Search Results (1,511)

Diagnosis of rickettsial infections is challenging due to nonspecific clinical symptoms and limitations of current diagnostic methods. Molecular assays allow early detection but are limited by cost and technical demands, whereas conventional serological tests often exhibit cross-reactivity and low sensitivity during the early stages of infection. This study aimed to develop and evaluate a recombinant-antigen sandwich ELISA for improved antibody detection against Rickettsia spp. Three Rickettsia akari proteins, rGroEL, rDnaK, and rA8GP63 (uncharacterized protein), were produced and validated for immunogenicity. The assay was evaluated using 94 patient serum samples, including those with positive, negative, and unknown clinical course. The optimized ELISA demonstrated high reproducibility, with IgG sensitivity of 89.47–95.39% and specificity of 90%. IgM detection, also assessed, showed lower sensitivity (42.11–82.89%) but maintained strong specificity (83.33%). The diagnostic performance was comparable to that of a commercial indirect immunofluorescence assay, with no cross-reactivity detected in sera from patients with unrelated infections. rDnaK and rA8GP63 represent newly explored diagnostic candidates. These findings highlight the potential of this recombinant protein-based ELISA as an accessible, sensitive and specific diagnostic tool, with a meaningful clinical impact for improving the early and accurate detection of rickettsial infections. Full article

Johne’s disease (JD) is a chronic infection of cattle that undermines herd productivity and profitability. While test-and-cull programs are commonly proposed for control, their effectiveness and economic feasibility remain uncertain in beef production systems. This study used an updated agent-based model (ABM) to simulate JD transmission in a representative 300-cow Western Canadian beef herd, coupled with a partial budget model to evaluate net present value (NPV) over a 10-year time horizon. Seven diagnostic test-and-cull strategies were compared, varying in test type (ELISA, individual PCR, and pooled PCR), sampling frequency (6, 12, or 24 mo), and risk-based sampling protocols. Results showed that, under baseline assumptions (6% starting prevalence; 1% prevalence in purchased stock), all strategies reduced JD prevalence relative to no testing, and six of seven yielded higher NPVs. Annual individual PCR testing provided the best balance between prevalence reduction and profitability, whereas semi-annual PCR most effectively reduced prevalence but at greater economic cost. Failure to implement control measures resulted in increasing prevalence and long-term economic losses. Sensitivity analyses demonstrated that strategy performance was consistent across variations in market conditions, cost of production, and replacement female management, although profitability declined substantially when JD prevalence in externally sourced stock was high (i.e., 10%). Collectively, these findings indicate that JD can be controlled economically in beef herds, with long-term application of various test-and-cull strategies offering robust options adaptable to management preferences. Full article

The role of ethylene in the adaptation of Arabidopsis thaliana to salt stress induced by 150 mM NaCl is investigated. The responses of wild-type (Columbia, WT) plants and ethylene-insensitive etr1-1 mutants to short-term daily salt treatments were compared. Parameters analyzed included growth, water status, chlorophyll content, and hormone levels (ABA, IAA, cytokinins) using ELISA and immunohistochemistry. The results revealed that in the WT, salt stress induced hormonal redistribution: accumulation of ABA, IAA, and zeatin in shoots, accompanied by decreased ABA in the root tips and cytokinins in the whole roots. These hormonal changes were associated with stomatal closure, maintained leaf hydration, and inhibition of root growth. The inhibition of root growth may contribute to reduced uptake of toxic ions from the environment. In contrast, etr1-1 mutants exhibited no changes in hormonal status, failed to close stomata—leading to decreased leaf water content—and showed a sharp decline in chlorophyll content accompanied by suppressed shoot growth. The conclusions emphasize that ethylene sensitivity is essential for initiating adaptive hormonal rearrangements that coordinate growth and stomatal responses to mitigate the effects of salt stress. Full article

