased the expression of inflammatory cytokines (IL-1β, IL-6, MCP-1, TNF-α). GUANKE augmented the early type I interferon response and activated the STING-TBK1-IRF3-IFN signaling pathway. Conclusions: GUANKE exerts anti-RSV effects by enhancing the early type I interferon response and activating the STING-TBK1-IRF3-IFN signaling pathway, thereby inhibiting RSV replication and alleviating pulmonary inflammatory responses. This suggests its potential value as an anti-RSV agent. Full article

Point-of-care diagnostics are revolutionizing the detection of plant pathogens by enabling rapid, on-site identification without the need for specialized laboratories. One of the tools used for this purpose is loop-mediated isothermal amplification (LAMP). LAMP is a powerful molecular technique increasingly used in pathogen control for its rapid, sensitive, and specific detection of plant pathogens. The aim of this study was the development of a novel, easy-to-use portable colorimetric LAMP (cLAMP) assay that could be used by inexperienced personnel for the detection of the pathogen Clavibacter michiganensis. The assay was combined with a newly constructed device in which LAMP can be performed in 30 min. Initially, a new set of LAMP primers targeting the micA gene was designed and evaluated the sensitivity (100 fg/reaction) and specificity of the assay. Next, the limit of detection (LoD) of two different commercial LAMP kits was compared with common laboratory detection techniques (DAS-ELISA, immunofluorescence, quantitative PCR, and PCR) using the same samples. Additionally, the LoD of the developed cLAMP assay was evaluated in bacterial suspensions and plant extracts spiked with C. michiganensis and validated the effect on the LoD of plant extracts from different tomato varieties. Lastly, its efficacy for C. michiganensis detection was assessed in experimentally inoculated tomato seedlings. The developed method for C. michiganensis detection can be used as a reliable tool for the early detection of the pathogen for field-based applications by untrained personnel. Full article

Objective: The aim of this study was to develop and evaluate non-antibiotic strategies against Helicobacter pylori by establishing a bovine immune milk platform and designing a synergistic multi-antigen immunogen to enhance humoral immune responses. Methods: Inactivated Helicobacter pylori (H. pylori) was used to immunize dairy cows, and the resulting immune milk was characterized for antibody specificity, acid stability, and target antigens via ELISA, Western blot, agglutination assays, and mass spectrometry. Key identified antigens (UreA, UreB, UreE, UreG, HypA, FlaA, and FlaB) were produced as recombinant proteins. Their immunogenicity was evaluated in a murine model, comparing single antigens with various protein combinations. Immune responses were assessed by antigen-specific IgG ELISA, bacterial agglutination titers, flow cytometry for T-cell activation, and histopathology for safety. Results: Immune milk contained high-titer, acid-stable IgG antibodies targeting multiple H. pylori virulence factors. In mice, while single proteins induced specific IgG, a multi-antigen cocktail (FlaA + FlaB + HypA + UreA + UreB + UreE + UreG) elicited significantly higher serum agglutination titers (~7 × 10³) than single antigens or inactivated whole-cell vaccine, alongside robust CD4⁺ T-cell activation. No formulations showed any hepatorenal or splenic toxicity. Conclusion: Bovine immune milk is a viable platform for acid-stable antibody delivery. A rationally designed multi-antigen cocktail synergistically enhances functional humoral immunity in vivo, providing a promising foundation for developing antibody-based or subunit vaccine strategies against H. pylori. Full article

