Search Results (480)

Vorinostat, an FDA-approved histone deacetylase inhibitor, was evaluated for its potential anti-inflammatory activity through modulation of TACE (ADAM17)-mediated TNF-α signaling. The study was conducted using LPS-stimulated RAW264.7 macrophages. TACE enzymatic activity was assessed by a fluorogenic assay, TNF-α release was measured by ELISA, and phosphorylation of MAPKs and NFκB signaling proteins was examined by a western blot. Molecular docking was performed using GNINA to evaluate binding affinity to ERK. Vorinostat was found to modestly inhibit TACE enzymatic activity in vitro, while significantly suppressing TNF-α secretion in cells, comparable to the selective TACE inhibitor BMS-561392. A concentration-dependent reduction in phosphorylated IκB and NFκB was observed, along with selective inhibition of ERK phosphorylation. Docking studies indicated a stable, albeit weaker, binding of vorinostat to ERK compared to reference ERK inhibitors. These findings suggest that vorinostat suppresses TNF-α production primarily through indirect mechanisms involving ERK and NF-κB signaling pathways, rather than by direct TACE inhibition. The repositioning of vorinostat as a modulator of inflammatory signaling is supported, offering potential therapeutic value in inflammatory disorders. Full article

The hyaluronidase enzyme derived from Vespa tropica (VesT2a) venom contains two putative catalytic residues. Herein, a double mutation was introduced into VesT2a at its catalytic sites by substituting Asp107 and Glu109 with Asn and Gln, respectively, to assess their essential roles in enzymatic function. We used Pichia pastoris to produce the mutated version of the VesT2a (mVesT2a) protein, and the process was more efficient when employing the methanol-inducible promoter (P_AOX1) compared to the constitutive promoter (P_GAP). In bioreactor scale-up, P. pastoris harboring the pAOX1-αMF-mVesT2a plasmid secreted 34.03 ± 2.31 mg/L of mVesT2a, with an apparent molecular mass of 46.6 kDa, retaining only 2.9% of hyaluronidase activity, thus indicating successful mutation. The newly developed indirect ELISA-based method using mVesT2a demonstrated its potential as an alternative approach for measuring hyaluronic acid (HA) at low concentrations and was also used to confirm HA-binding capacity. In silico docking and molecular dynamics simulations further supported the stable interaction of the mVesT2a–HA complex while suggested other surrounded acidic amino acid residues, which may play a minor role in HA degradation, supporting the remaining activity observed in the in vitro experiments. Full article

Due to the severe hazard of aflatoxins (AFs) to humans, it is of great significance to detect the key aflatoxins, aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁), in food and feed in simple, rapid, and semi-quantitative ways. The hybridoma clone 3A1 was prepared in this study, and anti-AFB₁ monoclonal antibody (mAb) with high specificity and affinity (9.38 × 10⁸ L/mol) from 3A1 was purified. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) demonstrated that the linear detection range for AFB₁ was 0.029–1.526 ng/mL with a limits of determination (LOD) of 0.023 ng/mL. A latex microsphere-based immunochromatographic test strip (LM-ICTS) was constructed based on 3A1, which showed that the strip could detect AFB₁ (LOD: lower than 1.79 ng/mL) and AFG₁ (LOD: lower than 8.08 ng/mL), and the linear detection ranges for AFB₁ and AFG₁ are 1.79–48.46 ng/mL and 8.08–107.40 ng/mL, respectively. The average recoveries of intra-assay and inter-assay for peanuts were (98.4 ± 4.7)% and (92.6 ± 7.6)%, and the average coefficient of variation (CVs) were 4.38% and 8.15%, respectively. For sunflower seeds, the intra-assay and inter-assay recoveries were (94.4 ± 7.2)% and (89.2 ± 4.3)%, and the average CVs were 6.6% and 4.9%, respectively. In summary, the developed LM-ICTS exhibited excellent sensitivity and specificity, which provided a rapidly stable on-site detection choice for AFB₁ and AFG₁ to contaminated agricultural samples, including grain and feed. Full article

