Precision engineering for localization, validation, and modification of allergenic epitopes.

The allergen-IgE interaction is essential for the genesis of allergic responses, yet investigation of the molecular basis of these interactions is in its infancy. Precision engineering has unveiled the molecular features of allergen-antibody interactions at the atomic level. High-resolution technologies, including x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy, determine allergen-antibody structures. X-ray crystallography of an allergen-antibody complex localizes in detail amino acid residues and interactions that define the epitope-paratope interface. Multiple structures involving murine IgG mAbs have recently been resolved. The number of amino acids forming the epitope broadly correlates with the epitope area. The production of human IgE mAbs from B cells of allergic subjects is an exciting recent development that has for the first time enabled an actual IgE epitope to be defined. The biologic activity of defined IgE epitopes can be validated in vivo in animal models or by measuring mediator release from engineered basophilic cell lines. Finally, gene-editing approaches using the Clustered Regularly Interspaced Short Palindromic Repeats technology to either remove allergen genes or make targeted epitope engineering at the source are on the horizon. This review presents an overview of the identification and validation of allergenic epitopes by precision engineering.

Structural analysis of human IgE monoclonal antibody epitopes on dust mite allergen Der p 2.

BACKGROUND: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire. OBJECTIVE: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2. METHODS: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis. RESULTS: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants. CONCLUSIONS: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Human Monoclonal IgE Antibodies-a Major Milestone in Allergy.

PURPOSE OF REVIEW: Bound to its high affinity receptor on mast cells and basophils, the IgE antibody molecule plays an integral role in the allergic reaction. Through interactions with the allergen, it provides the sensitivity and specificity parameters for cell activation and mediator release that produce allergic symptoms. Advancements in human hybridoma technologies allow for the generation and molecular definition of naturally occurring allergen-specific human IgE monoclonal antibodies. RECENT FINDINGS: A high-resolution structure of dust mite allergen Der p 2 in complex with Fab of the human IgE mAb 2F10 was recently determined using X-ray crystallography. The structure reveals the fine molecular details of IgE 2F10 binding its 750 Å2 conformational epitope on Der p 2. This review provides an overview of this major milestone in allergy, the first atomic resolution structure of an authentic human IgE epitope. The molecular insights that IgE epitopes provide will allow for structure-based design approaches to the development of novel diagnostics, antibody therapeutics, and immunotherapies.

Predictors of hospital-acquired pressure injuries in patients with complete spinal cord injury: a retrospective case-control study.

BACKGROUND: Despite current best practices, pressure injuries (PI) remain a devastating and prevalent hospital-acquired complication for patients with acute traumatic spinal cord injuries (SCIs). This study examined associations between risk factors for PI development in patients with complete SCI, such as norepinephrine dose and duration, and other demographic factors or lesion characteristics. METHODS: This case-control study included adults with acute complete SCIs ASIA-A, who were admitted to a level-one trauma center between 2014-18. A retrospective review was implement using data on patient and injury characteristics, including age, gender, level of SCI (cervical vs. thoracic), Injury Severity Score (ISS), length of stay (LOS) and mortality; presence/absence of PI during their acute hospital stay; and treatment factors such as spinal surgery, mean arterial pressure (MAP) targets, and vasopressor treatment. Multivariable logistic regression evaluated associations with PI. RESULTS: Eighty-two out of 103 eligible patients had complete data, and 30 (37%) developed PIs. Patient and injury characteristics, including age (Mean: 50.6; SD:21.3), location of SCI (48 cervical, 59%) and ISS (Mean 33.1; SD:11.8), did not differ between PI and non-PI groups. Logistic regression analysis revealed that male gender (OR:34.1; CI95:2.3-506.5, p = 0.010) and increased LOS (log-transformed; OR:20.5, CI95:2.8-149.9, p = 0.003) were associated with increased risk of PI. Having an order for a MAP > 80mmg (OR:0.05; CI95:0.01-0.30, p = 0.001) was associated with a reduced risk of PI. There were no significant associations between PI and duration of norepinephrine treatment. CONCLUSIONS: Norepinephrine treatment parameters were not associated with development of PI, suggesting that MAP targets should be a focus for future investigations for SCI management. Increasing LOS should highlight the need for high-risk PI prevention and vigilance.

