IgE and IgG4 binding to lentil epitopes in children with red and green lentil allergy.

BACKGROUND: The consumption of lentil is common in the Mediterranean area and is one of the causes of IgE-mediated food allergy in many countries. Len c 1 is a well-defined allergen of lentil and approximately 80% of the patients with lentil allergy recognize the purified Len c 1 protein. We sought to identify IgE and IgG4 sequential epitopes of Len c 1 in patients with red and/or green lentil allergy. We also aimed to determine IgE and IgG4 binding differences between those patients who had outgrown or remained reactive to lentil. METHODS: Children with IgE-mediated lentil allergy were included in the study. We applied a microarray immunoassay to determine the characterization of positive IgE and IgG4 binding to Len c 1 epitopes in the patients' sera. RESULTS: The peptides specifically recognized by IgE and IgG4 antibodies were mainly detected between peptides 107 and 135 of Len c 1. The signal intensities of positive epitopes were significantly greater in reactive patients than tolerant ones (P = .008 for IgE and P = .002 for IgG4). Moreover, IgE and IgG4 antibodies bound largely the same sequential epitopes in patients who remained reactive or outgrew their allergy. CONCLUSION: IgG4-binding epitopes in lentil allergy were identified and IgE and IgG4 binding to epitopes in both red and green lentils was compared. Our data regarding signal intensity differences between reactive and outgrown patients and overlap binding of IgE and IgG4 antibodies may be important for the development of more accurate diagnostic tests and understanding of natural tolerance development.

Predicting development of sustained unresponsiveness to milk oral immunotherapy using epitope-specific antibody binding profiles.

BACKGROUND: In a recent trial of milk oral immunotherapy (MOIT) with or without omalizumab in 55 patients with milk allergy treated for 28 months, 44 of 55 subjects passed a 10-g desensitization milk protein challenge; 23 of 55 subjects passed the 10-g sustained unresponsiveness (SU) challenge 8 weeks after discontinuing MOIT. OBJECTIVE: We sought to determine whether IgE and IgG4 antibody binding to allergenic milk protein epitopes changes with MOIT and whether this could predict the development of SU. METHODS: By using a novel high-throughput Luminex-based assay to quantitate IgE and IgG4 antibody binding to 66 sequential epitopes on 5 milk proteins, serum samples from 47 subjects were evaluated before and after MOIT. Machine learning strategies were used to predict whether a subject would have SU after 8 weeks of MOIT discontinuation. RESULTS: MOIT profoundly altered IgE and IgG4 binding to epitopes, regardless of treatment outcome. At the initiation of MOIT, subjects achieving SU exhibited significantly less antibody binding to 40 allergenic epitopes than subjects who were desensitized only (false discovery rate ≤ 0.05 and fold change > 1.5). Based on baseline epitope-specific antibody binding, we developed predictive models of SU. Using simulations, we show that, on average, IgE-binding epitopes alone perform significantly better than models using standard serum component proteins (average area under the curve, >97% vs 80%). The optimum model using 6 IgE-binding epitopes achieved a 95% area under the curve and 87% accuracy. CONCLUSION: Despite the relatively small sample size, we have shown that by measuring the epitope repertoire, we can build reliable models to predict the probability of SU after MOIT. Baseline epitope profiles appear more predictive of MOIT response than those based on serum component proteins.

A new Luminex-based peptide assay to identify reactivity to baked, fermented, and whole milk.

BACKGROUND: The majority of children with cow's milk allergy (CMA) tolerate baked milk. However, reactivity to fermented milk products such as yogurt/cheese has not been previously evaluated. We sought to determine whether children with CMA could tolerate yogurt/cheese and whether a patient's IgE and IgG4-binding pattern to milk protein epitopes could distinguish clinical reactivity. METHODS: Four groups of reactivity were identified by Oral food challenge: baked milk reactive, fermented milk reactive, whole milk reactive, and outgrown. sIgE and sIgG4 binding to milk protein epitopes were assessed with a novel Luminex-based peptide assay (LPA). Using machine learning techniques, a model was developed to predict different degrees of CMA. RESULTS: The baked milk reactive patients demonstrated the highest degree of IgE epitope binding, which was followed sequentially by fermented milk reactive, whole milk reactive, and outgrown. Data were randomly divided into two groups with 75% of the data utilized for model development (n = 68) and 25% for testing (n = 21). All 68 children used for training were correctly classified with models using IgE and IgG4 epitopes. The average cross-validation accuracy was much higher for models using IgE plus IgG4 epitopes by LPA (84.8%), twice the performance of the serum component proteins assayed by UniCAP (41.9%). The performance of the model on "unseen data" was tested using the 21 withheld patients, and the accuracy of IgE was 86% (AUC = 0.89) while of IgE+IgG4 model was 81% (AUC = 0.94). CONCLUSION: Using a novel high-throughput LPA, we were able to distinguish the diversity of IgE/IgG4 binding to epitopes in the varying CMA phenotypes. LPA is a promising tool to predict correctly different degrees of CMA.