Urine-based immunoassay is a non-invasive method with demonstrated utility in detecting anti-SARS-CoV-2 antibodies in unvaccinated patients with COVID-19. To evaluate urine’s potential for serological surveys in a real-world setting, SARS-CoV-2 serology was performed on urine samples from vaccinated individuals, both with and without prior confirmed COVID-19. (1) Methods: An in-house indirect ELISA was used to measure antibodies against recombinant spike (S) and nucleocapsid (N) proteins of SARS-CoV-2 in urine and paired serum from 149 individuals vaccinated with Janssen AD26.COV2.S, an S protein-based COVID-19 vaccine. (2) Results: Anti-S and anti-N levels were higher in the urine and serum of participants with confirmed prior COVID-19 compared to those without prior infection. Urinary anti-S effectively distinguished vaccinated individuals with (AUC = 0.96) and without (AUC = 0.88) prior infection from negative controls (non-vaccinated, non-previously infected individuals) (p < 0.0001). Among vaccinated participants, urinary anti-S and anti-N identified prior infection, with AUC values of 0.73 (p < 0.0001) and 0.60 (p = 0.03), respectively, being recorded. (3) Conclusions: Findings indicate that urinary anti-SARS-CoV-2 antibodies reflect AD26.COV2.S vaccination and previous COVID-19. To further advance the methodology, studies with larger sample sizes and a greater diversity of COVID-19 vaccines are required. Full article

African swine fever (ASF) is a highly pathogenic and hemorrhagic swine infectious disease caused by the African swine fever virus (ASFV). It encodes over 150 proteins, among which the CD2v protein plays multiple roles throughout the infection process. Single B-cell antibody technology is a cutting-edge method for preparing monoclonal antibodies (mAbs), which has the advantages of rapid, efficient, and high yield in antibody production, while possessing natural conformations. In this study, by cloning and expressing antibody genes in vitro, 14 murine-derived mAbs were prepared using recombinant CD2v proteins as immunogenic sources, which brings sufficient enrichment and selectivity for the development of antibodies based on the single B-cell antibody technique. All 14 mAbs demonstrated reactivity with CD2v protein by indirect ELISA, whereas 8 mAbs successfully detected CD2v in ASFV-infected PAM cells by IFA, indicating the tested mAbs can effectively recognize and bind to ASFV CD2v. Finally, a blocking ELISA method for detecting CD2v antibodies using CD2v mAb C89 was established, which holds significant potential for broad application in the serological diagnosis of ASFV with determination of the CD2v-blocking ELISA specificity, sensitivity, reproducibility, and compliance rate. It could be used for the rapid clinical detection of ASFV CD2v protein to provide a powerful tool for the monitoring of epidemics. Full article

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a significant infectious disease that affects small ruminants and poses economic challenges to livestock production. This study aimed to assess the seroprevalence of C. pseudotuberculosis in goats from Espírito Santo state, Brazil, and identify risk factors associated with infection by the bacterium. Serum samples from 145 goats were analyzed using an indirect enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence was found to be 34.5%. The risk factors significantly associated with infection included the presence of abscesses and the absence of veterinary assistance on farms. The findings emphasize the need for improved management practices and veterinary oversight to mitigate caseous lymphadenitis transmission. This research provides critical insights into the epidemiology of caseous lymphadenitis in goats from Espírito Santo, informing effective disease control strategies. Full article

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by vascular abnormalities, immune dysfunction, and progressive fibrosis. One of the most common manifestations of SSc is interstitial lung disease (ILD), known by a progressive course leading to significant morbidity and mortality. Aim: to investigate autoantibodies, cytokines, and genetic markers in SSc-ILD through a systematic review and analysis of a Kazakh cohort of SSc-ILD patients. Methods: A PubMed search over the past 10 years was performed with “SSc-ILD”, “autoantibodies”, “cytokines”, and “genes”. Thirty patients with SSc were assessed for lung involvement, EScSG score, and modified Rodnan skin score. IL-6 was measured by ELISA, antinuclear factor on HEp-2 cells by indirect immunofluorescence, and specific autoantibodies by immunoblotting. Genetic analysis was performed using a 120-gene AmpliSeq panel on the Ion Proton platform. Results: The literature review identified 361 articles, 26 addressed autoantibodies, 20 genetic variants, and 12 cytokine profiles. Elevated levels of IL-6, TGF-β, IL-33, and TNF-α were linked to SSc. Based on the results of the systemic review, we created a preliminary immunogenic panel for SSc-ILD with following analysis in Kazakh patients with SSc (n = 30). Fourteen of them (46.7%) demonstrated signs of ILD and/or lung hypertension, with frequent detection of antibodies such as Scl-70, U1-snRNP, SS-A, and genetic variants in SAMD9L, REL, IRAK1, LY96, IL6R, ITGA2B, AIRE, TREX1, and CD40 genes. Conclusions: Current research confirmed the presence of the broad range of autoantibodies and variations in IRAK1, TNFAIP3, SAMD9L, REL, IRAK1, LY96, IL6R, ITGA2B, AIRE, TREX1, CD40 genes in of Kazakhstani cohort of SSc-ILD patients. Full article