Proteomics-Based Approach for Detailing the Allergenic Profile of Cannabis Chemotypes.

Allergic sensitization to cannabis is an emerging public health concern and is difficult to clinically establish owing to lack of standardized diagnostic approaches. Attempts to develop diagnostic tools were largely hampered by the Schedule I restrictions on cannabis, which limited accessibility for research. Recently, however, hemp was removed from the classified list, and increased accessibility to hemp allows for the evaluation of its practical clinical value for allergy diagnosis. We hypothesized that the proteomic profile is preserved across different cannabis chemotypes and that hemp would be an ideal source of plant material for clinical testing. Using a proteomics-based approach, we examined whether distinct varieties of cannabis plant contain relevant allergens of cannabis. Cannabis extracts were generated from high tetrahydrocannabinol variety (Mx), high cannabidiol variety (V1-19) and mixed profile variety (B5) using a Plant Total Protein Extraction Kit. Hemp extracts were generated using other standardized methods. Protein samples were subjected to nanoscale tandem mass spectrometry. Acquired peptides sequences were examined against the Cannabis sativa database to establish protein identity. Non-specific lipid transfer protein (Can s 3) level was measured using a recently developed ELISA 2.0 assay. Proteomic analysis identified 49 distinct potential allergens in protein extracts from all chemotypes. Most importantly, clinically relevant and validated allergens, such as profilin (Can s 2), Can s 3 and Bet v 1-domain-containing protein 10 (Can s 5), were identified in all chemotypes at label-free quantification (LFP) intensities > 106. However, the oxygen evolving enhancer protein 2 (Can s 4) was not detected in any of the protein samples. Similarly, Can s 2, Can s 3 and Can s 5 peptides were also detected in hemp protein extracts. The validation of these findings using the ELISA 2.0 assay indicated that hemp extract contains 30-37 ng of Can s 3 allergen per µg of total protein. Our proteomic studies indicate that relevant cannabis allergens are consistently expressed across distinct cannabis chemotypes. Further, hemp may serve as an ideal practical substitute for clinical testing, since it expresses most allergens relevant to cannabis sensitization, including the validated major allergen Can s 3.

Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5.

BACKGROUND: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. METHODS: A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. RESULTS: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. CONCLUSIONS: Based on the collaborative study results, the assessed Phl p 5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.

Mapping Human Monoclonal IgE Epitopes on the Major Dust Mite Allergen Der p 2.

IgE Abs drive the symptoms of allergic disease upon cross-linking allergens on mast cells or basophils. If the IgE binding sites on the allergens could be identified, it may be useful for creating new forms of immunotherapy. However, direct knowledge of the human IgE (hIgE) epitopes is limited because of the very low frequency of IgE-producing B cells in blood. A new hybridoma technology using human B cells from house dust mite-allergic patients was used to identify four Der p 2-specific hIgE mAbs. Their relative binding sites were assessed and compared by immunoassays with three previously studied murine IgG mAbs. Immunoassays showed that the recognition of Der p 2 by the first three hIgE was inhibited by a single murine IgG, but the fourth hIgE recognized a different epitope from all the other mAbs. The functional ability of the hIgE that bind different epitopes to cross-link Der p 2 was demonstrated in a mouse model of passive systemic anaphylaxis. Nuclear magnetic resonance analyses of Der p 2 in complex with IgG and IgE Abs were used to identify specific residues in the epitopes. To our knowledge, the combination of immunoassays to distinguish overlapping epitopes and nuclear magnetic resonance analyses to identify specific residues involved in Ab binding provided the first epitope mapping of hIgE mAbs to an allergen. The technologies developed in this study will be useful in high-resolution mapping of human epitopes on other Ags and the design of improved therapeutics.

Bos d 11 in baked milk poses a risk for adverse reactions in milk-allergic patients.

