Utility of epitope-specific IgE, IgG4, and IgG1 antibodies for the diagnosis of wheat allergy.

BACKGROUND: The bead-based epitope assay has been used to identify epitope-specific (es) antibodies and successfully used to diagnose clinical allergy to milk, egg, and peanut. OBJECTIVE: We sought to identify es-IgE, es-IgG4, and es-IgG1 of wheat proteins and determine the optimal peptides to differentiate wheat-allergic from wheat-tolerant using the bead-based epitope assay. METHODS: Children and adolescents who underwent an oral food challenge to confirm their wheat allergy status were enrolled. Seventy-nine peptides from α-/ß-gliadin, γ-gliadin, ω-5-gliadin, and high- and low-molecular-weight glutenin were commercially synthesized and coupled to LumAvidin beads (Luminex Corporation, Austin, Tex). Machine learning methods were used to identify diagnostic epitopes, and performance was evaluated using the DeLong test. RESULTS: The analysis included 122 children (83 wheat-allergic and 39 wheat-tolerant; 57.4% male). Machine learning coupled with simulations identified wheat es-IgE, but not es-IgG4 or es-IgG1, to be the most informative for diagnosing wheat allergy. Higher es-IgE binding intensity correlated with the severity of allergy phenotypes, with wheat anaphylaxis exhibiting the highest es-IgE binding intensity. In contrast, wheat-dependent exercise-induced anaphylaxis showed lower es-IgG1 binding intensity than did all the other groups. A set of 4 informative epitopes from ω-5-gliadin and γ-gliadin were the best predictors of wheat allergy, with an area under the curve of 0.908 (sensitivity, 83.4%; specificity, 88.4%), higher than the performance exhibited by wheat-specific IgE (area under the curve = 0.646; P < .001). The predictive ability of our model was confirmed in an external cohort of 71 patients (29 allergic, 42 nonallergic), with an area under the curve of 0.908 (sensitivity, 75.9%; specificity, 90.5%). CONCLUSIONS: The wheat bead-based epitope assay demonstrated greater diagnostic accuracy compared with existing specific IgE tests for wheat allergy.

Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Genomic patterns of selection in morphometric traits across diverse Indian cattle breeds.

This study seeks a comprehensive exploration of genome-wide selective processes impacting morphometric traits across diverse cattle breeds, utilizing an array of statistical methods. Morphometric traits, encompassing both qualitative and quantitative variables, play a pivotal role in characterizing and selecting livestock breeds based on their external appearance, size, and physical attributes. While qualitative traits, such as color, horn structure, and coat type, contribute to adaptive features and breed identification, quantitative traits like body weight and conformation measurements bear a closer correlation with production characteristics. This study employs advanced genotyping technologies, including the Illumina BovineSNP50 Bead Chip and next-generation sequencing methods like Reduced Representation sequencing, to identify genomic signatures associated with these traits. We applied four intra-population methods to find evidence of selection, such as Tajima's D, CLR, iHS, and ROH. We found a total of 40 genes under the selection signature, that were associated with morphometric traits in five cattle breeds (Kankrej, Tharparkar, Nelore, Sahiwal, and Gir). Crucial genes such as ADIPDQ, DPP6, INSIG1, SLC35D2 in Kankrej, LPL, ATP6V1B2, CDC14B in Tharparkar, HPSE2, PLAG1 in Nelore, PCSK1, PRKD1 in Sahiwal, and GNAQ, HPCAL1 in Gir were identified in our study. This approach provides valuable insights into the genetic basis of variations in body weight and conformation traits, facilitating informed selection processes and offering a deeper understanding of the evolutionary and domestication processes in diverse cattle breeds.

De novo Semi-Synthetic Platform for Monitoring Protein degradation in Live Cells.

We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semi-synthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel-Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells.

A brief account of evolution of assays to study carbohydrate-protein interaction.

Molecular recognition remains one of the most desirable means of cellular communication. Each cell offers a unique surface pattern of biomolecules that makes it very specific about the nature of molecules that interact with the cell. Protein-glycan interaction has been one of the most common forms of cell signaling. Glycans expressed on the cell surface interact with an exogenous protein, and in many cases lead to a physiological response. These carbohydrate-binding proteins, commonly known as lectins, are very specific to the glycan they bind to. An exogenous lectin interacting with an animal cell surface glycan is generally studied using the classical hemagglutination assay. However, this method presents certain challenges that make it imperative to design and develop novel methods that are more specific and efficient in their interaction. In the last decade, a few methods have been developed to analyze more diverse reactions and use a lesser amount of sample. In some cases, the processing of the sample is also reduced. This review discusses how the methods have evolved over the decades and how they have reduced error while becoming more efficient.

Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

BACKGROUND: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. METHODS: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay. RESULTS: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration. CONCLUSION: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

Th2-predominant immune response underlies the pathogenesis of Dengue.