Oz virus, an emerging tick-borne thogotovirus, has been reported to cause fatal human infection in Japan. However, its ecology and geographic distribution remain largely unknown. In this study, we developed a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS ELISA) for detecting Oz virus antibodies in animals and used it to conduct a seroepidemiological survey of wild boars (Sus scrofa) in Miyazaki Prefecture, Japan. Recombinant Oz virus nucleoprotein was expressed in E. coli and used as both the capture and detection antigen. Relative to the neutralization test, the DAgS ELISA showed a sensitivity of 72.2%, a specificity of 88.2%, and an overall concordance rate of 79.0%. We used this assay to examine 1045 wild boar serum samples collected between November 2022 and May 2025, finding a seroprevalence of 33.5%. The seroprevalence did not significantly differ by sex, age, or region, but showed significant seasonal variation, peaking in summer (p < 0.0001). Oz virus RNA was detected by quantitative RT-PCR in one serum sample (0.09%). Phylogenetic analysis of the partial Oz virus glycoprotein gene showed that this strain shared 98.8% nucleotide identity with the EH8 strain, which was the first Oz virus isolate obtained from ticks in Ehime Prefecture. These findings suggest that wild boars in Miyazaki are frequently exposed to Oz virus and that ticks in the region harbor the virus. However, no human cases have been reported to date. The DAgS ELISA developed in this study provides a practical tool for serological surveillance in animals. Continuous monitoring of animal populations is warranted to clarify the epidemiology of Oz virus in the region and to identify potential reservoir species. Full article

Background/Objectives: Postoperative organ complications following open thoracoabdominal aortic aneurysm (TAAA) repair pose significant challenges during the early postoperative period, where prompt detection is crucial for improving patient outcomes. Sepsis is often a central factor in these complications. This study investigates the perioperative dynamics of soluble urokinase plasminogen activator receptor (suPAR) plasma levels in TAAA patients undergoing elective surgical repair and evaluates its diagnostic potential for early detection of postoperative sepsis. Methods: In this retrospective, single-center study, 28 patients (mean age 52.6 ± 13.4 years; 67.9% male) underwent elective open TAAA repair between 2022 and 2024. Blood samples were collected at five perioperative time points, and suPAR levels were measured using ELISA. The primary endpoint was the onset of postoperative sepsis, with secondary endpoints including other organ complications. The predictive performance of suPAR levels was evaluated using Receiver Operator Characteristics (ROC) analysis. Results: Postoperative sepsis developed in 7 of 28 patients (25%), with the diagnostic criteria met at a mean of 9.7 ± 6.9 days. Baseline suPAR levels did not differ between groups; however, from 12 h after surgery, the sepsis group exhibited significantly higher serum concentrations (14.43 ng/mL vs. 7.23 ng/mL; p = 0.004), a difference that persisted throughout the first 24 h. At 24 h, suPAR had the highest predictive accuracy for sepsis, with an AUC of 0.90, 90% sensitivity, and 86% specificity at a 9 ng/mL cut-off (p < 0.001). Conclusions: Elevated suPAR levels in the early postoperative period are strongly associated with the later onset of sepsis. Early monitoring may enable timely intervention, potentially improving outcomes in this high-risk patient population. Full article

Background/Objectives: Early detection of pancreatic ductal adenocarcinoma (PDAC) can improve patient survival and biomarkers to facilitate this are greatly needed. We recently reported a serum biomarker signature comprising tissue inhibitor of metalloproteinase 1 (TIMP1), intercellular adhesion molecule 1 (ICAM1), cathepsin D (CTSD), thrombospondin 1 (TSP1/THBS1), and carbohydrate antigen 19-9 (CA 19-9), that detected Stage I and II PDAC with high sensitivity and specificity. In this assay, CA 19-9 is measured with a commercial instrument and individual ELISAs were developed to measure TIMP1, ICAM1, CTSD, and THBS1. Here, we report the analytical performance of these four analytes in their ELISA formats. Methods: Biomarker precision, linearity, algorithm precision, matrix effects, hook effect, method comparison, interference, and analyte stability were evaluated against acceptance criteria per CLSI guidelines. Results: High, medium, and low concentrations of each biomarker met acceptance criteria for inter- and intra-day precision (%CVs < 14%) and for linearity (%CVs < 11%). Matrix effects did not impact quantitation of any analyte nor was hook effect present. All analytes met acceptance criteria for accuracy and stability (all biases < 11.2% and <16.5%, respectively). For interference, two CTSD measurements and one ICAM1 measurement in HAMA-spiked samples showed 20.7–29% biases, falling slightly outside of acceptance criteria (<20% bias). All other analyte concentrations met interference acceptance criteria. In total, 94.1% of all diagnostic calls were made with 100% certainty, indicating high precision of the assay’s algorithm. Conclusions: All analytes demonstrated acceptable analytical precision, linearity, accuracy, and stability, showing high overall analytical performance of each analyte. Full article