Exercise training has demonstrated potential benefits in addressing the adverse effects of cardiovascular diseases, particularly myocardial infarction (MI). This study analyzed the cardioprotective effects of moderate exercise before and after MI in rats subjected to isoproterenol (ISO)-induced heart damage. Wistar rats were assigned to five groups: controls (CTRL), isoproterenol-treated (ISO), swimming before ISO (PRE + ISO), swimming after ISO (ISO + POST), and swimming both before and after ISO (PRE + ISO + POST). Cardiac function was assessed through echocardiography, while oxidative stress markers, Heme Oxygenase-1 (HO-1) and Myeloperoxidase (MPO), were quantified using biochemical assays and enzyme-linked immunosorbent assay (ELISA). Statistical analyses were conducted by one-way analysis of variance (ANOVA), accompanied by Tukey’s post hoc test. Exercise performed post-MI and both pre- and post-MI significantly reduced ISO-induced infarct size and improved left ventricular function (stroke volume (SV), ejection fraction (EF), and Tei index). HO-1 protein concentration and HO enzyme activity were restored, while swim training reduced the activity of MPO compared to the ISO group. Moderate exercise training, when appropriately timed, provides cardioprotection against ISO-induced myocardial damage by reducing oxidative stress and cardiac dysfunction. Full article

Background: Vasicine (Vas) is a quinazoline alkaloid derived from Adhatoda vasica Nees, which has good anti-allergic asthma and anti-inflammatory effects. However, its specific functional mechanism on allergic asthma is unclear. This study aims to investigate the protective effect of Vas on allergic asthma and its underlying mechanisms. Methods: Initially, the therapeutic effects of Vas were assessed in ovalbumin-sensitized BALB/c mice using airway hyperresponsiveness (AHR), histopathological examinations, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA). Subsequently, a non-targeted metabolomic analysis was performed to examine the influence of Vas on lung metabolites, while molecular docking was utilized to clarify the mechanisms by which Vas intervenes in allergic asthma. Lastly, RBL-2H3 cells were employed in vitro to validate the metabolomic findings by measuring intracellular Ca²⁺ concentrations, in addition to conducting ELISA and Western blot analyses. Results: In vivo, Vas alleviates AHR in mice with allergic asthma, enhances histopathological conditions, and reduces inflammatory factors. Non-targeted metabolomics analyses indicate that the primary pathway implicated in its intervention in allergic asthma may be the FcεRI pathway. Furthermore, molecular docking techniques were utilized to evaluate the binding affinity between Vas and proteins associated with this pathway. In vitro, Vas effectively inhibits degranulation in RBL-2H3 cells and diminishes the release of inflammatory factors by modulating the FcεRI/Lyn + Syk/MAPK pathway. Conclusions: These findings indicate that Vas may effectively alleviate allergic asthma by reducing inflammatory responses, decreasing AHR, and improving histopathological features. Furthermore, Vas seems to inhibit mast cell degranulation and modulate the FcεRI/Lyn + Syk/MAPK pathway. Full article

Background/Objectives: Severe COVID-19 is marked by IL-6-driven inflammation, endothelial injury, and dysregulated coagulation. Although IL-6 antagonists improve clinical outcomes, their effects on the temporal evolution of coagulation and fibrinolysis remain insufficiently defined. This study characterizes inflammatory, endothelial, coagulation, and fibrinolytic trajectories following IL-6 receptor blockade in critically ill COVID-19 patients. Methods: In this prospective, exploratory multicenter case series (ClinicalTrials.gov NCT05218369), 15 ICU patients with PCR- or antigen-confirmed COVID-19 received tocilizumab per protocol. Serial sampling at five timepoints (T0–T4) included routine laboratories, comprehensive viscoelastic hemostatic assays (ClotPro^®), and ELISA-based endothelial and fibrinolytic biomarkers. Analyses were primarily descriptive, emphasizing temporal patterns through boxplots; paired Wilcoxon tests with FDR correction contextualized within-patient changes. Results: Patients exhibited marked inflammation, hyperfibrinogenemia, endothelial activation, and delayed fibrinolysis at baseline. IL-6 blockade induced rapid suppression of CRP and PCT, progressive declines in fibrinogen, and modest platelet increases. In contrast, vWF antigen and activity further increased, indicating persistent endothelial dysfunction. Viscoelastic testing showed preserved thrombin generation and sustained high clot firmness, while biochemical markers (rising PAI-1, modest PAP increase, and progressively increasing D-dimer) and VHA indices suggested ongoing antifibrinolytic activity despite resolution of systemic inflammation. Conclusions: IL-6 antagonism was associated with rapid attenuation of systemic inflammation but was not accompanied by normalization of endothelial activation or fibrinolytic resistance. The observed hemostatic profile was consistent with attenuation of inflammation-associated coagulation features, while endothelial and prothrombotic alterations appeared to persist during follow-up, warranting further investigation in larger controlled studies. Full article

