Search Results (3,135)

Although canine parvovirus (CPV) vaccination has been widely implemented, CPV continues to circulate in dog populations and poses a potential cross-species transmission risk to wildlife, including giant pandas. Recent increases in CPV-2c detection in China highlight the need for molecular surveillance and standardized immunoreagents for diagnosis and epitope mapping. This study aimed to isolate a representative CPV-2c strain from China and develop VP2-targeted nanobody-based recognition molecules to support antigen monitoring and detection optimization. Canine and giant panda samples were collected in Sichuan Province, and CPV was isolated in F81 cells, followed by VP2 gene sequencing and phylogenetic analysis. A secretion expression system in Bacillus subtilis was established to produce VP2-targeting nanobodies, and a canine Fc-fused format of Nb10 (Nb10-Fc) was constructed. Immunoreactivity was evaluated via immunoassays, and structural modeling and molecular docking were performed to predict binding interfaces. The results showed that CPV-2c was the dominant genotype in Sichuan, with CPV L4 being a representative strain that exhibited 100% identity in VP2 with a giant panda-derived CPV-2c strain. Nb10 and Nb10-Fc demonstrated strong reactivity in Western blotting and immunofluorescence assays. The Fc-fusion improved detection sensitivity, offering potential in vivo application benefits. This study provides a standardized VP2-specific nanobody and molecular system for CPV-2c surveillance, antigenic studies, and diagnostic optimization. Full article

Wild mushroom poisoning remains a major medical and toxicological challenge worldwide because of the diversity of toxic compounds, the broad spectrum of clinical manifestations, and the risk of severe hepatic or renal injury. Early differentiation between self-limiting gastrointestinal syndromes and potentially fatal intoxications with progressive organ failure remain a central clinical challenge. This review examines recent advances in the diagnosis, risk stratification, and therapeutic management of wild mushroom poisoning, with amatoxin intoxication serving as the principal clinical focus. Selected evidence from other mushroom toxic syndromes is also included to support differential diagnosis, highlight syndrome-specific variability, and provide comparative clinical and methodological context. The recent literature indicates a shift from predominantly symptom-based diagnosis toward integrated models combining clinical evaluation, laboratory biomarkers, toxicological testing, and analytical and molecular methods. Liquid chromatography, mass spectrometry, immunoassays, and the molecular identification of fungal species have improved diagnostic precision, particularly in cases with uncertain exposure history or delayed presentation. Current management relies on early multimodal strategies including intensive supportive care, targeted pharmacological interventions, extracorporeal detoxification, and, in selected severe cases, liver transplantation. Overall, clinical outcome depends not only on toxin profile, but also on timely diagnosis, accurate early risk stratification, and prompt coordinated treatment. Future research should prioritize standardized diagnostic pathways, validated prognostic models, and clinically applicable treatment algorithms that support earlier escalation of care in severe mushroom intoxication. Full article

Background: Osteoarthritis (OA) is a heterogeneous joint disorder traditionally considered mechanically driven; however, evidence indicates that inflammatory mechanisms contribute to symptom expression. Exploratory analyses of peripheral biomarkers may provide insights into systemic inflammation in symptomatic knee OA, but formal phenotypic validation requires dedicated clustering or longitudinal studies. Objective: To examine associations between clinical pain, functional impairment, and circulating inflammatory biomarkers in patients with knee OA compared with healthy controls. Methods: In this prospective, single-center study, patients aged 40–80 years with radiographically confirmed knee OA and chronic knee pain were compared with age- and sex-matched healthy controls. Pain intensity and functional status were assessed using the Visual Analogue Scale (VAS) and the Knee Injury and Osteoarthritis Outcome Score (KOOS). Circulating inflammatory biomarkers, including cytokines and matrix metalloproteinases, were quantified using multiplex immunoassays. Statistical analyses included adjusted linear regression models, with age and BMI as covariates, and multiple testing correction using the Benjamini–Hochberg procedure (FDR alpha error 5%). Results: OA patients exhibited higher circulating levels of TNF-α, IL-6, IL-8, MMP-1, MMP-3, TNFSF13, TNFSF13B, and pentraxin-3 compared with controls (p < 0.01). No significant sex differences were observed. KOOSs correlated with IL-6 and IL-10 levels, suggesting an association between systemic inflammatory activity and functional limitation. All findings are presented as exploratory and associative. Conclusions: Patients with knee OA display systemic inflammatory biomarker differences associated with pain and functional impairment. These results support the role of inflammation in OA symptoms within an exploratory framework. Larger, longitudinal studies are warranted to validate these observations. Full article