BACKGROUND: Patients are commonly challenged with foods containing baked milk, for example muffins, yet little is known about the specific allergen content of muffins used in milk challenges or of the effect that baking has on allergenicity. OBJECTIVE: Our objective was to compare the levels of major milk allergens in uncooked and baked muffins using monoclonal immunoassays and IgE antibody binding before and after baking. METHODS: Uncooked and baked muffins were prepared using recipes from Mount Sinai and Imperial College. Allergen levels were compared by ELISA for Bos d 5 (ß-lactoglobulin) and Bos d 11 (ß-casein). IgE reactivity was assessed using sera from milk-sensitized donors in direct binding and inhibition ELISA. RESULTS: Bos d 5 was reduced from 680 µg/g in uncooked muffin mix to 0.17 µg/g in baked muffins, representing a >99% decrease after baking. Conversely, Bos d 11 levels in baked muffin remained high and only decreased by 30% from a mean of 4249 µg/g in uncooked muffin mix to 2961 µg/g when baked (~181 mg Bos d 11 per muffin). Baked muffins retained ~70% of the IgE binding to uncooked muffin mix. Baked muffin extract inhibited IgE binding to uncooked muffin mix by up to 80%, demonstrating retention of in vitro IgE reactivity. CONCLUSIONS AND CLINICAL RELEVANCE: High levels of Bos d 11 in baked muffins pose a risk for adverse reactions, especially in patients who have high anti-casein IgE antibodies.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Protected Code Stroke: Hyperacute Stroke Management During the Coronavirus Disease 2019 (COVID-19) Pandemic.

Background and Purpose- Hyperacute assessment and management of patients with stroke, termed code stroke, is a time-sensitive and high-stakes clinical scenario. In the context of the current coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 virus, the ability to deliver timely and efficacious care must be balanced with the risk of infectious exposure to the clinical team. Furthermore, rapid and effective stroke care remains paramount to achieve maximal functional recovery for those needing admission and to triage care appropriately for those who may be presenting with neurological symptoms but have an alternative diagnosis. Methods- Available resources, COVID-19-specific infection prevention and control recommendations, and expert consensus were used to identify clinical screening criteria for patients and provide the required nuanced considerations for the healthcare team, thereby modifying the conventional code stroke processes to achieve a protected designation. Results- A protected code stroke algorithm was developed. Features specific to prenotification and clinical status of the patient were used to define precode screening. These include primary infectious symptoms, clinical, and examination features. A focused framework was then developed with regard to a protected code stroke. We outline the specifics of personal protective equipment use and considerations thereof including aspects of crisis resource management impacting team role designation and human performance factors during a protected code stroke. Conclusions- We introduce the concept of a protected code stroke during a pandemic, as in the case of COVID-19, and provide a framework for key considerations including screening, personal protective equipment, and crisis resource management. These considerations and suggested algorithms can be utilized and adapted for local practice.

Promotion of Regular Oesophageal Motility to Prevent Regurgitation and Enhance Nutrition Intake in Long-Stay ICU Patients. A Multicenter, Phase II, Sham-Controlled, Randomized Trial: The PROPEL Study.