Therapeutic strategies that fine-tune epithelial inflammatory responses are highly sought after in respiratory and mucosal disorders, but few molecules selectively target these pathways. Vernoniastrum migeodii (S. Moore) Isawumi (Asteraceae) represents a chemically promising but understudied source of bioactive small molecules. This study aimed to define the metabolite profile of V. migeodii and evaluate the modulation of inflammatory epithelial signaling of the constituents. From the methanolic extract of V. migeodii, five germacranolide sesquiterpenes, vernolide (1), 3′-hydroxylvernolide (2), pectorolide (3), 4′-hydroxypectorolide-14-O-acetate (4) and 4′-hydroxypectorolide (5), together with (6S,9R)-vomifoliol (6), eucarvone (7), luteolin (8), and luteolin-7-O-glucoside (9) were isolated by multiple chromatographic separations. The structures were determined by comprehensive 1D and 2D NMR spectroscopy. Isolated compounds 1 to 9 together with previously reported steroids (10–17) and tripeptide (18) were evaluated in LPS-activated A549 cells by quantitative PCR for interleukin-6 (IL6), interleukin-1β (IL1β), and prostaglandin-endoperoxide synthase 2 (PTGS2) and by enzyme-linked immunosorbent assay (ELISA) for IL-6 and IL-8. Compounds 2, 7, steroids 10–17 and aurantiamide acetate (18) reduced IL6 mRNA relative to the LPS control, while (6S,9R)-vomifoliol (6) increased IL-6 and elevated IL-8. In the assay IL1β and PTGS2 transcripts were not significantly altered. These findings highlight the potential of V. migeodii metabolites as modulators of epithelial inflammatory pathways. Combining chemical and biological evidence provides a clear basis for structure–activity- and pathway-focused studies. Full article

Ovarian cancer is frequently diagnosed at an advanced stage due to non-specific symptoms, contributing to high mortality. The limited diagnostic performance of current serum assays in early disease underscores the need for complementary circulating biomarkers. Circulating microRNAs and inflammation-related markers are promising candidates. Although miRNAs are implicated in cancer diagnostics, the role of miRNA-29a in ovarian cancer remains underexplored. Given that sST2 is elevated in several malignancies and is a direct target of miRNA-29a, concurrent evaluation may be informative. This pilot study compared serum miRNA-29a and sST2 levels in 23 ovarian cancer patients and 22 healthy female controls. miRNA-29a expression was quantified by real-time PCR (2^−ΔΔCt), and sST2 was measured by ELISA; diagnostic performance was assessed using ROC analysis. miRNA-29a levels were significantly reduced (p < 0.05), whereas sST2 concentrations were significantly increased (p < 0.001) in patients versus controls. ROC analysis showed modest discrimination for miRNA-29a (AUC 0.678) and higher performance for sST2 (AUC 0.825). No significant correlation was observed between the two markers. These findings suggest that circulating miRNA-29a and sST2 may have biomarker potential in ovarian cancer; larger, well-designed studies are required to confirm clinical utility. Full article