Functional antibody-dependent enhancement (ADE) represents a fundamental and context-dependent characteristic of antiviral antibody responses, reflecting the dual capacity of antibodies to mediate both the neutralization and Fc receptor-dependent enhancement of infection. In flavivirus research, this duality complicates the interpretation of conventional serological metrics and limits the reliability of single-parameter correlates of immunity, particularly in populations with complex exposure histories. Over the past decade, functional ADE assays have evolved from specialized mechanistic tools into integrated immune assessment platforms supporting translational immunology, vaccine evaluation, and population-level immune surveillance. These platforms incorporate Fcγ receptor-relevant target cell systems, standardized viral inputs, dilution series-based profiling, quantitative enhancement metrics, and structured quality control frameworks to enable reproducible, comparable, and context-aware functional measurements across cohorts and laboratories. A central concept emerging from these developments is that ADE reflects a dynamic functional immune state rather than an intrinsic property of antibodies or a direct indicator of pathological risk. Accordingly, functional ADE platforms support the contextual interpretation of antibody activity across physiologically relevant conditions, facilitating discrimination between transient functional enhancement and clinically meaningful immunological risk. By integrating functional ADE metrics with serological, cellular, and epidemiological data, these platforms provide a structured framework for interpreting immune profiles in vaccine evaluation, booster strategy design, and population-level risk stratification. This review synthesizes the development, standardization, and global dissemination of functional ADE platforms and discusses key principles governing biological relevance, analytical robustness, and inter-site transferability. Emerging directions integrating functional ADE profiling with systems immunology, immunogenomics, and computational modeling are highlighted as pathways toward predictive, decision-support-oriented frameworks. By positioning ADE platforms as immune assessment infrastructures rather than isolated assays, this review underscores their value for mechanistic inquiry, translational interpretation, and preparedness-oriented responses to emerging viral threats in the absence of definitive correlates of protection. Full article

Biomarker detection demands low cost, rapid turnaround, interference resistance, and wide dynamic range. However, traditional immunoassays and nucleic acid amplification methods remain constrained by complex matrices, batch stability, and portability limitations. Molecularly imprinted polymers (MIPs) exhibit “artificial antibody”-like specific recognition and high stability, while nanomaterials (NMs), depending on their composition, structure, and interfacial organization, can provide conductive pathways, catalytic activity, high-density loading sites, or mass-transfer-favorable architectures. Electrochemical biosensors synergistically constructed from these two components achieve complementary functions in recognition, mass transfer, and signal transduction. This paper systematically reviews key strategies and mechanisms for NM–MIP synergistic construction, focusing on six synergistic strategies that target key bottlenecks in mass transfer, signal generation, and interfacial stability: dynamic response regulation, hierarchical structural engineering, anti-fouling interfaces, multi-signal cross-validation, catalytic–recognition integration, and interfacial binding regulation. Representative biomarker cases are analyzed to illustrate how functional modules can coordinate across sample processing, signal generation, and recognition confirmation to improve analytical reliability and overall sensing performance. Finally, the review discusses challenges in clinical translation, including consistent manufacturing, matrix interference, long-term stability, and standardized validation, while outlining future directions toward mechanism-guided imprint design, intelligent data-assisted optimization, and integration with microfluidic and wearable platforms for multiplexed biomarker detection. Full article