OBJECTIVES: To evaluate the effect of esophageal stimulation on nutritional adequacy in critically ill patients at risk for enteral feeding intolerance. DESIGN: A multicenter randomized sham-controlled clinical trial. SETTING: Twelve ICUs in Canada. PATIENTS: We included mechanically ventilated ICU patients who were given moderate-to-high doses of opioids and expected to remain alive and ventilated for an additional 48 hours and who were receiving enteral nutrition or expected to start imminently. INTERVENTIONS: Patients were randomly assigned 1:1 to esophageal stimulation via an esophageal stimulating catheter (E-Motion Tube; E-Motion Medical, Tel Aviv, Israel) or sham treatment. All patients were fed via these catheters using a standardized feeding protocol. MEASUREMENTS AND MAIN RESULTS: The co-primary outcomes were proportion of caloric and protein prescription received enterally over the initial 7 days following randomization. Among 159 patients randomized, the modified intention-to-treat analysis included 155 patients: 73 patients in the active treatment group and 82 in the sham treatment group. Over the 7-day study period, the percent of prescribed caloric intake (± SE) received by the enteral route was 64% ± 2 in the active group and 65% ± 2 in sham patients for calories (difference, -1; 95% CI, -8 to 6; p = 0.74). For protein, it was 57% ± 3 in the active group and 60% ± 3 in the sham group (difference, -3; 95% CI, -10 to 3; p = 0.30). Compared to the sham group, there were more serious adverse events reported in the active treatment group (13 vs 6; p = 0.053). Clinically important arrhythmias were detected by Holter monitoring in 36 out of 70 (51%) in the active group versus 22 out of 76 (29%) in the sham group (p = 0.006). CONCLUSIONS: Esophageal stimulation via a special feeding catheter did not improve nutritional adequacy and was associated with increase risk of harm in critically ill patients.

Anesthesia considerations of magnetic resonance imaging-guided focused ultrasound thalamotomy for essential tremor: a case series.

PURPOSE: Essential tremor (ET) is a common movement disorder with disability in voluntary actions such as eating and writing. First-line treatment involves pharmacological agents, although efficacy is limited by side effects. In these patients, functional neurosurgery can be considered. Magnetic resonance imaging-guided focused ultrasound (MRgFUS) thalamotomy offers a non-invasive solution for treatment. This paper examines an original cohort of ET patients undergoing MRgFUS thalamotomy and discusses the anesthetic management of these cases. METHODS: We retrospectively reviewed the anesthetic records of all MRgFUS thalamotomy cases from 15 May 2012 to 16 July 2015 at our centre (Sunnybrook Health Sciences Centre, Toronto, Canada) to expand a data set provided by the focused ultrasound system manufacturer (Insightec, Tirat Carmel, Israel) from a prior phase-II regulatory approval study. Specific drug and procedural details were listed including aspects of the patients' experience. RESULTS: A total of 82 patients were included in the analysis, 78 from a phase-II trial (16 were from the local site) and four local non-trial cases. No patient required general anesthesia and only 29% of cases required sedation to tolerate the procedure. The most frequent medications required were antiemetics and analgesics. Headache (31%) was the most frequent perioperative symptom. Transient intra-procedural paresthesia symptoms were a common occurrence (32%). CONCLUSIONS: The use of MRgFUS for thalamotomy provides a non-invasive and well-tolerated method for treating ET, which usually only requires monitored anesthesia care sedation. Nevertheless, there are several predictable side effects that require contingency planning including the personnel and means to resolve them.

RéSUMé: OBJECTIF: Le tremblement essentiel (TE) est un trouble moteur courant qui affecte les mouvements volontaires tels que se nourrir ou écrire. Le traitement de première ligne est basé sur des agents pharmacologiques, mais son efficacité est limitée par les effets secondaires. Chez de tels patients, une neurochirurgie fonctionnelle peut être envisagée. La thalamotomie par ultrasons focalisés guidés par imagerie de résonance magnétique (ou MRgFUS) offre une solution de traitement non invasive. Cet article examine une cohorte initiale de patients souffrant de TE et subissant une thalamotomie par MRgFUS et discute de la prise en charge anesthésique de ces cas. MéTHODE: Nous avons évalué de manière rétrospective les dossiers anesthésiques de toutes les interventions de thalamotomie par MRgFUS réalisées entre le 15 mai 2012 et le 16 juillet 2015 dans notre centre (Sunnybrook Health Sciences Centre, Toronto, Canada) afin d'élargir un ensemble de données fourni par le fabricant de systèmes d'ultrasons focalisés (Insightec, Tirat Carmel, Israël) basé sur une étude d'approbation règlementaire de phase II. Les médicaments spécifiques et détails procéduraux ont été enregistrés, y compris divers aspects de l'expérience des patients. RéSULTATS: Un total de 82 patients ont été inclus dans l'analyse, dont 78 participants de l'étude de phase II (16 au site local) et quatre cas locaux non inclus dans l'étude. Aucun patient n'a nécessité d'anesthésie générale et seulement 29 % des cas ont nécessité une sédation pour tolérer l'intervention. Les médicaments les plus fréquemment requis étaient les antiémétiques et analgésiques. Les céphalées (31 %) constituaient le symptôme périopératoire le plus fréquent. Des symptômes de paresthésie intra-procédurale transitoire étaient fréquents (32 %). CONCLUSION: L'utilisation de la MRgFUS pour la thalamotomie offre une méthode non invasive et bien tolérée pour le traitement du tremblement essentiel, qui ne nécessite habituellement qu'une sédation sous surveillance comme soin anesthésique. Toutefois, cette intervention a plusieurs effets secondaires prévisibles qui nécessitent un plan de contingence incluant le personnel et les moyens pour les traiter.