Chemical Stability of Mesoporous Oxide Thin Film Electrodes under Electrochemical Cycling: from Dissolution to Stabilization.

Mesoporous oxide thin films (MOTF) present very high surface areas and highly controlled monodisperse pores in the nanometer range. These features spurred their possible applications in separation membranes and permselective electrodes. However, their performance in real applications is limited by their reactivity. Here, we perform a basic study of the stability of MOTF toward dissolution in aqueous media using a variety of characterization techniques. In particular, we focus in their stability behavior under the influence of ionic strength, adsorption of electrochemical probes, and applied electrode potential. Mesoporous silica thin films present a limited chemical stability after electrochemical cycling, particularly under high ionic strength, due to their high specific surface area and the interactions between the electrochemical probes and the surface. In contrast, TiO2 or Si0.9Zr0.1O2 matrices present higher stability; thus, they are an adequate alternative to produce accessible, sensitive, and robust permselective electrodes or membranes that perform under a wide variety of conditions.

TiO2 mesoporous thin film architecture as a tool to control Au nanoparticles growth and sensing capabilities.

In this paper, a systematic study regarding the effect of the mesoporous structure over Au nanoparticles (NPs) growth inside and through the pores of mesoporous TiO2 thin films (MTTFs) is presented, and the effect of such characteristics over the composites' sensing capabilities is evaluated. Highly stable MTTFs with different pore diameters (range: 4-8 nm) and pore arrangements (body- and face-centered cubic) were synthesized and characterized. Au NPs were grown inside the pores, and it was demonstrated-through a careful physicochemical characterization-that the amount of incorporated Au and NP size depends on the pore array; being higher for bigger pore diameters and face-centered cubic structures. The same structure allows the growth of more and longer tips over Au NPs deposited at the thin film-substrate interface. Finally, to confirm the effect of the structural characteristics of the composites over their possible applications, the materials were tested as surface-enhanced Raman scattering (SERS)-based substrates. The composites with a higher amount of Au and more ramified NPs were the ones that presented better sensitivity in the detection of a probe molecule (4-nitrothiophenol). Overall, this work demonstrates that the pore size and ordering in MTTFs determine the materials' accessibility and connectivity, and therefore, have a clear impact on their potential applications.

Use of IgE and IgG4 epitope binding to predict the outcome of oral immunotherapy in cow's milk allergy.

BACKGROUND: Oral immunotherapy (OIT) with cow's milk (CM) has been reported to induce a number of specific antibody responses, but these remain to be fully characterized. Our objective was to explore whether IgE and IgG4 epitope binding profiles could predict the risk of side effects during CM OIT. METHODS: The study population consisted of 32 children (6-17 yr of age) with CM allergy: 26 children who successfully completed OIT and six children who discontinued therapy due to adverse reactions. We investigated sera drawn before and after OIT. We analyzed specific IgE and IgG4 binding to CM protein-derived peptides with a microarray-based immunoassay. Antibody binding affinity was analyzed with a competition assay where CM proteins in solution competed with peptides printed on the microarray. RESULTS: IgE binding to CM peptides decreased and IgG4 binding increased following the OIT in children who attained desensitization. Compared with children who successfully completed OIT, those who discontinued OIT due to adverse reactions developed increased quantities and affinity of epitope-specific IgE antibodies and a broader diversity of IgE and IgG4 binding, but less overlap in IgE and IgG4 binding to CM peptides. CONCLUSIONS: Detailed analysis of IgE and IgG4 binding to CM peptides may help in predicting whether CM OIT will be tolerated successfully. It may thus improve the safety of the therapy.

Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens.

BACKGROUND: Patients with peanut allergy have highly stable pathologic antibody repertoires to the immunodominant B-cell epitopes of the major peanut allergens Ara h 1 to 3. OBJECTIVE: We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires. METHODS: Measurements of total peanut-specific IgE (psIgE) and peanut-specific IgG(4) (psIgG(4)) were made with CAP-FEIA. We analyzed sera from 22 patients with OIT and 6 control subjects and measured serum specific IgE and IgG(4) binding to epitopes of Ara h 1 to 3 using a high-throughput peptide microarray technique. Antibody affinity was measured by using a competitive peptide microarray, as previously described. RESULTS: At baseline, psIgE and psIgG(4) diversity was similar between patients and control subjects, and there was broad variation in epitope recognition. After a median of 41 months of OIT, polyclonal psIgG(4) levels increased from a median of 0.3 µg/mL (interquartile range [25% to 75%], 0.1-0.43 µg/mL) at baseline to 10.5 µg/mL (interquartile range [25% to 75%], 3.95-45.48 µg/mL; P < .0001) and included de novo specificities. psIgE levels were reduced from a median baseline of 85.45 kU(A)/L (23.05-101.0 kU(A)/L) to 7.75 kU(A)/L (2.58-30.55 kU(A)/L, P < .0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated patients, several subjects generated new IgE specificities, even as the total psIgE level decreased. Global epitope-specific shifts from IgE to IgG(4) binding occurred, including at an informative epitope of Ara h 2. CONCLUSION: OIT differentially alters Ara h 1 to 3 binding patterns. These changes are variable between patients, are not observed in control subjects, and include a progressive polyclonal increase in IgG(4) levels, with concurrent reduction in IgE amount and diversity.

A bioinformatics approach to identify patients with symptomatic peanut allergy using peptide microarray immunoassay.

BACKGROUND: Peanut allergy is relatively common, typically permanent, and often severe. Double-blind, placebo-controlled food challenge is considered the gold standard for the diagnosis of food allergy-related disorders. However, the complexity and potential of double-blind, placebo-controlled food challenge to cause life-threatening allergic reactions affects its clinical application. A laboratory test that could accurately diagnose symptomatic peanut allergy would greatly facilitate clinical practice. OBJECTIVE: We sought to develop an allergy diagnostic method that could correctly predict symptomatic peanut allergy by using peptide microarray immunoassays and bioinformatic methods. METHODS: Microarray immunoassays were performed by using the sera from 62 patients (31 with symptomatic peanut allergy and 31 who had outgrown their peanut allergy or were sensitized but were clinically tolerant to peanut). Specific IgE and IgG(4) binding to 419 overlapping peptides (15 mers, 3 offset) covering the amino acid sequences of Ara h 1, Ara h 2, and Ara h 3 were measured by using a peptide microarray immunoassay. Bioinformatic methods were applied for data analysis. RESULTS: Individuals with peanut allergy showed significantly greater IgE binding and broader epitope diversity than did peanut-tolerant individuals. No significant difference in IgG(4) binding was found between groups. By using machine learning methods, 4 peptide biomarkers were identified and prediction models that can predict the outcome of double-blind, placebo-controlled food challenges with high accuracy were developed by using a combination of the biomarkers. CONCLUSIONS: In this study, we developed a novel diagnostic approach that can predict peanut allergy with high accuracy by combining the results of a peptide microarray immunoassay and bioinformatic methods. Further studies are needed to validate the efficacy of this assay in clinical practice.

Ordered Mesoporous Electrodes for Sensing Applications.