Background: Thyroid hormones influence bone metabolism, and autoimmune thyroid diseases may further impact skeletal homeostasis. Wnt signaling inhibitors, including Dickkopf-1 (DKK-1) and sclerostin (SOST), as well as osteoprotegerin (OPG), play key roles in regulating bone formation and resorption. This study aimed to evaluate circulating DKK-1, SOST, and OPG in women with newly diagnosed overt thyroid dysfunction. Methods: This cross-sectional study included 62 women with newly diagnosed, untreated overt thyroid dysfunction (35 hypothyroid and 27 hyperthyroid) and 33 age- and BMI-matched healthy controls. Serum levels of DKK-1, sclerostin, and OPG were measured using ELISA. Thyroid function and autoantibodies were assessed using automated immunoassays. Correlation analysis was performed to evaluate associations between variables. Results: Serum DKK-1 levels were significantly elevated in both hypothyroid and hyperthyroid women compared with controls (p < 0.001). Sclerostin levels showed a non-significant trend toward higher values. OPG levels were significantly increased in hyperthyroid patients and moderately elevated in hypothyroid patients. Significant positive correlations were observed between OPG and FT3 (r = 0.42, p = 0.001) and FT4 (r = 0.43, p = 0.001). In hypothyroid patients, OPG correlated positively with TgAb (r = 0.46, p = 0.007). A strong positive correlation was found between DKK-1 and SOST (p < 0.001), while DKK-1 was negatively associated with age (p < 0.05). Conclusions: Overt thyroid dysfunction is associated with significant alterations in circulating Wnt signaling inhibitors and OPG. These findings suggest a potential role of Wnt signaling and immune–bone interactions in thyroid-related changes in bone metabolism. Full article

Rapid antigen tests targeting SARS-CoV-2 nucleocapsid protein were essential for decentralised testing during the COVID-19 pandemic. Independent performance evaluations are essential to support regulatory approval and inform clinical implementation, particularly in resource-limited settings. This study presents a retrospective analytical and operational evaluation of two instrument-based fluorescent immunoassays (FIAs): the PCL COVID-19 Ag Rapid FIA and LumiraDx SARS-CoV-2 Ag Test. Analytical sensitivity was determined using recombinant nucleocapsid protein and viral cultures. Clinical performance was assessed using residual clinical specimens (n = 110) with RT-PCR as a reference, stratified by cycle threshold (Ct). Operational characteristics were assessed using a structured Likert framework. Overall sensitivity was 63% (51–73) for PCL and 95% (88–99) for LumiraDx. For Ct ≤ 25, sensitivity increased to 93% and 100%. Specificity was ≥97% for both. LumiraDx maintained sensitivity (83–94%) at Ct 25–30, whereas PCL did not detect any positives in this range. The limit of detection was 39 pM (PCL) and 0.6 pM (LumiraDx). Operational usability was high for both (90% PCL, 87% LumiraDx). LumiraDx showed higher analytical sensitivity across a broader viral load range, supporting primary diagnostic use, whereas PCL was limited to high viral loads. This evaluation provides a reproducible framework for rapid diagnostic assessment during emerging outbreaks. Full article

The COVID-19 pandemic highlighted the critical role of serological assays in understanding antiviral immune responses, monitoring vaccine efficacy, and informing public health strategies. This review provides a comprehensive overview of commonly used SARS-CoV-2 antibody detection methods, focusing on binding and neutralization assays. Antibody binding assays, including enzyme-linked immunosorbent assays (ELISAs), chemiluminescence immunoassays (CLIAs), lateral flow immunoassays (LFAs), and multiplex platforms, enable the rapid and high-throughput detection of immunoglobulin isotypes against various viral antigens. Neutralization assays, including live-virus, pseudovirus (PsV), and surrogate assays, offer functional insights into the ability of antibodies to prevent viral entry, though they often require higher biosafety levels and optimization. Serological assays, primarily antibody binding assays and several surrogate neutralization assays, received Emergency Use Authorization (EUA) during the pandemic, supporting seroprevalence efforts. Antibody binding assays and neutralization assays were also widely used in vaccine immunogenicity studies. Despite many standardization initiatives, assay standardization and data harmonization remain challenging and require further efforts. The choice of assay should be guided by study goals: antibody binding assays are preferred for high-throughput monitoring and epidemiological studies, while neutralization assays are essential for assessing functional immunity and variant-specific neutralization and protection. Full article