Allergen content in German cockroach extracts and sensitization profiles to a new expanded set of cockroach allergens determine in vitro extract potency for IgE reactivity.

BACKGROUND: Cockroach allergens are an important cause of IgE-mediated sensitization in inner-city asthmatic patients. However, cockroach extracts used for diagnosis and immunotherapy are not standardized. OBJECTIVE: We sought to determine the allergen content of nonstandardized German cockroach extracts and the levels of sensitization to an expanded set of cockroach allergens as determinants of in vitro extract potency for IgE reactivity. METHODS: Twelve German cockroach extracts were compared for allergen content and potency of IgE reactivity. Bla g 1, Bla g 2, and Bla g 5 were measured by using immunoassays. IgE antibody levels to 8 purified recombinant allergens from groups 1, 2, 4, 5, 6, 7, 9, and 11 were measured by using ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract in vitro potencies (concentration inhibiting 30% of the total IgE antibody-binding inhibition) relative to an arbitrarily selected reference extract in 5 patients with cockroach allergy. RESULTS: Allergen levels were highly variable. Three new major allergens (groups 6, 9, and 11), were identified among highly cockroach-sensitized subjects (CAP class ≥ 3). Sensitization profiles were unique per subject without immunodominant allergens. The sum of IgE to 8 allergen components showed a good correlation with cockroach-specific IgE levels (r = 0.88, P < .001). In vitro potencies varied among different extracts per subject and among subjects for each extract. CONCLUSIONS: The in vitro potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen-specific IgE titers of patients with cockroach allergy. These factors are relevant for selection of potent extracts to be used for immunotherapy and for the design and interpretation of data from immunotherapy trials.

Antiepileptic Drugs to Prevent Seizures After Spontaneous Intracerebral Hemorrhage.

Background and Purpose- We sought to evaluate the available literature to determine whether primary seizure prevention with antiepileptic drugs reduces the risk of poor outcomes and clinically relevant seizures among adult patients with spontaneous intracerebral hemorrhage. Methods- Meta-analysis of observational studies and randomized controlled trials evaluating the use of any antiepileptic drug for primary seizure prevention among adult (≥18 years) patients with spontaneous intracerebral hemorrhage. The primary end point was poor clinical outcome at the longest recorded follow-up, defined as either a high (>3) modified Rankin Scale score or all-cause mortality during follow-up if the modified Rankin Scale score was not recorded. Early and late seizures were secondary outcomes. A random mixed effects model was used to estimate the pooled odds ratio of outcomes and associated 95% CI. Results- We identified 7 studies with a total of 3241 patients for analysis of the primary outcome and 4 studies with a total of 1861 patients for analysis of the secondary outcomes. Overall, the use of antiepileptic drugs was not associated with a high Rankin Scale or all-cause mortality (odds ratio: 0.99; 95% CI, 0.66-1.49) or incident seizures (odds ratio: 0.89; 95% CI, 0.52-1.51) at the longest recorded follow-up time. Conclusions- The use of antiepileptic drugs as primary prevention among adult patients with spontaneous intracerebral hemorrhage is not associated with improved neurological function during long-term follow-up. Future studies should focus on the preventive use of distinct antiepileptic agents among patients at high risk of both seizures and poor outcomes.