Via a One Health approach, this study concomitantly assessed the susceptibility of humans and dogs to Trypanosoma cruzi infections on three islands and in two mainland seashore areas of southern Brazil. Human serum samples were tested using an enzyme-linked immunosorbent assay (ELISA) to detect anti-T. cruzi antibodies, while dog serum samples were tested using indirect fluorescent antibodies in an immunofluorescence assay (IFA). Seropositive human and dog individuals were also tested using quantitative polymerase chain reaction (qPCR) in corresponding blood samples. Overall, 2/304 (0.6%) human and 1/292 dog samples tested seropositive for T. cruzi by ELISA and IFA, respectively, and these cases were also molecularly positive for T. cruzi by qPCR. Although a relatively low positivity rate was observed herein, these cases were likely autochthonous, and the individuals may have been infected as a consequence of isolated events of disturbance in the natural peridomicile areas nearby. Such a disturbance could come in the form of a fire or deforestation event, which can cause stress and parasitemia in wild reservoirs and, consequently, lead to positive triatomines. In conclusion, T. cruzi monitoring should always be conducted in suspicious areas to ensure a Chagas disease-free status over time. Further studies should also consider entomological and wildlife surveillance to fully capture the transmission and spread of T. cruzi on islands and in seashore mainland areas of Brazil and other endemic countries. Full article

Background/Objectives: Anti-p200 pemphigoid is a rare and likely underdiagnosed autoimmune blistering disorder. Laminin γ1 and laminin β4 have been implicated as potential target antigens in its pathogenesis. Recently, a novel indirect immunofluorescence assay targeting anti-laminin β4 antibodies has been developed, demonstrating high sensitivity and specificity, and offering a valuable tool for improved diagnosis. Methods: Of the 451 patients, 21 were selected for further laboratory analysis based on medical records. Sera from 10 patients, which showed a positive direct immunofluorescence (DIF) result and negative results in multiplex enzyme-linked immunosorbent assays (ELISAs) and/or mosaic six-parameter indirect immunofluorescence (IIF) for various autoimmune bullous diseases, were tested for the presence of anti-laminin β4 antibodies. Additionally, sera from 11 patients with positive DIF and positive ELISA for antibodies against BP180 and/or BP230 were analyzed. Results: Among the 10 patients with positive DIF and negative ELISA and/or mosaic six-parameter IIF, 6 sera were positive for anti-laminin β4 antibodies. These patients presented with atypical clinical features. In contrast, all 11 sera from patients with both positive DIF and positive ELISA for BP180 and/or BP230 were negative for anti-laminin β4 antibodies. Conclusions: In patients with a positive DIF result but negative ELISA and/or mosaic six-parameter IIF findings, testing for anti-laminin β4 antibodies should be considered. Furthermore, in cases presenting with atypical clinical features—such as acral distribution of lesions, intense pruritus, or erythematous–edematous plaques—the possibility of anti-p200 pemphigoid should be included in the differential diagnosis. Full article