Background: Despite hybrid and vaccine-induced immunity, SARS-CoV-2 continues to cause disease. The characterization of humoral and cellular immune responses is essential for guiding prevention strategies and booster dose policies; Methods: A prospective cohort study was conducted with 131 hospitalized patients with confirmed COVID-19 in the Aburrá Metropolitan Valley, Colombia. Clinical and immunological data were evaluated on days 1–3, days 5–7, days 8–12, and 4–5 months after diagnosis. Humoral immunity was assessed by enzyme-linked immunosorbent assay (ELISA), chemiluminescent microparticle immunoassay (CMIA), and neutralization testing, and cellular immunity by CD4⁺/CD8 T-cell responses. Results: vaccinated patients had higher baseline levels of IgG and neutralizing antibody positivity than unvaccinated patients (ELISA 89.1% vs. 60.0%; CMIA 86.4% vs. 50.0%; neutralizing antibodies 88.2% vs. 65.0%), but cases of severe disease occurred in both groups. Adults aged ≥65 years had higher antibody positivity, but severe disease persisted. Mortality at 28 days was 7.6%, mainly among critically ill patients with comorbidities. Antibodies persisted at 4–5 months but were lower in those with severe acute disease. Those who received the booster dose showed stronger CD4⁺/CD8⁺ activation (notably against the Omicron variant) than unvaccinated/partially vaccinated patients. Conclusions: Vaccination improved humoral and cellular responses, but severe breakthrough infections still occurred, particularly in high-risk patients. Full article

Background: Pancreatic cancer is an aggressive malignancy with poor survival. Most patients are diagnosed at advanced or metastatic stages because early disease is often asymptomatic and effective screening tools are lacking. We evaluated a three-marker model comprising serum CA19-9 in combination with the plasma proteins ITIH3 and CEACAM1 for pancreatic ductal adenocarcinoma (PDAC) detection. Methods: Matched plasma and serum samples were collected from 649 participants (250 PDAC cases and 399 controls). Plasma proteins were enriched using high-surface area magnetic covalent organic framework (COF) polymers. Serum CA19-9 was measured using the Tosoh Bioscience immunoassay. The marker panel was trained using a radial-based SVM with repeated 10-fold cross-validation using a set-aside training sample set. The derived model along with a fixed cutoff corresponding to 95% sensitivity in training samples were independently validated using a blinded sample set. Results: In the independent blinded validation, the combined panel of serum CA19-9 with plasma ITIH3 and CEACAM1 achieved an AUC of 0.917 indicating that the three-marker panel maintained strong performance in distinguishing PDAC from controls. At the prefixed threshold, the three-marker panel had a specificity of 53.3% (95% CI: 46.8–59.7%), significantly outperforming CA19-9 alone at 14.5% (95% CI: 10.4–19.7%). Conclusions: In independently blinded validation, combining plasma ITIH3 and CEACAM1 with serum CA19-9 substantially improved diagnostic performance for PDAC, achieving high specificity while maintaining 95% sensitivity compared with serum CA19-9 alone. These findings support further validation of this three-marker panel as a potential PDAC monitoring and detection approach in larger, multicenter studies. Full article