Background: Hepatitis B vaccination is the most effective strategy for preventing chronic hepatitis B virus (HBV) infection; however, interindividual variability in vaccine-induced antibody responses remains a significant challenge in the field. Innate immune components, particularly lectin complement pathway proteins such as mannose-binding lectin (MBL), mannose-associated serine protease 1 (MASP-1), and mannose-associated serine protease 2 (MASP-2), may contribute to this variability in outcomes. This study aimed to evaluate the association between serum MBL, MASP-1, and MASP-2 levels, birth weight, and humoral response to hepatitis B vaccination in infants. Methods: This single-center prospective observational study included 37 term infants who received hepatitis B vaccinations at birth, 1 month, and 6 months of age according to the national immunization schedule. Venous blood samples were collected at month 6, before, and month 7 after the 3rd vaccine dose. Serum MBL, MASP-1, MASP-2, and antiHB levels were measured using commercial ELISA and chemiluminescence assays. Data were analyzed using non-parametric statistical tests and Spearman’s correlation analysis. Results: AntiHB levels increased significantly following vaccination (median Pre-3rdVac: 125.8 mIU/mL; Post-3rdVac: 609.7 mIU/mL; p < 0.001). MASP-1 concentrations also showed a significant Post-3rdVac increase (median Pre-3rdVac: 809.52 ng/mL; Post-3rdVac: 1133.93 ng/mL; p = 0.019). Birth weight was positively correlated with both MASP-1 levels (r_s = 0.492, p = 0.004) and changes in MASP-1 concentrations (r_s = 0.524, p = 0.002) after the third dose. In addition, MASP-1 levels were positively associated with antiHB levels (r_s = 0.385, p = 0.030) and Post-3rdVac antiHB titers (r_s = 0.493, p = 0.004). In contrast, serum MBL and MASP-2 concentrations were not significantly associated with antiHB levels before or after vaccination. Conclusions: MASP-1, but not MBL or MASP-2, is associated with the magnitude of the antibody response to hepatitis B vaccination in infants. These findings suggest that specific components of the lectin pathway may influence vaccine-induced immunity, independent of MBL. Further large-scale studies incorporating genetic and functional analyses are warranted to clarify the mechanisms by which lectin pathway proteins shape hepatitis B vaccine response. Full article

Tetanus is a zoonotic disease posing significant threats to both humans and animals, particularly horses, sheep, and ruminants. Current antitoxin therapies rely on animal-derived immunoglobulins, presenting challenges including animal welfare concerns, pathogen contamination risks, and manufacturing complexity. Alpaca-derived nanobodies (VHH) are promising alternatives owing to their high antigen-binding affinity, thermostability, and potential for microbial production. We developed highly active trivalent VHH antibodies (tVHH) that target multiple epitopes of tetanus neurotoxin (TeNT). Following alpaca immunization with tetanus toxoid, 41 VHH clones were isolated using phage display. Six VHH clones were selected through in vivo neutralization assays, from which three clones of VHH (8, 11, 36) were selected to construct tVHH-8/11/36 and tVHH-8/36/11. Using an improved 21-day mouse neutralization assay, tVHH-8/11/36 demonstrated exceptional neutralizing activity of approximately 1580 IU/mg against 4000 LD₅₀ of toxin, substantially exceeding current human and veterinary anti-tetanus immunoglobulin preparations. Surface plasmon resonance and ELISA confirmed that each VHH recognizes different TeNT domains, producing synergistic neutralizing effects through multimerization. Since antitoxin therapy challenges are common to both animals and humans, this tVHH technology supports One Health by providing a unified therapeutic platform applicable across species through sustainable microbial production. Full article