Maedi-visna virus (MVV), a small ruminant lentivirus causing chronic multisystemic disease in sheep, poses significant economic burdens due to reduced productivity and a lack of effective treatments. Despite its worldwide prevalence, epidemiological data from Algeria remain absent. This first national seroprevalence study aimed to elucidate MVV distribution, risk factors, and transmission dynamics in Algerian sheep herds. A cross-sectional survey of 1400 sheep across four regions (East, Center, West, South) was conducted, with sera analyzed via indirect ELISA (IDvet). Risk factors (geography, age, sex, breed, farming system) were evaluated using chi-square tests and Cramer’s V. Overall seroprevalence was 9.07% (95% CI: 7.57–10.57), with significant variation by sex (females: 20.44% vs. males: 3.68%; p < 0.05), age (1–5 years: 6.86% vs. <1 year: 0.29%; p = 0.01), and region (Central: 3.36% vs. Eastern: 0.86%; p < 0.05). Notably, no association was found with breed or farming system (p ≥ 0.08), contrasting prior studies and suggesting region-specific transmission dynamics. Females exhibited heightened seropositivity, implicating prolonged herd retention and vertical transmission risks. Geographic disparities highlighted industrialized farming in central Algeria as a potential transmission amplifier. Strikingly, seronegative animals in high-prevalence herds hinted at genetic resistance, warranting further investigation. This study provides foundational insights into MVV epidemiology in North Africa, underscoring the need for targeted surveillance, ewe-focused control measures, and genetic research to mitigate transmission. The absence of prior national data elevates its significance, offering actionable frameworks for resource-limited settings and enriching the global understanding of SRLV heterogeneity. Full article

Colorectal cancer (CRC) is characterized by complex interactions between inflammation, oxidative stress, and extracellular matrix remodeling. Recent studies have highlighted the significance of the reactive oxygen species (ROS)–nuclear factor kappa B (NF-κB)–matrix metalloproteinase-9 (MMP-9) axis in promoting tumor invasion and metastasis in CRC, linking oxidative stress with inflammatory signaling and extracellular matrix degradation. In this study, we analyzed the concentration of advanced oxidation protein products (AOPPs), expression of NF-κB, and the activity of MMP-9 in tumor tissue, adjacent tissue, and healthy control colon tissue. Tissue specimens were collected from 50 patients with primary CRC following surgical resection. The analyses were performed using appropriate and validated biochemical methods, including ELISA, spectrophotometry, and indirect immunofluorescence. Significantly higher levels of all three markers were observed in tumor tissue compared to controls. Additionally, adjacent tissue exhibited elevated NF-κB expression and MMP-9 activity when compared to healthy colon tissue. AOPP levels correlated strongly with MMP-9 activity, highlighting the role of oxidative stress in the activation of MMP-9. MMP-9 demonstrated the highest predictive value for CRC, emphasizing its potential as a diagnostic and theranostic marker. Our findings support the hypothesis that the ROS–NF-κB–MMP-9 axis plays an important role in CRC progression, particularly during stages T2 and T3. Targeting this pathway may offer new therapeutic strategies for limiting tumor invasion and recurrence. Moreover, ensuring adequate surgical resection margins is crucial to optimizing treatment outcomes. Full article

Background/Objectives: Beak and feather disease virus (BFDV) is the causative agent of psittacine beak and feather disease (PBFD), affecting psittacine birds. There is currently no commercial vaccine or treatment for this disease. This study developed a novel BFDV coat protein mRNA vaccine encapsidated by TMV coat protein to form pseudovirions (PsVs) and tested its immunogenicity alongside BFDV coat protein (CP) subunit and DNA vaccine candidates. Methods: mRNA and BFDV CP subunit vaccine candidates were produced in Nicotiana benthamiana and subsequently purified using PEG precipitation and gradient ultracentrifugation, respectively. The DNA vaccine candidate was produced in E. coli cells harbouring a plasmid with a BFDV1.1mer pseudogenome. Immunogenicity of the vaccine candidates was evaluated in African grey parrot chicks. Results: Successful purification of TMV PsVs harbouring the mRNA vaccine, and of the BFDV-CP subunit vaccine, was confirmed by SDS-PAGE and western blot analysis. TEM analyses confirmed formation of TMV PsVs, while RT-PCR and RT-qPCR cDNA amplification confirmed encapsidation of the mRNA vaccine candidate within TMV particles. Restriction digests verified presence of the BFDV1.1mer genome in the plasmid. Four groups of 5 ten-week-old African grey parrot (Psittacus erithacus) chicks were vaccinated and received two boost vaccinations 2 weeks apart. Blood samples were collected from all four groups on day 14, 28 and 42, and sera were analysed using indirect ELISA, which showed that all vaccine candidates successfully elicited specific anti-BFDV-CP immune responses. The subunit vaccine candidate showed the strongest immune response, indicated by higher binding titres (>6400), followed by the mRNA and DNA vaccine candidates. Conclusions: The candidate vaccines present an important milestone in the search for a protective vaccine against PBFD, and their inexpensive manufacture could considerably aid commercial vaccine development. Full article