Background/Objectives: Glycated hemoglobin (HbA1c) is widely used to monitor glycemic control, but the accuracy and flag detection rates of HbA1c assays can vary in the presence of Hb variants such as Hb G-Coushatta (Coushatta) and Hb Queens (Queens), which are common in the Korean population. Methods: We evaluated four HbA1c platforms—Arkray ADAMS HA-8190V (HA-8190V) fast/variant modes; Tosoh HLC-723 G11 (G11) standard/variant modes; Bio-Rad D-100 (D-100); Sebia Capillarys 2 Flex Piercing (Capillarys)—using 33 Hb variant samples (26 Coushatta, 7 Queens). The Roche Tina-quant HbA1c Gen. 3 immunoassay was used as the comparative method. With UPLC-MS/MS used as the reference for variant identification, analytical performance was assessed by calculating mean % differences in HbA1c and evaluating Hb variant flag detection rates. Results: The mean % differences in HbA1c compared to the comparative method were −1.5% and −0.9% for Coushatta and Queens, respectively, with HA-8190V variant mode; −28.4% and −17.6% with HA-8190V fast mode; +33.5% and +2.2% with the G11 variant mode; −28.1% and −14.3% with G11 standard mode; −5.9% and −3.2% with D-100; and +1.0% and −6.2% with Capillarys. For flag detection, rates were 100% and 0% with HA-8190V fast mode; 84.6% and 28.6% with G11 standard mode; and 100% and 100% with all other platforms. Conclusions: Coushatta caused severe underestimation in rapid modes, while Queens hindered automated detection. While variant modes significantly improved detection, they showed platform-dependent accuracy. When Hb variant status is unknown, use of the variant mode is recommended to ensure reliable results. Full article

Endometriosis is a chronic inflammatory disease in which transforming growth factor-beta (TGF-β) has been implicated in immune dysregulation, extracellular matrix remodeling, and fibrosis. Data on baseline secretion of TGF-β isoforms by systemic immune cells remain limited. This pilot study quantified unstimulated secretion of TGF-β1, TGF-β2, and TGF-β3 by peripheral blood mononuclear cell (PBMC) cultures from women with and without endometriosis and explored stage-related patterns. In this prospective case–control study, PBMCs from 50 women with surgically confirmed endometriosis and 30 controls were cultured for 24 h without exogenous stimulation. Supernatant concentrations were measured using a multiplex bead-based immunoassay (Bio-Plex, Bio-Rad) and expressed as pg/mL; between-group and stage-related differences were assessed using non-parametric tests. Median 24 h secretion was similar between groups (TGF-β1: 103,816 vs. 114,700 pg/mL, p = 0.25; TGF-β2: 3735 vs. 3732 pg/mL, p = 0.32; TGF-β3: 3280 vs. 3284 pg/mL, p = 0.70). Within the endometriosis cohort, TGF-β2 was significantly higher in moderate/advanced disease (rASRM stages III–IV) than in minimal/mild disease (stages I–II), whereas TGF-β1 and TGF-β3 did not reach statistical significance for a stage-dependent pattern in this pilot cohort (p = 0.42 and p = 0.41, respectively; Kruskal–Wallis), and a type II error cannot be excluded given the small sample size per rASRM (revised American Society of Reproductive Medicine)stage (n = 11–14). These findings suggest that TGF-β dysregulation is compartmentalized to the peritoneal environment rather than systemically imprinted in circulating immune cells. The stage-dependent elevation of TGF-β2 supports its role in progressive fibrogenesis and as a candidate severity biomarker, warranting confirmation in larger, stimulus-augmented studies. Full article

Background: Linezolid is widely used for the empirical and targeted treatment of Gram-positive infections. Elderly patients frequently exhibit substantial pharmacokinetic variability due to age-related physiological changes and high comorbidity burden, which may predispose to drug accumulation and toxicity. This study aimed to develop and evaluate a population pharmacokinetic (PopPK) model of intravenous (IV) linezolid in elderly patients (65–87 years) to support therapeutic drug monitoring and explore exposure-risk scenarios associated with overexposure. Methods: A retrospective single-center study including 149 patients and 293 serum trough concentrations was conducted. Patients were randomly assigned to development (n = 103) and independent validation (n = 46) cohorts. Linezolid concentrations were quantified using an enzyme immunoassay. The PopPK model was developed in NONMEM^® using FOCE-I. Model performance was evaluated using standard diagnostic plots, bootstrap analysis, visual predictive checks, and validation metrics (mean prediction error [MPE] and mean absolute prediction error [MAPE]). Monte Carlo simulations assessed the probability of overexposure (Cmin > 8 mg/L) and the probability of target attainment (PTA; AUC24/MIC ≥ 100) under standard dosing (600 mg IV every 12 h). Results: Linezolid pharmacokinetics were best described by a one-compartment model with first-order elimination. Estimated glomerular filtration rate, treatment duration, and age were identified as significant predictors of clearance. Internal and independent validation confirmed the robustness and predictive performance of the model. Simulations showed a high probability of overexposure in patients with impaired renal function, particularly during prolonged treatment. Conclusions: Renal function, age, and treatment duration are major determinants of linezolid exposure in elderly patients. Standard dosing frequently results in overexposure, supporting early therapeutic drug monitoring and individualized dose adjustment in this vulnerable population. Full article