BACKGROUND: Dengue is a rapidly emerging pandemic-prone disease, whose manifestations range from asymptomatic infection to life-threatening complications like Dengue Hemorrhagic Fever and Dengue Shock Syndrome. This study investigates and compares the immune response in clinically defined cohorts of Dengue with and without warning signs, with the aim of identifying immunological correlates of clinical disease and potential markers of disease severity. METHODS: Blood samples, collected from study participants fulfilling the WHO definition of Dengue with and without warning signs and healthy volunteers, were analyzed using flow cell-based fluorometric methods for cytokines and chemokines. Gene expression analysis, using RT-PCR, was conducted on T helper cell subset-specific transcription factors and cytokines. Demographic details, virological markers, serotype distribution, and hematological parameters were also investigated in all the subjects. RESULTS: The 35 participants recruited in the study, included 11 healthy volunteers and 12 patients each fulfilling the WHO criteria of Dengue with and without warning signs. While the demographic characteristics and serotype distribution was similar in Dengue with and without warning signs cohorts of the disease, platelet counts and Aspartate Aminotransferase (AST) levels changed significantly between Dengue with and without warning signs patients. Plasma cytokine analysis showed up-regulation of IL-4, IL-10, IP-10, and MCP-1 in Dengue patients compared to healthy volunteers. Disease severity was associated with elevated levels of IL-10, IP-10, IL-4, MCP-1, and MIP-1α. IL-8 and MIP-1α were significantly up-regulated in Dengue with warning sign compared to Dengue without warning signs cases. Transcription factor analysis indicated increased expression of RORα, FoxP3, and GATA3 in Dengue patients. mRNA expression of TGFß and IL-4 was also elevated in Dengue patients. A positive correlation between mRNA expression of IL-4 and plasma IL-4 was observed. CONCLUSION: The study reveals a Th2-predominant immune response in all Dengue patients, regardless of disease severity, with overexpression of IL-8 and MIP-1α being observed in patients with warning signs.

Arterial-Portal Venous Shunt after Drug-Eluting Bead Transarterial Chemoembolization for Hepatocellular Carcinoma Patients: Risk factors and Impact on Patient Survival.

INTRODUCTION: The effectiveness of transarterial chemoembolization (TACE) in treating hepatocellular carcinoma (HCC) has been well established. The differential impacts of drug-eluting bead TACE (DEB-TACE) as opposed to conventional TACE (cTACE) on vascular changes, such as arterial-portal venous shunts (APSs), have been recognized. However, their subsequent effects on treatment outcomes have not been fully explored. This study aims to identify risk factors associated with the occurrence of APS in HCC patients treated with DEB-TACE and to evaluate its impact on patient survival. METHODS: A retrospective analysis was conducted from January 2012 to December 2018 including 74 HCC patients receiving DEB-TACE as initial treatment and a 1:1 cTACE. Kaplan-Meier analysis estimated overall survival (OS) and progression-free survival (PFS). Logistic regression identified significant risk factors for APS occurrence after DEB-TACE. RESULTS: APS incidence was significantly higher after DEB-TACE than cTACE (46.0% vs. 16.2%, p < 0.001). There was no significant difference in median OS between APS and non-APS groups after DEB-TACE: 50 months (24.6-75.4) versus 26.9 months (19.5-43.2), p = 0.111; median PFS was 15.6 months (4.1-27.1) and 9.5 months (6.8-12.1) for the two groups, respectively, p = 0.065. Risk factors for APS occurrence after DEB-TACE were more than two feeding arteries (OR: 7.25, 95% CI: 1.82-28.95, p = 0.005) and non-selective embolization (OR: 8.02, 95% CI: 2.30-27.95, p = 0.001). CONCLUSION: APS occurrence was higher in DEB-TACE-treated HCC patients, but it did not significantly affect OS and PFS. More than two feeding arteries and non-selective embolization were significant risk factors for APS occurrence after DEB-TACE.

Is DEB-TACE as locoregional therapy before liver transplantation for hepatocellular carcinoma effective?

BACKGROUND: The objectives were to assess the safety and efficacy of drug-eluting bead transarterial chemoembolization (DEB-TACE) as locoregional therapy (LRT) in hepatocellular carcinoma (HCC) before liver transplantation (LT) beyond Hangzhou criteria (HC) and to analyze the prognostic factors. METHODS: Forty patients with HCC beyond HC who received DEB-TACE only before LT were retrospectively analyzed between January 2017 and December 2022. Data on patient demographics, disease characteristics, treatment response, and adverse events (AE) were collected. Overall survival (OS) and recurrence-free survival (RFS) were evaluated with Kaplan-Meier curves. Univariate and multivariate Cox regression analyses were performed to identify factors independently associated with RFS and OS. RESULTS: All patients successfully underwent LT following DEB-TACE with a mean interval of 2.3 months. The objective response rates (ORRs) for these patients following DEB-TACE was 82.5%. The primary AE was post-embolization syndrome (PES), with affected patients experiencing grades I and II. The median RFS and OS were 12.0 months (95%CI: 0.0-30.1) and 52.0 months (95%CI: 11.8-92.2) over the follow-up period until December 2022. The 2-year RFS and OS rates were 42.5%, and 67.5%. Multivariate analyses revealed Child-Pugh classification (HR = 6.24; 95%CI,1.83-21.24; P = 0.01) and macrovascular invasion (MAV) (HR = 3.89; 95%CI,1.07-14.15; P = 0.04) were both significant independent predictors of OS. CONCLUSIONS: DEB-TACE can serve as a safe and effective LRT in HCC patients beyond HC before LT, and can improve the prognosis of patients, especially without MAV. The higher Child-Pugh classification and MAV are independent prognostic factors after LT.