Background/Objectives: Toxocara spp. infection has been associated with severe asthma and allergic manifestations due to the activation of eosinophils by the release of Th2 cell cytokines. The aim of this study was to investigate the association between Toxocara spp. infection and eosinophil levels in severe asthmatic patients. Methods: The socio-demographic, peripheral blood eosinophils counting total IgE, sIgE to aeroallergens and FEV1 results were acquired from the Program of Asthma and Rhinitis Control (ProAR) at the Salvador–Brazil databank; IgG anti-Toxocara spp. levels were measured in 176 severely asthmatic patients by indirect ELISA. Results: The Toxocara spp. seroprevalence was 50.6%. Eosinophilia was present in 54% of the population. The correlation between IgG anti-Toxocara spp. levels and eosinophils levels was positive. Eosinophilic individuals with SPT, sIgE for D. pteronyssinus, D. farinae and B. tropicalis showed positive results; IgE ≥ 160 UI/dL and uncontrolled asthma presented more positive results for IgG anti-Toxocara spp. Conclusions: Our findings suggest that eosinophil levels are influenced by the presence of IgG antibodies against Toxocara spp. Additionally, helminth infection may modulate immunological responses in allergies and uncontrolled asthma, which could help explain the exacerbation of asthma symptoms. Full article

Toxoplasma gondii, a pathogen of significant concern in animal production, companion animal health, and public health, particularly affects immunocompromised individuals and pregnant women. Current diagnostic techniques employ both direct and indirect methods, with serological assays widely used for detecting T. gondii infections in humans and animals. In this study, the TIM-barrel structure of Br2 β-glucosidase was engineered to create 10 chimeric multi-epitope proteins for T. gondii serological detection. Indirect ELISA screening identified three promising candidate proteins, V4Z, SFF, and S7V-V4Z-SFF, with sensitivities ranging from 71–86% and specificities ranging from 68–76%. Among these, ELISA-V4Z achieved the highest concordance with the reference IFAT method (Kappa = 0.58, 95% CI = 0.32–0.84) and demonstrated a moderate positive predictive value (PPV, 67%) and strong negative predictive value (NPV, 90%). These results suggest that the V4Z chimeric protein demonstrated the strongest performance among the tested candidates for T. gondii detection, exhibiting the highest sensitivity and specificity along with moderate agreement with the reference IFAT. However, its overall diagnostic performance remains limited. These findings highlight the need for further refinement and validation to enhance its diagnostic potential and assess its applicability for broader serological testing. Full article

Effective surveillance of viral disease in fish populations is critical for disease control and the sustainable development of global aquaculture. Here, we evaluated the application and performance of pooled serum samples using an indirect ELISA based on recombinant segment 4 protein to assess farm-level immunity in tilapia infected with Tilapia lake virus (TiLV). The TiLV-S4 ELISA was developed using a recombinant nucleoprotein (segment 4) antigen, optimized through checkerboard titration, and validated for repeatability and reproducibility, with intra- and inter-assay coefficients of variation below 10%. A pooling strategy was used to combine multiple serum samples before testing for the presence of TiLV-specific antibodies using an enzyme-linked immunosorbent assay (ELISA). Our results showed that pooling five serum samples was effective for detecting TiLV-specific antibodies, particularly when multiple seropositive individuals were presented in the pool, supporting its application for population-level surveillance. However, ELISA sensitivity may be reduced when only one seropositive sample is included in the pool, due to the dilution effects. Despite this limitation, pooled testing yielded a high proportion of positive results, suggesting similar detection performance in many cases. Overall, the pooling strategy provides a cost-effective and time-efficient approach for large-scale monitoring of immune status in tilapia populations. Full article