Electrochemical sensors have become increasingly relevant in fields such as medicine, environmental monitoring, and industrial process control. Selectivity, specificity, sensitivity, signal reproducibility, and robustness are among the most important challenges for their development, especially when the target compound is present in low concentrations or in complex analytical matrices. In this context, electrode modification with Mesoporous Thin Films (MTFs) has aroused great interest in the past years. MTFs present high surface area, uniform pore distribution, and tunable pore size. Furthermore, they offer a wide variety of electrochemical signal modulation possibilities through molecular sieving, electrostatic or steric exclusion, and preconcentration effects which are due to mesopore confinement and surface functionalization. In order to fully exploit these advantages, it is central to develop reproducible routes for sensitive, selective, and robust MTF-modified electrodes. In addition, it is necessary to understand the complex mass and charge transport processes that take place through the film (particularly in the mesopores, pore surfaces, and interfaces) and on the electrode in order to design future intelligent and adaptive sensors. We present here an overview of MTFs applied to electrochemical sensing, in which we address their fabrication methods and the transport processes that are critical to the electrode response. We also summarize the current applications in biosensing and electroanalysis, as well as the challenges and opportunities brought by integrating MTF synthesis with electrode microfabrication, which is critical when moving from laboratory work to in situ sensing in the field of interest.

Field Performance of Resistant Potato Genotypes Transformed with the EFR Receptor from Arabidopsis thaliana in the Absence of Bacterial Wilt (Ralstonia solanacearum).

Bacterial wilt caused by the pathogen Ralstonia solanacearum is a devastating disease of potato crops. Harmonizing immunity to pathogens and crop yield is a balance between productive, economic, and environmental interests. In this work, the agronomic performance of two events of potato cultivar INIA Iporá expressing the Arabidopsis thaliana EFR gene (Iporá EFR 3 and Iporá EFR 12) previously selected for their high resistance to bacterial wilt was evaluated under pathogen-free conditions. During two cultivation cycles, the evaluated phenotypic characteristics were emergence, beginning of flowering, vigor, growth, leaf morphology, yield, number and size of tubers, analyzed under biosecurity standards. The phenotypic characteristics evaluated did not show differences, except in the morphology of the leaf with a more globose appearance and a shortening of the rachis in the transformation events with respect to untransformed Iporá. The Iporá EFR 3 genotype showed a ~40% yield decrease in reference to untransformed Iporá in the two trials, while Iporá EFR 12 did not differ statistically from untransformed Iporá. Iporá EFR 12 shows performance stability in the absence of the pathogen, compared to the untransformed control, positioning it as an interesting candidate for regions where the presence of the pathogen is endemic and bacterial wilt has a high economic impact.

Author Correction: Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in food allergy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combined Vaccination with NY-ESO-1 Protein, Poly-ICLC, and Montanide Improves Humoral and Cellular Immune Responses in Patients with High-Risk Melanoma.

Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1-specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in food allergy.

Identification of allergenic IgE epitopes is instrumental for the development of novel diagnostic and prognostic methods in food allergy. In this work, we present the quantification and validation of a Bead-Based Epitope Assay (BBEA) that through multiplexing of epitopes and multiple sample processing enables completion of large experiments in a short period of time, using minimal quantities of patients' blood. Peptides that are uniquely coupled to beads are incubated with serum or plasma samples, and after a secondary fluorophore-labeled antibody is added, the level of fluorescence is quantified with a Luminex reader. The signal is then normalized and converted to epitope-specific antibody binding values. We show that the effect of technical artifacts, i.e. well position or reading order, is minimal; and batch effects - different individual microplate runs - can be easily estimated and eliminated from the data. Epitope-specific antibody binding quantified with BBEA is highly reliable, reproducible and has greater sensitivity of epitope detection compared to peptide microarrays. IgE directed at allergenic epitopes is a sensitive biomarker of food allergy and can be used to predict allergy severity and phenotypes; and quantification of the relationship between epitope-specific IgE and IgG4 can further improve our understanding of the immune mechanisms behind allergic sensitization.

Modeling of behavioral responses for successful selection of easy-to-train rams for semen collection with an artificial vagina.