Background/Objectives: Work stress (WS), occupational fatigue (OF), and Burnout syndrome (BS) among administrative workers are associated with negative psychosocial and metabolic effects. Although antioxidant-rich nutritional strategies have been proposed to help manage stress, evidence from real-world occupational settings is still limited. This study evaluated the total antioxidant capacity (TAC) of a multi-antioxidant dietary supplement 2.0 (DS2.0; apple polyphenols, [APP], astaxanthin [AXT], and fucoxanthin [FXT]; 387:12:1 ratio) and explored its association with metabolic parameters, OF, psychosocial outcomes, and hair cortisol concentration (HCC) in administrative workers with and without obesity. Methods: A quasi-experimental pilot study was conducted among 22 workers, who received DS2.0 (52.13 mg/day, n = 17) or a placebo (n = 5) for 30 days. TAC was analytically assessed using standardized assays. Metabolic outcomes (lipid profile, fasting plasma glucose), psychosocial variables (SOFI-SM, CESQT/SBI, and IMSS tests), and HCC (competitive immunoassay) were evaluated before and after supplementation. Statistical analyses included within-group pre–post comparisons, independent-sample tests, and effect size estimation. Results: DS2.0 demonstrated high TAC. Supplementation was associated with reductions in total lipids, total cholesterol, and non-HDL cholesterol, as well as decreases in OF, BS, and WS scores. HCC decreased in the overall sample (217.19 vs. 31.64 pg/mg; p = 0.000) and among workers with obesity (276.80 vs. 34.13 pg/mg; p = 0.002). Stress-related symptoms, including sleep deprivation, exhaustion, appetite changes, difficulty waking, and palpitations, also improved (p ≤ 0.05). Conclusions: An antioxidant-rich DS2.0 supplement may be associated with psychosocial and stress-related biomarkers; however, these exploratory findings require confirmation in larger randomized controlled trials. Trial registration: ISRCTN 12762846. Full article

Background/Objectives: The prevalence of allergic diseases has markedly increased in developed countries, with environmental and dietary factors considered important contributors. Folic acid is an essential micronutrient involved in one-carbon metabolism and DNA methylation, playing a key role in epigenetic regulation of immune function. Both high and low folate exposure have been associated with allergic outcomes, but the data on postnatal folate status in paediatric populations remain limited. This study aimed at assessing serum folate status in children with atopic diseases compared with children without chronic allergic disease in Croatia. Methods: This cross-sectional study included 292 paediatric patients from the University Hospital in Split and a paediatric primary care practice between January 2024 and January 2025. Serum folic acid concentrations were measured using electrochemiluminescence immunoassay. Additional laboratory parameters included vitamin B12, total IgE levels, and eosinophil counts. Demographic and clinical data were obtained from medical records. Statistical analyses included Chi-square tests, Mann–Whitney U tests, linear regression modelling, and analysis of covariance with statistical significance set at p < 0.05. Results: Folic acid deficiency was present in 66.4% of all participants. Children with atopic diseases were significantly more likely to have folate deficiency and had lower mean serum folate concentrations compared to children without allergic disease. There were no significant differences in folate levels between children with and without asthma. Lower folate levels were associated with higher IgE levels, higher eosinophil counts, and older age. When controlling for the effects of age on folic acid levels, the differences between participants with and without atopic diseases remained significant. Conclusions: Folic acid deficiency is highly prevalent among children in the Mediterranean region of Croatia and is significantly associated with atopic diseases and markers of allergic inflammation. These findings highlight a potential role of folate status in paediatric allergic disease and support the need for longitudinal studies to clarify causality and potential clinical implications. Full article