Objective: To investigate the mechanism by which sphingosine-1-phosphatase 2 (SGPP2) modulates endoplasmic reticulum stress (ERS) through the SIRT1/AMPK pathway to improve ischemic cardiomyopathy-induced chronic heart failure (IHF). Methods: Key genes of IHF and ERS were identified through bioinformatics analysis, and significantly associated pathways of the key genes were obtained via single-gene enrichment analysis. In vivo, IHF was induced in Sprague–Dawley (male) rats via ligation of the left anterior descending coronary artery, with cardiac function examined through echocardiography. Myocardial tissue injury and fibrosis were evaluated utilizing hematoxylin-eosin, Masson, and TUNEL staining. Serum levels of NT-proBNP and cTnT were measured via ELISA. SGPP2 protein expression was assessed via immunohistochemistry and Western blotting (WB). In vitro, neonatal rat cardiomyocytes (NRCMs) were isolated and underwent oxygen-glucose deprivation (OGD) to establish an IHF model. SGPP2-overexpressing NRCMs were constructed and treated with the ERS inducer tunicamycin (Tu) or the SIRT1 inhibitor EX527. Cell injury was evaluated using Cell Counting Kit-8 and lactate dehydrogenase release assays, as well as flow cytometry. Endoplasmic reticulum structure was examined by transmission electron microscopy. The endoplasmic reticulum was labeled with the ER-Tracker Red molecular probe. WB was utilized to detect the expression of apoptosis- and ERS-linked proteins, and the activity of the SIRT1/AMPK signaling pathway. Results: Six key genes (CTSK, FURIN, SLC2A1, RSAD2, SGPP2, and STAT3) were identified through bioinformatics analysis, with SGPP2 showing the most significant differential expression. Additionally, SGPP2 was found to be downregulated in IHF. Single-gene enrichment analysis showed that SGPP2 exhibited a significant association with the AMPK signaling pathway. Animal experiments demonstrated that rats with IHF exhibited significantly impaired cardiac function, marked myocardial tissue injury and fibrosis, ERS in myocardial tissue, lowered SGPP2 expression, and decreased SIRT1/AMPK signaling pathway activity. In vitro experiments confirmed that SGPP2 overexpression alleviated OGD-induced cardiomyocyte injury by inhibiting ERS and simultaneously activating the SIRT1/AMPK signaling pathway. Rescue experiments further demonstrated that both Tu and EX527 significantly promoted ERS and cellular injury, thereby counteracting the protective effects of SGPP2. Conclusions: SGPP2 alleviates IHF by inhibiting ERS modulated by the SIRT1/AMPK pathway. Full article

Reticuloendotheliosis virus (REV) is an immunosuppressive virus in poultry that can cause acute reticular neoplasms, chronic lymphoid tumors, stunting syndrome, and secondary infections. In many countries, the lack of effective vaccines has resulted in a high prevalence of REV infections and substantial economic losses. Enzyme-linked immunosorbent assay (ELISA)-based antibody detection is an important tool for monitoring the REV prevalence in poultry farms. ELISA coating antigens generally consist of either whole virus or viral protein; however, most commercially available REV antibody ELISA detection kits use whole virus as the coating antigen, which limits their applicability in certain diagnostic and research settings. In this study, the gp90 protein from a dominant REV strain was expressed and purified using 293F suspension cell eukaryotic expression system. Using recombinant gp90 protein as the coating antigen, an indirect ELISA for detecting gp90 antibodies (gp90-ELISA) was developed. After optimization, the optimal conditions were as follows: coating antigen concentration of 4 µg/mL with overnight incubation at 4 °C; blocking with 5% skim milk at 37 °C for 1.5 h; serum dilution of 1:200 with incubation at 37 °C for 45 min; secondary antibody dilution of 1:1000 with incubation at 37 °C for 30 min; and color development using TMB substrate at room temperature in the dark for 10 min. The cut-off value was defined as an OD₄₅₀ ≥ 0.22 for positive samples and <0.22 for negative samples. The developed gp90-ELISA specifically detected REV-positive sera at a maximum serum dilution ratio of 1:3200. Intra- and inter-assay variation coefficients were ≤10%, indicating that the gp90-ELISA had good specificity, sensitivity, and reproducibility. Laboratory serum testing showed that the gp90-ELISA successfully detected sera from chickens immunized with the gp90 protein or infected with REV. Furthermore, analysis of clinical serum samples demonstrated 100% concordance between the gp90-ELISA results and a commercial whole-virus-coated ELISA kit. These results indicate that the gp90-ELISA is a reliable supplementary method to whole-virus-coated ELISA and has potential utility in disease surveillance and evaluation of immune responses. Full article