The aim of this study was to analyze the reproductive behavioral responses in Australian Merino rams, to identify those related to a faster training for semen collection with an artificial vagina. Eight Australian Merino rams, aged 1.5 years and with no prior sexual experience, were randomly selected from an extensively grazed flock. One immobilized ewe with no hormone stimulation was used for rams to sexually interact and mount. The frequencies of approaching, sniffing, flehmen, pushing, pawing with chin resting, and tongue flicking were recorded during eight training and three post-training assessments periods. In addition, the duration of sniffing and flehmen responses, as well as the time from when the ram started to approach the ewe until the mount with ejaculation (completed mount) were recorded. Descriptive, correlation, and modeling analyses were performed. Amongst the rams, four mounted the ewe and ejaculated for the first time during the training phase, and three mounted and ejaculated for the first time after the training phase. The remaining ram mounted the ewe and ejaculated for the first time during the post-training evaluation in the following year. A great variability in the behavior repertoire was observed among rams. The correlation analysis indicated that the completed mount was associated with the behaviors during the approaching response. The expression of the sniffing response decreased between the training phase and post-training evaluation, while the responses of pushing the ewe and tongue flicking ceased to occur. Pawing the side of the ewe with the chin resting on the back of the ewe and flehmen responses, however, continued between the training and post-training phases. This led to a decrease in the time from when the ram started to approach the ewe until the completed mount. It is concluded that the responses of approaching the ewe, pawing the side of the ewe with chin resting on the ewe, and sniffing of the ewe (the latter occurring only during the training phase) are behavioral indicators that could be used for selection of easy-to-train rams for purposes of semen collection with an artificial vagina.

Evaluación del sistema de vigilancia epidemiológica universal de las meningoencefalitis en Paraguay, 2016 / Evaluation of the universal epidemiological surveillance system for meningoencephalitis in Paraguay, 2016

Introducción.Las patologías neurológicas bacterianas constituyen un problema de salud mundial. En Paraguay, se emplean dos tipos de vigilancia epidemiológica para las infecciones meningocócicas: universal y centinela. Objetivo. Evaluar el sistema de vigilancia epidemiológica universal de las meningoencefalitis. Metodología.Estudio descriptivo transversal, basado en las directrices del Updated Guidelines for Evaluating Public Health Surveillance Systems-CDC-2001. Se analizó la base de datos del sistema nacional del 2016 seleccionando en forma aleatoria una muestra de 240 casos sospechosos de un total de 640 casos para evaluar los atributos de calidad de datos, aceptabilidad, oportunidad, sensibilidad y valor predictivo positivo (VPP), mediante una escala de calificaciones. Las medidas de frecuencias y tendencia central (media, mediana) fueron calculadas en Excel. Resultado.La evaluación de la calidad de los datos mostró una completitud de 95% e inconsistencia 4,2%. La aceptabilidad de los actores alcanzó 92%. La oportunidad de toma de muestra ≤ 24h (mediana= 1 día; rango= -31-35 días), el de diagnóstico ≤72h (mediana= 0 día; rango= 0-61 días) y la de notificación ha superado las 24h requeridas (mediana= 3 días; rango= 0-41 días). La sensibilidad del sistema fue 90% y elVPP 7,5%. Conclusión. El sistema es sensible en cuanto a la vigilancia clínica con un VPP bajo, la calidad de datos es excelente, aceptable por los actores, oportuna para la toma de muestras y de diagnóstico. Sin embargo, se debe mejorar la oportunidad de notificación.

Introduction. Bacterial neurological diseases are a global health problem. In Paraguay, two types of epidemiological surveillance are used for meningococcal infections: universal and sentinel. Objetive.The objective was to evaluate the universal epidemiological surveillance system for meningoencephalitis. Methodology:Cross-sectional descriptive study, following the guidelines of the Updated Guidelines for Evaluating Public Health Surveillance Systems-CDC-2001. The database of the national system of 2016 was analyzed. To evaluate the attributes: data quality, acceptability, timeliness, sensitivity and positive predictive value (PPV) the sample size of a total population of 640 was calculated using EpiInfo7.2.2.2, confidence level 95%. The minimum size studied was 240 suspected cases. The grading scale was developed. Random samples of records, frequency measurements and central tendency (mean, median) were calculated in Excel. Results:Epidemiological files were evaluated, the quality of the data in terms of completeness was 95% and inconsistency 4.2%. The acceptability of the actors reached 92%. The opportunity to take a sample ≤ 24 hours (Me= 1 day; R= -31-35 days), diagnosis ≤72h (Me= 0 days; R= 0-61 days) and notification has exceeded the required 24h (Me= 3 days; R= 0-41 days). The sensitivity of thesystem was found to be 90% with a PPV of 7.5%. Conclusion.In conclusion, the system is sensitive in terms of clinical surveillance with a low PPV, the data quality is excellent, acceptable by the actors, timely for sampling and diagnosis. However, the timeliness of notification needs to be improved.