Enzyme-Linked-Immunosorbent-Spot (ELISpot) is a highly sensitive technique capable of detecting low-level immune responses, offering critical insights into therapy-induced immune activation. Our mouse interferon-gamma (mIFN-γ) ELISpot assay was originally based on a monoclonal capture antibody and a rabbit polyclonal detection antibody. The objective of our study was to replace the polyclonal detection antibody with a monoclonal alternative, using a llama immune library and phage display technology. A llama was immunized with recombinant mIFN-γ, and an immune VHH library was constructed. The library underwent two rounds of panning using the recombinant antigen. Subsequently, 190 clones were screened by Enzyme-Linked-Immunosorbent Assay (ELISA), yielding 27 specific binders to mIFN-γ. Sequence analysis revealed 24 unique clones grouped into four families based on their CDR3-VH sequences. One representative clone from each family was reformatted as VHH-Human Fragment Crystallizable (VHH-hFc) fusion and produced recombinantly for testing in the ELISpot assay. The purified candidates were evaluated in pairs on native mIFN-γ from mouse splenocytes. Two candidates, H3 and G4, were selected for further trial. Comparative analysis of ELISpot performance showed that G4 is a promising substitute for the original rabbit polyclonal antibody, enhancing the overall performance of the mIFN-γ ELISpot assay. This study highlights the potential of VHH antibodies in ELISpot applications and supports their use as a robust, reproducible alternative to polyclonal antibodies. Full article

Background: Periodontitis is associated with systemic inflammation; however, the relationship between disease severity and systemic inflammatory biomarkers remains unclear. This study aimed to evaluate the association between periodontitis stage and grade with systemic levels of C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) and assess changes following standardized non-surgical periodontal therapy. Methods: Patient records from the University Dentistry Clinical Center of Kosovo were reviewed. Periodontitis was classified using the 2018 staging and grading system. Periodontal parameters (probing pocket depth, clinical attachment loss, bleeding on probing, plaque index, and gingival index) were assessed at six sites per tooth (excluding third molars). Serum levels of IL-1β, IL-6, TNF-α, and high-sensitivity CRP were measured before and after therapy using high-sensitivity ELISA. Blood samples were centrifuged, and serum was stored at −20 °C. All patients underwent standardized non-surgical periodontal therapy, including full-mouth scaling and root planning, without systemic antibiotics. Data were analyzed using SPSS v22.0. Results: Among the patients, 28.0% had Stage I–II, 40.0% Stage III, and 32.0% Stage IV periodontitis; 29.3% were Grade A, 45.3% Grade B, and 25.3% Grade C. At baseline, all systemic inflammatory biomarkers (CRP, IL-1β, IL-6, and TNF-α) were significantly higher in periodontitis patients compared with the control group, indicating an increased systemic inflammatory burden before therapy. After therapy, significant reductions in CRP, IL-1β, IL-6, and TNF-α were observed across all stages and grades (all p < 0.01), indicating a decrease in systemic inflammatory burden. Conclusions: Non-surgical periodontal therapy significantly lowers systemic inflammatory biomarkers regardless of periodontitis severity, supporting their role as indicators of disease activity and treatment response. Full article