Antigenic Determinants of Der p 1: Specificity and Cross-Reactivity Associated with IgE Antibody Recognition.

Der p 1 and Der f 1 are major allergens from Dermatophagoides pteronyssinus and D. farinae, respectively. An analysis of antigenic determinants on both allergens was performed by site-directed mutagenesis. The analysis was based on the x-ray crystal structures of the allergens in complex with Fab fragments of three murine mAbs that interfere with IgE Ab binding: the two Der p 1-specific mAbs 5H8 and 10B9, and the cross-reactive mAb 4C1. On one hand, selected residues in the epitopes for mAb 5H8 and mAb 4C1 were substituted with amino acids that resulted in impaired Ab binding to Der p 1. On the other hand, an epitope for the Der p 1-specific mAb 10B9, which partially overlaps with mAb 4C1, was created in Der f 1. The mutation of 1-3 aa residues in Der f 1 was sufficient to bind mAb 10B9. These residues form hydrogen bonds with CDRs of the Ab other than H CDR3. This observation unveils an exception to the dominant role of H CDR3 commonly observed in Ag recognition. Overall, this study resulted in the identification of important residues for mAb and IgE Ab recognition in group 1 mite allergens. This information can be used to engineer allergen mutants with reduced IgE Ab binding for immunotherapy.

Specific allergen profiles of peanut foods and diagnostic or therapeutic allergenic products.

BACKGROUND: Generic immunoassays for peanut cannot discriminate between allergen levels in peanut-derived food products or therapeutics. Clinical trials of oral immunotherapy (OIT) are strengthened by using standardized peanut preparations with defined doses of major allergens. OBJECTIVE: This article describes measurement of Ara h 1, Ara h 2, and Ara h 6 in peanut foods and in peanut flour extracts used for allergy diagnosis and OIT. METHODS: Monoclonal antibody-based enzyme immunoassays for Ara h 1, Ara h 2, and Ara h 6 were used to compare allergen levels in peanut (n = 16) and tree nut (n = 16) butter, peanut flour (n = 11), oils (n = 8), extracts used for diagnosis and OIT (n = 5), and the National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387. RESULTS: Roasted peanut butters contained 991 to 21,406 µg/g Ara h 1 and exceeded Ara h 2 and Ara h 6 levels by 2- to 4-fold. Similarly, National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387 contained 11,275 µg/g Ara h 1, 2,522 µg/g Ara h 2, and 2,036 µg/g Ara h 6. In contrast, peanut flours contained 787 to 14,631 µg/g Ara h 2 and exceeded Ara h 1 levels by 2- to 20-fold. Flour extracts used for OIT contained 394 to 505 µg/mL Ara h 1, 1,187 to 5,270 µg/mL Ara h 2, and 1,104 to 8,092 µg/mL Ara h 6. In most cases specific peanut allergens were not detected in tree nut butters or peanut oils. CONCLUSIONS: The results show marked differences in specific peanut allergen profiles in peanut butter and flour and peanut preparations for clinical use. Roasting can increase Ara h 1 levels in peanut butter. Variability in allergen levels could affect the outcome of clinical trials of peanut OIT, especially with respect to Ara h 1. Specific allergen measurements will improve standardization and provide accurate dosing of peanut preparations that are being used for OIT.

Half-life of IgE in serum and skin: Consequences for anti-IgE therapy in patients with allergic disease.

We present results from clinical studies on plasma infusion done in the late 1970s in patients with hypogammaglobulinemia in which we documented the short half-life of both total and allergen-specific IgE in serum. The development of specific allergic sensitization in the skin of those patients followed by the gradual decrease in sensitization over 50 days was also documented. The data are included here along with a discussion of the existing literature about the half-life of IgE in both the circulation and skin. This rostrum reinterprets the earlier clinical studies in light of new insights and mechanisms that could explain the rapid removal of IgE from the circulation. These mechanisms have clinical implications that relate to the increasing use of anti-IgE mAbs for the treatment of allergic disease.