Background: Heparin-induced thrombocytopenia (HIT) is a rapid, life-threatening adverse drug reaction, with the widely used 4T score being the critical clinical tool guiding the need for serological confirmation. This study aimed to validate the diagnostic utility of the 4T score and evaluate its application in a tertiary hospital in Saudi Arabia. Methods: In this retrospective cohort study, we analyzed 449 patients with suspected HIT, comparing their initially recorded clinical 4T scores with those recalculated by trained specialists, using the anti-PF4/heparin IgG ELISA as the reference standard for HIT confirmation. Results: Of the 292 patients who underwent laboratory testing, the HIT positivity rate was 6.5% (n = 19). The primary finding was a markedly low agreement (15.4%) between the system-recorded and the standardized physician-calculated 4T scores, indicating significant inter-observer variability. Despite this, higher calculated 4T scores remained significantly associated with positive HIT test results, and the anti-PF4/heparin IgG ELISA demonstrated 100% sensitivity and 100% negative predictive value (NPV). Conclusions: While the 4T score is indispensable for guiding the diagnostic pathway, the observed profound inter-observer variability highlights an urgent need for standardized scoring training and protocol refinement to enhance diagnostic accuracy and reduce inappropriate therapeutic interventions. Full article

Background and Objectives: Endometrial carcinoma is among the most common gynecological malignancies, with recurrence and chemoresistance remaining major clinical challenges. This study aimed to evaluate the combined effects of Boswellic acid (BA), a natural pentacyclic triterpene, and Gemcitabine (GEM), a nucleoside analog chemotherapeutic, on hypoxia, angiogenesis, and apoptosis in human endometrial cancer cells. Materials and Methods: ECC-1 cells were treated with BA, GEM, or their combination under normoxic and hypoxic conditions. Cell viability (MTT assay); nuclear morphology (NucBlue staining); cell cycle distribution (PI flow cytometry); angiogenesis (VEGF ELISA expression); apoptosis (Caspase-3/7 activity; Bax; Bcl-2 expression); inflammatory cytokines (IL-1β; IL-6; TNF-α); and gene ontology enrichment were analyzed. Results: Both BA and GEM reduced cell viability in a dose- and time-dependent manner, with the combination producing synergistic cytotoxicity and lower IC₅₀ values. Hypoxia enhanced drug sensitivity, particularly in combination therapy. BA and GEM significantly suppressed HIF-1α and VEGF expression, with maximal inhibition observed in the combination group. Apoptotic induction was confirmed by increased Bax and Caspase-3 and decreased Bcl-2 expression, together with elevated Caspase-3/7, -8, and -9 activity. Pro-inflammatory cytokine levels were markedly reduced, and gene ontology analysis revealed enrichment of apoptotic, anti-proliferative, and anti-angiogenic pathways. Conclusions: BA + GEM combination synergistically suppresses hypoxia-driven angiogenesis and promotes apoptosis in endometrial cancer cells. These findings support its potential as an adjuvant therapeutic approach, warranting further preclinical and clinical validation. Full article

Molecularly Imprinted Polymer (MIP)-based sensors have gained increasing attention in the field of food safety analysis due to their unique ability to selectively recognize and quantify chemical contaminants and allergens with interesting sensitivity. These synthetic receptors, often referred to as “plastic antibodies,” offer several advantages over conventional analytical methods, including high stability, cost-effectiveness, reusability, and compatibility with miniaturized sensor platforms. This review provides a comprehensive overview of recent advances in the design, fabrication, and application of MIP-based sensors for the detection of a broad range of food contaminants, including pesticides, antibiotics, mycotoxins, heavy metals, acrylamide, heterocyclic amines, allergens, viruses, and bacteria. Various transduction mechanisms—electrochemical, optical, thermal, and mass-sensitive—are discussed in relation to their integration with MIP recognition elements. The review also highlights the advantages and limitations of MIPs in comparison with traditional techniques such as ELISA and HPLC. Finally, we explore current challenges and emerging trends, including nanomaterial integration, multiplexed detection, and smartphone-based platforms, which are expected to drive future developments toward real-time, point-of-need, and regulatory-compliant food safety monitoring tools. Full article

Although vitamin D3 (VD3) deficiency has been recognized as a harmful agent in several respiratory diseases, the present study is the first one to investigate its influence on the development of hypersensitivity pneumonitis (HP). This research was conducted in a murine model of HP, wherein pulmonary fibrosis was induced by antigen of Pantoea agglomerans. VD3 deficiency was provoked by diet with 10-times less cholecalciferol than feed given to VD3-sufficient mice. Before and after 14 and 28 days of nebulization, lung function was evaluated. Moreover, at indicated time points, lungs were collected and subjected to histological assessment, flow cytometry, gene expression assays, and ELISA. The performed research showed a higher sensitivity of VD3-deficient mice to fibrosis response to P. agglomerans antigen, which was strongly associated with enhanced epithelial-to-mesenchymal transition, the signs of which were over-expression of EMT-transcription factors (Snail2, Zeb1, Zeb2) and mesenchymal cell markers (Cdh2/N-cadherin, Acta2/SMA, Fn1/Fibronectin, Vim/Vimentin). Indicated negative changes in VD3-deficient mice with developed HP were supported by deepening calcitriol deficiency and worsening respiratory functions, including the frequency of breathing, minute volume, total cycle times, expiratory and inspiratory time. Moreover, typical for VD3-deficient mice with HP, there was also an increased influx of immune cells into the lungs (especially neutrophils, macrophages, dendritic cells and lymphocytes Tc), a disturbed cytokine profile with over-production of growth factors favoring fibrosis (FGF2 and TGFβ), and lowered synthesis of several cytokines (IL1β, IL6, IL12, IL4 IL10, IL13). The present study reveals that VD3 deficiency promotes the development of pulmonary fibrosis in the murine model of HP. Full article

Cancer is the disease found to be the reason for the largest portion of deaths in the world annually and these mortality values are expected to increase in the future. Early detection of cancer biomarkers may help save millions of lives, particularly by implementing non-invasive and economical detection methods. In this review, we tabulated and quantitatively compared the data collected in 173 rows from 124 publications, which describe the clinical application of various methods in detection of cancer biomarkers. Those methods include mass spectrometry (MS), immunoassays (IAs), enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), surface-enhanced Raman spectroscopy (SERS), and Fourier-transform infrared spectroscopy (FTIR). We found that direct methods may have an advantage over indirect methods. Direct SERS reported in clinical applications can also achieve a higher area under the curve, higher sensitivity, and specificity than those parameters for ELISA, PCR, MS, and FTIR applications. Based on the average area under the curve (AUC) values reported in the last 6–7 years for each method, the performance of the analytical methods for the clinical cancer detection increases from IAs (0.76), ELISA (0.83), MS (0.87), and PCR (0.89) to FTIR (0.95) and SERS (0.97). Full article

Background: Pediatric patients with beta-thalassemia (BT) face unique immunologic challenges due to chronic transfusions and viral exposure. Hepatitis C virus (HCV), a common infection in polytransfused individuals, may influence immune polarization. However, the combined effect of chronic HCV and host immunogenetics on allergic sensitization remains incompletely understood. Objective: To assess total serum IgE levels and allergic manifestations in HCV-positive vs. HCV-negative BT patients, and explore associations with common polymorphisms in IL10, TLR7, IL4, and IFNG genes Methods: This cross-sectional observational study enrolled 46 BT patients (37 HCV-positive, 9 HCV-negative) and 50 healthy controls. Clinical allergy history, total IgE levels (ELISA), and skin prick tests (SPT) for aeroallergens were collected. Genotyping for IL10 −1082, TLR7 rs179008, IL4 −589, and IFNG +874 polymorphisms was performed. Associations between genotypes, HCV status, and IgE levels were analyzed descriptively due to small sample size Results: HCV-positive BT patients had lower mean IgE levels (18.73 ± 4.2 IU/mL) and fewer reported allergic symptoms (21.6%) compared to HCV-negative counterparts (118.76 ± 7.9 IU/mL; 55.5%). The IL10 −1082 AA and TLR7 rs179008 TT genotypes were more common in the HCV-positive group and were associated with lower IgE levels. No associations were noted for IL4 or IFNG variants. Splenectomy appeared to further modify IgE levels in HCV-negative patients. Due to limited power and absence of multivariate analysis, findings are exploratory. These preliminary observations may inform future studies of immune deviation in chronically infected pediatric cohorts. Conclusions: Chronic HCV infection may contribute to immune tolerance and reduced allergic expression in BT patients, potentially modulated by IL10 and TLR7 genotypes. Further studies with functional immune profiling and larger cohorts are required. 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